Method for efficiently preparing cyclodepsipeptide, cyclodepsipeptide and application
阅读说明：本技术 高效制备环缩肽的方法、环缩肽及应用 (Method for efficiently preparing cyclodepsipeptide, cyclodepsipeptide and application ) 是由 尹文兵 蔡磊 梁敏 李伟 于 2021-01-22 设计创作，主要内容包括：本申请涉及药用化合物制备领域,尤其涉及一种高效制备环缩肽的方法、环缩肽及应用。该方法包括：将蝙蝠粪便寄生真菌Amphichorda guana LC5815接种于培养基中发酵,得到发酵培养物；从所述发酵培养物中,分离得到环缩肽化合物。(The application relates to the field of preparation of medicinal compounds, in particular to a method for efficiently preparing cyclodepsipeptide, the cyclodepsipeptide and application. The method comprises the following steps: inoculating bat fecal parasitic fungus Amphichorda guana LC5815 into culture medium for fermentation to obtain fermentation culture; isolating the cyclodepsipeptide compound from the fermentation culture.)
1. A method of making a cyclodepsipeptide compound comprising:
inoculating bat fecal parasitic fungus Amphichorda guana LC5815 into culture medium for fermentation to obtain fermentation culture;
isolating the cyclodepsipeptide compound from the fermentation culture.
2. The method of claim 1, wherein the culture medium is obtained by mixing 1.5L of water per 1kg of rice.
3. The method of claim 1, wherein said inoculating a bat fecal parasitic fungus, amphickorda guana LC5815, in a culture medium for fermentation comprises:
inoculating Amphickora guana LC5815 parasitic fungus in bat feces onto potato glucose agar culture medium, and culturing at 25-28 deg.C for 5-7 days to obtain activated strain;
collecting spores of the activated strain by using 0.1% (w/v) Tween-80 to obtain seed liquid;
inoculating the seed solution to the culture medium, and culturing at 25-28 deg.C for 25-35 days for fermentation.
4. The method of any one of claims 1-3, wherein the cyclodepsipeptide compound comprises one or more cyclodepsipeptides of the formula (1) to (8);
5. the method of any one of claims 1-3, wherein the cyclic depsipeptide compound has a structural formula as shown in formula (5);
said isolating from said fermentation culture a cyclodepsipeptide compound comprising:
adding ethyl acetate into the fermentation culture, and soaking to obtain a leaching solution;
evaporating and drying the leaching liquor under vacuum to obtain a crude extract;
carrying out chromatographic separation on the crude extract by using a 200-mesh 300-mesh silica gel column, eluting by using acetone, and evaporating and drying the obtained fraction under vacuum to obtain a dry substance;
purifying the dry matter using a small pore resin; wherein, methanol water solution is used for elution to obtain a first elution product; in the methanol aqueous solution, the volume ratio of methanol to water is 7: 3;
separating and purifying the first eluted product by using a high performance liquid chromatography column; performing gradient elution by using acetonitrile water solution, and collecting a chromatographic peak with retention time of 26.7min to obtain the cyclodepsipeptide shown in the formula (5); in the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 85: 15.
6. the method of any one of claims 1-3, wherein the cyclic depsipeptide compound has a structural formula as shown in formula (1);
said isolating from said fermentation culture a cyclodepsipeptide compound comprising:
adding ethyl acetate into the fermentation culture, and soaking to obtain a leaching solution;
evaporating and drying the leaching liquor under vacuum to obtain a crude extract;
carrying out chromatographic separation on the crude extract by using a 200-mesh 300-mesh silica gel column, eluting by using acetone, and evaporating and drying the obtained fraction under vacuum to obtain a dry substance;
purifying the dry matter using a small pore resin; wherein, methanol water solution is used for elution to obtain a first elution product; in the methanol aqueous solution, the volume ratio of methanol to water is 9: 1;
separating and purifying the first eluted product using a high performance liquid chromatography column; performing gradient elution by using acetonitrile water solution, and collecting a chromatographic peak with retention time of 17.5min to obtain the cyclodepsipeptide shown in the formula (1); in the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 85: 15.
7. a cyclodepsipeptide, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the cyclodepsipeptide has a structural formula shown in formula (1):
8. the cyclic depsipeptide according to claim 7, wherein the cyclic depsipeptide is obtained by fermentation using bat fecal parasitic fungus Amphickorda guana LC5815 as a zymogen; the medium for the fermentation was obtained by mixing 1.5L of water per 1kg of rice.
