CN112680363A - Preparation and application of beauveria bassiana BB-7 metabolite - Google Patents
Preparation and application of beauveria bassiana BB-7 metabolite Download PDFInfo
- Publication number
- CN112680363A CN112680363A CN202110085335.8A CN202110085335A CN112680363A CN 112680363 A CN112680363 A CN 112680363A CN 202110085335 A CN202110085335 A CN 202110085335A CN 112680363 A CN112680363 A CN 112680363A
- Authority
- CN
- China
- Prior art keywords
- beauveria bassiana
- extract
- fermentation
- liquor
- fermentation broth
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 241000751139 Beauveria bassiana Species 0.000 title claims abstract description 64
- 238000002360 preparation method Methods 0.000 title claims description 11
- 239000002207 metabolite Substances 0.000 title abstract description 10
- 241000233866 Fungi Species 0.000 claims abstract description 19
- 208000017520 skin disease Diseases 0.000 claims abstract description 8
- 235000009579 balsamo Nutrition 0.000 claims abstract description 4
- 238000000855 fermentation Methods 0.000 claims description 57
- 230000004151 fermentation Effects 0.000 claims description 57
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 230000000843 anti-fungal effect Effects 0.000 claims description 20
- 239000000284 extract Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 14
- 239000012153 distilled water Substances 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 239000003208 petroleum Substances 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 9
- 238000001914 filtration Methods 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 206010017533 Fungal infection Diseases 0.000 claims description 4
- 208000031888 Mycoses Diseases 0.000 claims description 4
- 239000002026 chloroform extract Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 239000012259 ether extract Substances 0.000 claims description 4
- 239000002024 ethyl acetate extract Substances 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 238000010009 beating Methods 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 239000003937 drug carrier Substances 0.000 claims description 2
- 238000004806 packaging method and process Methods 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 12
- 230000002401 inhibitory effect Effects 0.000 abstract description 9
- 241001045770 Trichophyton mentagrophytes Species 0.000 abstract description 7
- 230000003385 bacteriostatic effect Effects 0.000 abstract description 7
- 241000893976 Nannizzia gypsea Species 0.000 abstract description 6
- 238000004321 preservation Methods 0.000 abstract description 6
- 244000052616 bacterial pathogen Species 0.000 abstract description 5
- 241000243190 Microsporidia Species 0.000 abstract description 3
- 206010048768 Dermatosis Diseases 0.000 abstract description 2
- 241000223238 Trichophyton Species 0.000 abstract description 2
- 230000002538 fungal effect Effects 0.000 abstract description 2
- 201000009642 tinea barbae Diseases 0.000 abstract description 2
- 241000282465 Canis Species 0.000 abstract 1
- 238000012136 culture method Methods 0.000 abstract 1
- 238000002955 isolation Methods 0.000 abstract 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 10
- 239000000243 solution Substances 0.000 description 8
- 229940121375 antifungal agent Drugs 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 241000893980 Microsporum canis Species 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000005507 spraying Methods 0.000 description 5
- 206010040844 Skin exfoliation Diseases 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 241001482471 Hosta plantaginea Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000002844 melting Methods 0.000 description 3
- 230000008018 melting Effects 0.000 description 3
- 206010040882 skin lesion Diseases 0.000 description 3
- 231100000444 skin lesion Toxicity 0.000 description 3
- 238000000638 solvent extraction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 206010015150 Erythema Diseases 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 description 2
- 206010039509 Scab Diseases 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229940124350 antibacterial drug Drugs 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 230000035618 desquamation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000003628 erosive effect Effects 0.000 description 2
- 231100000321 erythema Toxicity 0.000 description 2
- 244000053095 fungal pathogen Species 0.000 description 2
- 230000009422 growth inhibiting effect Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- DOMXUEMWDBAQBQ-WEVVVXLNSA-N terbinafine Chemical compound C1=CC=C2C(CN(C\C=C\C#CC(C)(C)C)C)=CC=CC2=C1 DOMXUEMWDBAQBQ-WEVVVXLNSA-N 0.000 description 2
- 229960000699 terbinafine hydrochloride Drugs 0.000 description 2
- 239000000273 veterinary drug Substances 0.000 description 2
- 241001302652 Bassiana Species 0.000 description 1
- 208000001840 Dandruff Diseases 0.000 description 1
- 208000007163 Dermatomycoses Diseases 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241001480037 Microsporum Species 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 229920003081 Povidone K 30 Polymers 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000002223 anti-pathogen Effects 0.000 description 1
- 230000001775 anti-pathogenic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000003749 cleanliness Effects 0.000 description 1
- 230000035617 depilation Effects 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 229960002344 dexamethasone sodium phosphate Drugs 0.000 description 1
- PLCQGRYPOISRTQ-FCJDYXGNSA-L dexamethasone sodium phosphate Chemical compound [Na+].[Na+].C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)COP([O-])([O-])=O)(O)[C@@]1(C)C[C@@H]2O PLCQGRYPOISRTQ-FCJDYXGNSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 125000001033 ether group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000001408 fungistatic effect Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000003779 hair growth Effects 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 229910052979 sodium sulfide Inorganic materials 0.000 description 1
- GRVFOGOEDUUMBP-UHFFFAOYSA-N sodium sulfide (anhydrous) Chemical compound [Na+].[Na+].[S-2] GRVFOGOEDUUMBP-UHFFFAOYSA-N 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Images
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to beauveria bassiana (Beauveria bassiana (balsamo) Vuillemin)Beauveria bassiana) The BB-7 strain isolation culture method and the metabolite thereof have the function of inhibiting the animal fungal dermatosis. The strain is preserved in China center for type culture Collection with the address: china, wuhan university; the zip code 430072 has a preservation date of 2019, month 4 and 30 and a preservation number of: CTCCNO: m2019260; the strain has been whole genome sequenced. The fungus metabolite is microsporidian canis (C)Sabourauditeslanosus) Gypsum-like microsporidia (A), (B), (C), (Microsporumgypseum) Tinea barbae trichophyton (A. mentagrophytes) ((A. mentagrophytes))Trichophyton mentagrophytes) The 3 animal pathogenic bacteria have good bacteriostatic activity.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of a metabolite of beauveria bassiana in preparation of antifungal drugs.
Background
In recent years, the prevalence rate of fungus-derived pet skin diseases is increasing year by year, and the fungus-derived pet skin diseases become a trouble and a difficult problem in the pet market. More importantly, the disease belongs to zoonosis and directly poses serious threat to human health. However, at present, the treatment of pet skin diseases is mainly antibiotic external ointment or spray, and the problems of strong toxicity, frequent use, short effect, easy generation of drug resistance and the like exist in the treatment process. The development of a novel antifungal pet medicine source with high efficiency, low toxicity and long acting is imminent.
In 2016, the department of agriculture specifically pointed out in the development program of national veterinary health service (2016-. In the next year, the Ministry of agriculture has brought strong advantages to research institutions and leading enterprises in national animal resistance action plan (2017-2020), and has developed the importance of product research and development and technical innovation in the fields of antibacterial drug substitution varieties such as special antibacterial drugs for animals and traditional Chinese veterinary drugs. Therefore, in response to the call of Ministry of agriculture, the development of novel natural antibacterial veterinary drugs is imperative.
Disclosure of Invention
In order to solve the above problems, the present invention provides an endophytic fungus BB-7 strain producing an antipathogenic fungus, a metabolite thereof, and a fermentation broth of the metabolite, the metabolite of the strain being applicable to the treatment of skin diseases.
The purpose of the invention is realized by the following technical scheme:
the invention provides a novel strain, which is named as BB-7 and belongs to endogenous beauveria bassiana (Beauveria bassiana (Bb.) (Beauveria bassiana) BB-7, which is deposited in the China center for type culture Collection, address: china, wuhan university; the zip code 430072 has a preservation date of 2019, month 4 and 30 and a preservation number of: CTCCNO: m2019260; the strain has been sequenced whole genome.
The beauveria bassiana (Beauveria bassiana) The BB-7 strain can be used for preparing an antipathogen fungus fermentation liquid.
The beauveria bassiana BB-7 fermentation liquor extract with antifungal activity is selected from one of a petroleum ether extract, a chloroform extract, an ethyl acetate extract and a water extract, or any combination thereof.