9. Use of a cyclodepsipeptide according to any one of claims 7 or 8 in the preparation of a fungal or bacterial fungicide.
10. The bat fecal parasitic fungus amphickorda guana LC5815 is used in the production of cyclic depsipeptide compounds.
Technical Field
The application relates to the field of preparation of medicinal compounds, in particular to a method for efficiently preparing cyclodepsipeptide, the cyclodepsipeptide and application.
Background
The fungus pesticide is a bioactive preparation for controlling harmful insects in nature by using fungi as a biological control means. The main fungal insecticides at present are beauveria bassiana, metarhizium anisopliae, paecilomyces lilacinus and the like. Although the fungal pesticide has the advantages of wide insecticidal spectrum, capability of being spread among target pests and the like, the development of the fungal pesticide industry is slow for a long time, and high-yield strains are also important except the problems of industrial policy, application concept, industrial production and the like.
Many reports have been made in the literature on the insecticidal activity of cyclic depsipeptide isarins compounds, such as against Plasmodium falciparum, greater wax moth larvae (Galleria mellonella larvae), and insect pests (Sitophilus spp.). The cyclic depsipeptide isaridins compound has the effects of resisting bacteria, plant pathogenic fungi, inflammatory factor and the like. To date, it has been found that 20 more species of isariins and isariidins cyclodepsipeptides are isolated primarily from Isaria cretacea and I.felina of the genus Corynebacterium and the marine fungus Beauveria felina.
At present, the yield of the cyclodepsipeptide is low, and the yield of the cyclodepsipeptide is improved, so that the cyclodepsipeptide cyclic synthesis method has important significance for biological control.
Disclosure of Invention
The application provides a method for efficiently preparing cyclodepsipeptide, the cyclodepsipeptide and application, and can efficiently prepare active cyclodepsipeptide.
In a first aspect, the present application provides a method of preparing a cyclodepsipeptide compound comprising: inoculating bat fecal parasitic fungus Amphichorda guana LC5815 into culture medium for fermentation to obtain fermentation culture; isolating the cyclodepsipeptide compound from the fermentation culture.
In one example, the medium is obtained by mixing 1.5L of water per 1kg of rice.
In one embodiment, the bat fecal parasitic fungus amphickorda guana LC5815 is inoculated into a culture medium for fermentation, comprising:
inoculating Amphickora guana LC5815 parasitic fungus in bat feces onto potato glucose agar culture medium, and culturing at 25-28 deg.C for 5-7 days to obtain activated strain;
collecting spores of the activated strain by using 0.1% (w/v) Tween-80 to obtain seed liquid;
inoculating the seed solution to the culture medium, and culturing at 25-28 deg.C for 25-35 days for fermentation.
In one embodiment, the cyclodepsipeptide compound comprises one or more cyclodepsipeptides having the structural formula shown in formulas (1) - (8);
in one embodiment, the cyclodepsipeptide compound has a structure as shown in formula (5);
said isolating from said fermentation culture a cyclodepsipeptide compound comprising:
adding ethyl acetate into the fermentation culture, and soaking to obtain a leaching solution;
evaporating and drying the leaching liquor under vacuum to obtain a crude extract;
carrying out chromatographic separation on the crude extract by using a 200-mesh 300-mesh silica gel column, eluting by using acetone, and evaporating and drying the obtained fraction under vacuum to obtain a dry substance;
purifying the dry matter using a small pore resin; wherein, methanol water solution is used for elution to obtain a first elution product; in the methanol aqueous solution, the volume ratio of methanol to water is 7: 3;
separating and purifying the first eluted product by using a high performance liquid chromatography column; performing gradient elution by using acetonitrile water solution, and collecting a chromatographic peak with retention time of 26.7min to obtain the cyclodepsipeptide shown in the formula (5); in the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 85: 15.
in one embodiment, the cyclodepsipeptide compound has the structural formula shown in formula (1);
said isolating from said fermentation culture a cyclodepsipeptide compound comprising:
adding ethyl acetate into the fermentation culture, and soaking to obtain a leaching solution;
evaporating and drying the leaching liquor under vacuum to obtain a crude extract;
carrying out chromatographic separation on the crude extract by using a 200-mesh 300-mesh silica gel column, eluting by using acetone, and evaporating and drying the obtained fraction under vacuum to obtain a dry substance;
purifying the dry matter using a small pore resin; wherein, methanol water solution is used for elution to obtain a first elution product; in the methanol aqueous solution, the volume ratio of methanol to water is 9: 1;
separating and purifying the first eluted product using a high performance liquid chromatography column; performing gradient elution by using acetonitrile water solution, and collecting a chromatographic peak with retention time of 17.5min to obtain the cyclodepsipeptide shown in the formula (1); in the aqueous acetonitrile solution, the volume ratio of acetonitrile to water is 85: 15.