The extract is prepared by the following method:
(1) cleaning fresh potato, peeling, cutting into small pieces, boiling with distilled water, filtering with gauze, adding glucose into the filtrate, stirring, diluting with distilled water, packaging, and sterilizing to obtain liquid fermentation culture medium;
(2) subpackaging the liquid fermentation culture medium in the step (1) in conical flasks, beating the preserved beauveria bassiana BB-7 into fungus cakes in the conical flasks by using a puncher, culturing, centrifuging the cultured fermentation liquor, taking the supernatant, and filtering by using a microporous filter membrane to obtain the beauveria bassiana BB-7 fermentation liquor;
(3) and (3) freeze-drying the Beauveria bassiana BB-7 fermentation liquor obtained in the step (2), dispersing the fermentation liquor in distilled water, sequentially extracting the fermentation liquor with petroleum ether, chloroform and ethyl acetate, combining the extraction liquor with the same solvent, and performing vacuum concentration and drying on the extraction liquor to respectively obtain a petroleum ether extract, a chloroform extract, an ethyl acetate extract and a water extract.
A pharmaceutical composition comprises the Beauveria bassiana BB-7 fermentation liquor extract and a pharmaceutically acceptable carrier.
The application of the beauveria bassiana BB-7 fermentation liquor extract or the pharmaceutical composition in preparing antifungal or fungal infection prevention and treatment medicines.
The application of the beauveria bassiana BB-7 fermentation liquor extract or the pharmaceutical composition in preparing medicines for preventing and treating skin diseases.
The fungus is selected from a Beauveria bassiana BB-7 strain.
The specific implementation mode is as follows: the beauveria bassiana BB-7 fermentation liquor extract specifically comprises the following steps:
(1) preparation of a fermentation medium: cleaning fresh potatoes, peeling, cutting into small pieces, weighing 200g, adding 1000 mL of distilled water, boiling for 30min, filtering with 4 layers of gauze, adding glucose (20 g) into the filtrate, stirring uniformly, adding 1000 mL of distilled water, subpackaging into a 250mL conical flask with the volume of 100mL, and sterilizing at 121 ℃ for 30 min;
(2) fermentation culture of Beauveria bassiana BB-7: subpackaging the liquid fermentation medium in the step (1) in a 250mL conical flask with the loading of 100mL, beating the preserved beauveria bassiana into a bacterial cake with the diameter of 6mm in the conical flask by using a puncher, culturing at 25 ℃ at 170 r/min for 15d, centrifuging the fermentation liquid at 3500r/min for 15min, taking the supernatant, and filtering by using a microfiltration membrane (0.22 mu m) to obtain the beauveria bassiana fermentation liquid;
(3) freeze-drying Beauveria bassiana BB-7 fermentation liquor, dispersing the freeze-dried Beauveria bassiana BB-7 fermentation liquor by using a proper amount of distilled water, placing a dispersed sample in a separating funnel, sequentially extracting the sample by using petroleum ether, chloroform and ethyl acetate at a ratio of 1:1 (v/v), repeating the extraction for 3 times, combining extraction liquid, and performing vacuum concentration and drying on the extraction liquid at 40 ℃ to respectively obtain petroleum ether, a chloroform part, an ethyl acetate part and a water part (freeze-drying).
(4) Adding fermentation liquor and different solvent extracts thereof into melting PDA culture medium (40-50 deg.C) at a ratio of 1:9, inoculating pathogenic bacteria cake (6 mm), placing in a constant temperature incubator at 25 deg.C, culturing, calculating the inhibition rate of each effective extract on pathogenic bacteria to be tested, and screening out antibacterial activity extract
The application of beauveria bassiana BB-7 metabolite in treating animal fungal dermatosis comprises the following steps: the fermentation liquor of Beauveria bassiana BB-7 and the polar parts of different solvents are matched with proper amount of pharmaceutical excipients to prepare the antifungal medicines with various dosage forms.
The pathogenic bacterium to be tested in the present invention (4) is Microsporum canisSabourauditeslanosus) Gypsum-like microsporidia (A), (B), (C), (Microsporumgypseum) Tinea barbae trichophyton (A. mentagrophytes) ((A. mentagrophytes))Trichophyton mentagrophytes)。
Description of the drawings:
FIG. 1: beauveria bassiana BB-7 colony morphology
FIG. 2: the whole genome comparison result of Beauveria bassiana BB-7;
FIG. 3: the result of the antifungal activity of the beauveria bassiana BB-7 fermentation liquor is shown;
FIG. 4: the pharmacodynamics result of the Beauveria bassiana BB-7 fermentation liquor film spraying agent;
Detailed Description
The invention will be further described with reference to the following examples.