in a second aspect, the present application provides a cyclodepsipeptide, a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the cyclodepsipeptide has a structural formula as shown in formula (1):
in one embodiment, the cyclic depsipeptide is obtained by fermenting with a bat fecal parasitic fungus amphicora guiana LC5815 as a zymogen; the fermentation medium for the fermentation was obtained by mixing 1.5L of water per 1kg of rice.
In a third aspect, the present application provides the use of a cyclodepsipeptide of the second aspect in the preparation of a fungal or bacterial fungicide.
In a fourth aspect, the present application provides the use of the bat fecal parasitic fungus amphickorda guana LC5815 in the manufacture of a cyclic depsipeptide compound.
In the embodiment of the application, bat fecal parasitic fungus Amphickorda guana LC5815 is used as a fermentation strain, a rice culture medium is used as a fermentation culture medium, and fermentation is carried out, so that various cyclodepsipeptides can be produced, and particularly, the yield of cyclodepsipeptide iso-isarin B is over 14 percent; and can produce a novel cyclic depsipeptide isaridin H having fungal and bacterial inhibitory ability.
Drawings
FIG. 1 shows the results of LC5815 solid fermentation cultures of the bat fecal parasitic fungus Amphickora guana;
FIG. 2 shows the HPLC analysis results of active cyclodepsipeptide fractions produced by the present application using the bat fecal parasitic fungus Amphichora guana LC 5815;
FIG. 3 shows the HPLC-MS/MS analysis of the cyclodepsipeptide isarii A prepared in the present application;
FIG. 4 shows the HPLC-MS/MS analysis of the cyclodepsipeptide iso-isarin B prepared in the present application;
FIG. 5 is the results of analysis of secondary metabolites of the bat fecal parasitic fungus Amphichora guana LC5815 in different media;
FIG. 6 shows the antifungal ability of cyclodepsipeptide compounds prepared in accordance with the present application;
FIG. 7 shows the antibacterial ability of cyclodepsipeptide compounds prepared in the present application.
Detailed Description
In the following, in specific examples, the solutions provided in the present specification are described by way of example.
Fungi that grow in special environments often have special physiological and metabolic characteristics, such as endophytic fungi, marine fungi, and copromogenous fungi. Some reports indicate that endophytic fungi are capable of biosynthesizing important medicinal "phytochemicals", and that many new bioactive natural products have also been discovered from marine fungi. For example, taxol from the endophytic fungus Taxomyces andreanae and destruxin from the marine fungus Beauveria felina play important roles in pharmaceuticals and agrochemicals. In view of the inherent microbial competition in the feces of short lived animals, fecal fungi are a source of abundant natural products. In recent years, coprophilic fungi have received increasing attention due to their widespread existence and research convenience. Amphickord guana LC5815 is a fungus parasitic on bat feces, only two species of Amphickord felina and Amphickord guana exist in the genus of Amphickord, and active cyclodepsipeptide immunosuppressant cyclosporin has been isolated from Amphickord felina, while we have studied the chemical composition of Amphickord guana LC5815 for the first time and mined 8 active cyclodepsipeptide compounds from it, including a new cyclodepsipeptide and a cyclodepsipeptide iso-isarin B with a yield of more than 14%. Therefore, the discovery of the high-producing strain of the active cyclodepsipeptide compound has extremely important effect on the fields of agriculture and medicine.
The present examples provide an active cyclodepsipeptide high producing strain which is the bat fecal parasitic fungus amphickorda guana LC 5815. The strain is separated from the Suiuyang of Guizhou, a natural protection area of a broad water country and a famous cave. The strain has transparent, smooth and septate vegetative hyphae with diameter of 1.5-3.5 μm, and sometimes 7.0 μm. The catena is generated in the center of colony on Potato Dextrose Agar (PDA) plate, has height of 15mm, width of 1-3mm, white, cylindrical shape, and is filled with villus and branches at the top. Conidiophores are grown laterally on hyphae, straight or slightly curved. Spore-forming cells are mostly born on conidiophores, occasionally on hyphae, fusiform or oval, straight or irregularly curved, 7-10x2-3 μm. Conidium is in an exogeminal type, is single or clustered, is transparent, smooth, wide in an ellipsoid shape to a nearly spherical shape, and is single-cell.
The bat fecal parasitic fungus Amphickorda guana LC5815 has been preserved in China general microbiological culture Collection center (CGMCC, No. 3 of the institute of microbiology, China academy of sciences) at 2016, 29/1/29/2016, and the preservation number is CGMCC 3.17908.
Hereinafter, the bat fecal parasitic fungus amphickorda guana LC5815 may be referred to simply as bat fecal parasitic fungus a.guana, or, bat fecal parasitic fungus, or, a.guana.
The embodiment of the application utilizes bat fecal parasitic fungus Amphichorda guana LC5815 to ferment and obtain 8 cyclic depsipeptides with structural formulas shown as formulas (1) to (8) in sequence.
Specifically, the compound represented by the formula (1) is referred to as isaridin H, the compound represented by the formula (2) is referred to as desmethylisasarin E, the compound represented by the formula (3) is referred to as isaridin E, the compound represented by the formula (4) is referred to as isaridin a, the compound represented by the formula (5) is referred to as iso-isaridin B, the compound represented by the formula (6) is referred to as iso-isaridin D, the compound represented by the formula (7) is referred to as isaridin E, and the compound represented by the formula (8) is referred to as nodipetide.
Next, the production process and properties of the cyclodepsipeptide will be described.
Example 1 preliminary analysis of the bat fecal parasitic fungus Amphichorda guana LC5815 solid fermentate by liquid chromatography-mass spectrometry (LC-MS)
Inoculating the parasitic fungus A. guana of bat feces to glucose agar culture medium (PDA) for activating the strain at 28 deg.C for 5-7 days. Collecting spores of the activated strain by using 0.1% (w/v) Tween-80, inoculating a proper amount of seed liquid for solid fermentation, wherein a culture medium adopted by the solid fermentation consists of 20g of rice and 30mL of water, and the culture condition of the solid fermentation is 28 ℃, and standing and culturing for 7 days in a dark place. For convenience of description, a medium consisting of 30mL of water mixed per 20g of rice may be referred to as a rice medium. The above experiment was repeated 3 times. Extracting the fermented product with ethyl acetate (specifically, extracting with 7.5L ethyl acetate three times per 1kg of fermentation medium, directly adding ethyl acetate after fermentation culture, extracting with 50mL ethyl acetate in 250mL triangular flask, repeating for 3 times, and 150mL ethyl acetate in total), ultrasonic treating for 1 hr, filtering with glass funnel, collecting filtrate, drying with rotary vacuum concentrator, dissolving the dried product with 1.5mL methanol, collecting 1mL in HPLC liquid phase analysis bottle, and performing LC-MS liquid quality analysis. The elution was carried out using a mixed solution of acetonitrile and water as a mobile phase, and the specific analysis method is shown in Table 1. In table 1, the volume ratio of acetonitrile to water is shown.
TABLE 1
Time (minutes)
Acetonitrile%
Water%
5.0
95.0
5.00
5.0
95.0
35.00
100.0
0.0
40.00
100.0
0.0
40.10
5.0
95.0
45.00
5.0
95.0
The LC-MS analysis results are shown in FIG. 1. The results show that the strain Amphichorda guana LC5815 used in the examples of the present application can produce abundant secondary metabolites with large molecular weight. The analysis of the fermentation culture product by using a high performance liquid chromatography-tandem mass spectrometry (LC-MS-MS) instrument further proves that M/z 637.4[ M + H]+And M/z 596.4[ M + H]+There is a continuous cleavage of the amino acids. Wherein, M/z 637.4[ M + H ] is identified by amino acid ion fragment]+Is the ion peak of cyclodepsipeptide isariin A (shown in FIG. 3), M/z 596.4[ M + H [ ]]+The ion peak of the cyclodepsipeptide iso-isarinB (FIG. 4).
The inventors of the present application performed fermentation using Amphickora guana LC5815 with PDA medium as the fermentation medium, and fermentation using Amphickora guana LC5815 with Potato Dextrose Broth (PDB) medium as the fermentation medium. The fermentation conditions are as described above.