Example 1: screening of beauveria bassiana metabolite fungistatic activity
1. Beauveria bassiana (balsamo) Vuillemin: (B)Beauveria bassiana) BB-7 is an antifungal endophytic fungus derived from root separation of Hosta plantaginea. Collecting fresh and healthy root of fragrant plantain lily, disinfecting the surface of the root tissue of fragrant plantain lily, checking cleanliness of a clean bench, checking rinsing liquid, and screening sterile tissue blocks by a plant tissue imprinting method; cutting the sterile tissue into small blocks with the diameter of about 0.5 cm on a sterile workbench, placing the small blocks into a PDA culture medium, carrying out inverted culture in a constant-temperature fungus incubator at 25 ℃, adopting a tip hypha picking method when the endophytic fungus hypha grows outwards along a tissue cut, picking bacterial colonies with different forms, repeatedly purifying and then transferring the bacterial colonies to a PDA slant culture medium, and respectively purifying, numbering and screening the bacterial colonies for resisting the activity of the pathogenic fungi. The strain provided in this example was named strain BB-7, and colonies of this strain were transferred to a new PDA medium plate and grown well, and the colony morphology is shown in FIG. 1.
(2) Sequencing the purified endophyte strain through the whole gene sequence to determine the endophyte strain species, which is shown in figure 2.
(3) According to the whole gene sequencing result, the strain is determined to be beauveria bassiana (Beauveria bassiana (balsamo)Beauveria bassiana）。
(4) The applicant reserves the strain in China center for type culture Collection, address: china, wuhan university; the zip code 430072 has a preservation date of 2019, month 4 and 30 and a preservation number of: CTCCNO: and M2019260.
2. And (3) recovering and activating the strain: beauveria bassiana (Beauveria bassiana) to be stored in a refrigerator at 4 ℃Beauveria bassiana) Inoculating to PDA culture medium, culturing in 25 deg.C fungus incubator for 7d, activating, selecting mycelium around activated colony, inoculating to PDA culture medium, and culturing in 25 deg.C fungus incubator for 7d to restore normal growth of fungus.
3. Preparation of a beauveria bassiana fermentation product: the strain was inoculated on PDA plates and cultured at 25 ℃ for 7 days. In a workbench, a punch is used for punching a fungus cake with the diameter of 6mm along the edge of a colony, and the fungus cake is inoculated into a 250mL conical flask containing 100mL of PDB culture medium; culturing at 25 deg.C and 170 r/min for 15d, stopping fermentation after mycelium completely covers the liquid surface of the culture solution, and simultaneously leaving a bottle of non-inoculated culture solution as blank control. Filtering the fermentation liquid with 4 layers of gauze, separating mycelium and fermentation liquid, centrifuging fermentation liquid at 3500r/min for 15min, collecting supernatant, and filtering with microporous membrane (0.22 μm) at-20 deg.C.
4. Bacteriostatic experiments: and (3) determining the antibacterial activity by adopting a hypha growth rate method: 150 mg of the freeze-dried fermentation broth is weighed and dissolved in 3 mL of distilled water to prepare 50 mg/mL of stock solution for testing. Filtering the aqueous solution with a microporous filter membrane (0.22 mu m), adding a melting PDA culture medium (40-50 ℃) according to a ratio of 1:9, making the final concentration to be 5mg/mL, pouring the solution into a flat plate (15 mL per dish), using sterile distilled water as a blank control, inoculating a pathogenic bacteria cake (6 mm) after solidification, repeating the treatment and the control for 3 times respectively, placing the solution into a 25 ℃ fungus constant-temperature incubator for culturing for 5 days, measuring the diameter of a bacterial colony by a cross method, and taking the average value. The inhibition was calculated as follows:
hypha growth inhibition = (control colony diameter-diameter of treated colony) × 100%/(control colony diameter-6)
5. MIC determination of antifungal activity polar site: and (3) measuring by a micro method: adding 100 mu L of a freshly prepared PDB culture medium into each of No. 1-8 wells of a 96-well plate, adding 100 mu L (5 mg/mL) of a prepared sample into the No. 1 well, diluting the medicament in 2-8 wells by adopting a multiple dilution method, and adding 100 mu L (2 multiplied by 10) of a freshly prepared bacterial liquid into each of 1-8 wells6CFU/mL), only 200. mu.L of the inoculum was added to the 9 th well, only 200. mu.L of the broth was added to the 10 th well, 100. mu.L of 1% DMSO solution and 100. mu.L of the inoculum were added to the 11 th well, and 100. mu.L of 1% terbinafine hydrochloride was added to the 12 th well, respectively, as a control. After each plate was cultured in an incubator at 35 ℃ for 48 hours, the growth inhibitory effect of the drug on the test bacteria was observed in comparison with the control group. Each set of experiments was repeated 3 times.
The experimental results are as follows:
the results of the beauveria bassiana BB-7 whole genome alignment are shown in FIG. 2.
The antifungal activity of the Beauveria bassiana (Beauveria bassiana, BB-7) fermentation liquor is shown in figure 3, and the antifungal activity of the Beauveria bassiana BB-7 fermentation liquor on 3 animal pathogenic fungi, namely microsporum canis, microsporum gypseum and trichophyton mentagrophytes can be known from figure 3, the bacteriostatic rates respectively reach 76.0%, 66.3% and 65.7%, and the broad-spectrum antifungal activity is realized.
The minimum inhibitory concentration of the beauveria bassiana fermentation broth is determined by adopting a micro-method, and test results show that the beauveria bassiana fermentation broth has the minimum inhibitory concentration of 312.5-625 mu g/mL against microsporum canis, 625-1250 mu g/mL against microsporum gypseum and 625-1250 mu g/mL against trichophyton mentagrophytes.
Example 2: active antifungal polar part screening of beauveria bassiana fermentation liquor
1. Preparing different solvent extraction parts of beauveria bassiana fermentation liquor: freeze drying the beauveria bassiana fermentation liquor, dispersing the beauveria bassiana fermentation liquor by using a proper amount of distilled water, placing the dispersed sample in a separating funnel, sequentially extracting the sample by using petroleum ether, chloroform and ethyl acetate at a ratio of 1:1 (v/v), repeating the extraction for 3 times, combining the extract liquor, and performing vacuum concentration and drying on the extract liquor at 40 ℃ to respectively obtain petroleum ether, a chloroform part, an ethyl acetate part and a water part (freeze-drying).
2. Determination of antifungal activity of different solvent extraction parts of beauveria bassiana fermentation liquor: dissolving the four polar parts by using 500 mu LDMSO, diluting the solution to 10 mg/mL by using distilled water, adding the solution into a PDA culture medium (40-50 ℃) in a melting state according to a ratio of 1:9 to enable the final concentration to be 1 mg/mL, and measuring the antibacterial activity by using a hypha growth rate method by using DMSO as a blank control.
3. MIC determination of antifungal activity polar site: and (3) measuring by a micro method: adding 100 mu L of a freshly prepared PDB culture medium into each of No. 1-8 wells of a 96-well plate, adding 100 mu L (5 mg/mL) of a prepared sample into the No. 1 well, diluting the medicament in 2-8 wells by adopting a multiple dilution method, and adding 100 mu L (2 multiplied by 10) of a freshly prepared bacterial liquid into each of 1-8 wells6CFU/mL), 200. mu.L of the inoculum alone was added to well 9, 200. mu.L of the broth alone was added to well 10, 100. mu.L of 1% DMSO solution and 100. mu.L of the inoculum were added to well 11, and100 μ L of 1% terbinafine hydrochloride was added to each of the 12 wells as a control. After each plate was cultured in an incubator at 35 ℃ for 48 hours, the growth inhibitory effect of the drug on the test bacteria was observed in comparison with the control group. Each set of experiments was repeated 3 times.
The experimental results are as follows:
1. antifungal activity results of different solvent extraction parts of beauveria bassiana fermentation liquor are as follows:
antifungal activity results of various polar parts show that DMSO and ethyl acetate parts have no bacteriostatic activity on 3 indicator bacteria under test concentration, and chloroform parts and water parts show broad-spectrum bacteriostatic activity. The bacteriostatic activity (31%) of the chloroform part on the microsporum canis is obviously higher than that of the petroleum ether part and the water part; the water part shows stronger bacteriostatic activity on gypseous sporophytes and trichophyton mentagrophytes of 27 percent and 26 percent respectively, which are higher than the chloroform part (23 percent and 21 percent). Therefore, the effective antibacterial polar parts of the beauveria bassiana fermentation liquor are the chloroform part and the water part.