After completion of the fermentation, the fermentation product using the PDA medium, the PDB medium, and the rice medium as the fermentation medium was analyzed by High Performance Liquid Chromatography (HPLC), and the results are shown in fig. 5. Wherein LC5815-PDB-7d represents the analysis result of the fermentation product using PDB medium as the fermentation medium, LC5815-PDB-7d represents the analysis result of the fermentation product using PDA medium as the fermentation medium, and LC5815-Rice-7d represents the analysis result of the fermentation product using Rice medium as the fermentation medium. As shown in FIG. 5, no large molecule secondary metabolites shown in FIG. 1 were detected in the fermentation products using PDA medium and PDB medium as the fermentation medium. Thus, it was confirmed that the rice medium is a suitable medium for producing the macromolecular secondary metabolites shown in FIG. 1.
Example 2 isolation and purification of active Cyclodepsipeptide fractions
Preliminary analysis of the rice fermentation product of a. guana LC5815 by LC-MS of example 1 has shown that it produces abundant high molecular weight compounds. Culturing the strain in 20kg rice culture medium (obtained by mixing 20kg rice and 30L water) for large-scale fermentation, and standing at 28 deg.C in dark place for 30 days to obtain rice fermentation culture. Adding ethyl acetate into the rice fermentation culture, extracting for 3 times, and soaking overnight each time to obtain leaching solution. Specifically, the rice was extracted with 7.5L of ethyl acetate per 1kg of rice, and ethyl acetate was added directly after completion of the culture. In the specific experiment, since 250mL triangular flasks of 20g rice per flask were used and 50mL ethyl acetate per flask were used for extraction, the procedure was repeated 3 times, and a total of 150mL ethyl acetate was required.
The resulting extract was evaporated to dryness under vacuum to give about 60g of crude extract.
The obtained crude extract is separated by chromatography with 200-300 mesh silica gel column, and is eluted with 4 eluents with different polarities of dichloromethane, ethyl acetate, acetone and methanol respectively to obtain 4 fractions. The 4 fractions obtained were evaporated to dryness in vacuo to yield 8g dry matter A, 21g dry matter B, 15g dry matter C and 10.2g dry matter D respectively. Further purifying dry matter C obtained from acetone fraction by using small pore resin (MCI), using a mixed solution of methanol and water as an eluent, and sequentially adding 5: 5. 7: 3. 9: 1. 10: elution was performed with an elution gradient of 0 and the elution products were collected for 4 elution gradients.
Separating and purifying the elution product of 70% methanol and 90% methanol by a preparative HPLC column, and performing gradient elution by using acetonitrile-water (volume ratio is 85: 15) as an eluent at a flow rate of 2 mL/min. Further separation with 85% acetonitrile separated 5 cyclodepsipeptide compounds from the above elution with 70% methanol. Collecting chromatographic peak with retention time of 11.9min, and performing rotary evaporation to obtain 25mg of cyclodepsipeptide iso-isarin D; collecting chromatographic peak with retention time of 13.1min, and performing rotary evaporation to obtain 10mg of cyclodepsipeptide Desmethylethyllisarindin E; collecting chromatographic peak with retention time of 17.5min, and performing rotary evaporation to obtain 30mg of cyclodepsipeptide isarii E; collecting chromatographic peak with retention time of 20.1min, and rotary evaporating to obtain 35mg cyclodipeptide nodipeetide; the chromatographic peak with retention time of 26.7min was collected and crystallized from methanol to give 8.5g of cyclodepsipeptide iso-isarii B. 3 cyclodepsipeptide compounds are isolated from the 90% methanol eluate, which comprises 25mg of a novel cyclodepsipeptide isaridin H with a peak time of 17.5 min; a cyclodepsipeptide isaridin E10 mg with a peak time of 19.2 min; a cyclodepsipeptide isariin A with a peak time of 25.6 min.
In particular, from 60g of crude extract, 8.5g of cyclodepsipeptide iso-isarii B can be isolated. That is, according to the scheme provided in the examples of the present application, the yield of cyclodepsipeptide iso-isarii B exceeds 14%, and efficient preparation of cyclodepsipeptide iso-isarii B is achieved.
Example 3 verification of the biological Activity of the cyclodepsipeptides prepared in example 2
3.1 verification of the Activity against phytopathogenic fungi
The antifungal activity was measured using a 90mm dish containing 10mL of PDA medium, and using 3 plant pathogenic fungi, Alternaria solani, Botrytis cinerea, Fusarium oxysporum as an indicator, the plant pathogenic fungi were cultured using the PDA medium, and when they grew about 2cm in diameter, a sterile blank filter paper sheet of the same size (0.625 cm in diameter) was placed 1cm from the edge of the hyphal colony. Distribution the isaridin H, desmethyllisarindin E, isaridin E prepared in example 2 were dissolved in dimethyl sulfoxide (DMSO) to give samples each at a final concentration of 1mg/mL, and 10uL of each was added to a filter paper sheet. In FIG. 6, in addition, isaridin H is added to the sheet 1, desmethyllisarindin E is added to the sheet 2, and isaridin E is added to the sheet 3. The culture dish was placed at 28 ℃ and cultured in the dark, and the hypha diameter was measured 3 days later, and the hypha inhibition rate was calculated according to the following calculation formula. The calculation results are shown in table 2.
TABLE 2 antifungal Activity of Isaridin H (1)
The results shown in Table 2 and FIG. 6 indicate that the novel cyclodepsipeptide compound isaridin H (1) prepared in example 2 has a significant inhibitory activity against Alternaria solani and Botrytis cinerea.
3.2 verification of antibacterial Activity by diffusion on Filter paper
3.2.1, using filter paper sheets
Test bacteria (Bacillus subtilis, Escherichia coli and Staphylococcus aureus) were activated by a 90 mm-diameter plate containing a lysis broth Luria-Bertani (LB) solid medium, cultured overnight at 37 ℃, and a single colony of the activated test bacteria was inoculated into a test tube containing 5mL of LB liquid medium, cultured overnight at 37 ℃ and 200rpm on a shaker. To 20mL of LB medium at 55 ℃ was added 10. mu.L of each test bacterium suspension (10. mu.L)10cfu mL-1) After mixing well, the medium containing the bacteria was poured into a petri dish with a diameter of 90 mm. 4 pieces of sterile filter paper (diameter 0.625cm) were placed on a petri dish containing the above mixture, and 10. mu.L of 1mg mL of a solution was added to each piece of filter paper using a pipette-1The isaridins cyclodepsipeptides (isaridin H (panel 1 in FIG. 7), desmethyllisarindin E (panel 2 in FIG. 7), isaridin E (panel 3 in FIG. 7)) isolated from this strain were treated with 10uL of dimethyl sulfoxideAs a negative control (a paper indicated by "-" in FIG. 7), the 10uL concentration was 1mg mL-1Ampicillin as a positive control (in the paper indicated by "+" in FIG. 7), was incubated overnight at 37 ℃ and the zone size was recorded. In 3 replicates of each experiment, the results showed that the novel cyclic depsipeptide compound isaridin H had antibacterial activity against bacillus subtilis (fig. 7).
3.2.2 MIC value determination-fold dilution method
A Minimum Inhibitory Concentration (MIC) test is performed by using a 96-well plate, a multiple dilution method is adopted, bacillus subtilis is used as an indicator strain, ampicillin is used as a positive control, and dimethyl sulfoxide is used as a negative control. The cyclic depsipeptide compound isaridn H used for the test was prepared in a stock solution of 10.24mg mL of the following concentration-1，5.12mg mL-1，2.56mg mL-1，1.28mg mL-1，0.64mg mL-1，0.32mg mL-1，0.16mg mL-1，0.08mg mL-15uL of each was placed in a 96-well plate containing 95uL of LB liquid medium. Culturing at 37 deg.C, and detecting absorbance with microplate reader at 12h and 24 h. Each experiment was repeated 3 times and the data is shown in table 3.
TABLE 3 antibacterial Effect of the cyclodepsipeptide isaridin H
The result shows that when the concentration of isaridin H is more than or equal to 8 mug/mL, the isaridin H has an antibacterial effect on bacillus subtilis.
In the embodiment of the application, bat fecal parasitic fungus Amphickorda guana LC5815 is used as a fermentation strain, a rice culture medium is used as a fermentation culture medium, and fermentation is carried out, so that various cyclodepsipeptides can be produced, and particularly, the yield of cyclodepsipeptide iso-isarin B is over 14 percent; and can produce a novel cyclic depsipeptide isaridin H having fungal and bacterial inhibitory ability.
The above-mentioned embodiments, objects, technical solutions and advantages of the present application are further described in detail, it should be understood that the above-mentioned embodiments are only examples of the present application, and are not intended to limit the scope of the present application, and any modifications, equivalent substitutions, improvements and the like made on the basis of the technical solutions of the present application should be included in the scope of the present application.