3. Results of MIC determination of antifungal Activity polar site:
the MIC measurement result of the antifungal activity polar part shows that the minimum inhibitory concentration of the chloroform part to the microsporum canis is 156.25 to 312.50 mu g/mL, the minimum inhibitory concentration to the microsporum gypseum is 312.50 to 625.00 mu g/mL, and the minimum inhibitory concentration to the trichophyton mentagrophytes is 625.00 to 1250.00 mu g/mL; the minimum inhibitory concentration of the water part to the microsporum canis is 312.50-625.00 mug/mL, the minimum inhibitory concentration to the microsporum gypseum is 625.00-1250.00 mug/mL, and the minimum inhibitory concentration to the trichophyton mentagrophytes is 312.50-625.00 mug/mL.
Example 3: preparation of beauveria bassiana fermentation liquor film spraying agent and antifungal animal pharmacodynamic evaluation thereof
1. Preparing a film spraying agent: selecting pharmaceutically commonly used film-forming materials polyvinyl alcohol (PVA 17-88) and polyvinylpyrrolidone (PVP-K30) as film-forming materials, taking water as a solvent, taking glycerin as a humectant, adding an antioxidant and a preservative, adding a certain amount of freeze-dried powder of beauveria bassiana fermentation broth, and preparing the film spraying agent after screening by a preparation process.
2. Test of drug effect of film-spraying agent for animals
2.1 preparation of test animals
3 days before the inoculation of the bacterial liquid, the white rabbits are injected with dexamethasone sodium phosphate immunosuppressant subcutaneously according to the dose of 2.5mg/kg, are inoculated once a day and are observed.
2.2 establishment of animal models of fungal infection
The white rabbits were anesthetized by ether inhalation, and the hair was shaved off from the backs of the animals with a shaver at a shaving area of 3 cm × 3 cm. The center part was marked with a marker in a square area of 2 cm × 2 cm, and the hair in this area was removed by depilation using 8% sodium sulfide, taking care not to scrape the skin. The central mark is polished with sterile sandpaper, preferably for punctate bleeding. A1 mL syringe was used to aspirate 104 conidia/mL of the bacterial suspension, gently prick the skin and inject the bacterial suspension to evenly coat the areas of bleeding. Observations were made daily after inoculation.
And (4) judging the standard: and 7 days after the animal skin is infected, if the following conditions are met, the animal model of dermatophyte infection is successfully made. Erythema, desquamation, erosion and incrustation appear on the local part of the skin damage; collecting skin lesions, and directly performing microscopic examination on the skin lesions to obtain visible hyphae and spores; collecting skin lesion, culturing to obtain primary infection fungus, and observing hypha and/or spore in cuticle or around or in hair (as basis but not as criterion) by histopathological examination
The experimental results are as follows:
the administration method comprises the following steps: on the 7 th day after the animal skin was infected, the administration was started after the judgment model was successfully established, 1 time each day, and 10 days were continued. The formulations were observed for treatment. After 10 days of administration, the affected parts with mild symptoms such as erythema, desquamation, and a little scab healed, and hair growth at the affected parts has already begun, as shown in FIG. 4A, B, C, A ', B ', and C '. Severe infection with extensive scabbing and slight erosion occurred, the infected site was substantially healed with a small amount of dandruff, and a small amount of villi grew from the infected site, as shown in fig. 4D, D'. The preparation has good therapeutic effect on pet fungal infection.
Claims (6)
1. Beauveria bassiana (balsamo) Vuillemin ((B))Beauveria bassiana) BB-7 strain deposited in the Chinese dictionaryType culture collection, its deposit number is: CTCCNO: and M2019260.
2. A Beauveria bassiana fermentation broth extract with antifungal activity is characterized in that one of petroleum ether extract, chloroform extract, ethyl acetate extract and water extract of the fermentation broth, or any combination thereof.
3. A fermentation broth extract according to claim 2, characterized in that the extract is prepared by the following method:
(1) cleaning fresh potato, peeling, cutting into small pieces, boiling with distilled water, filtering with gauze, adding glucose into the filtrate, stirring, diluting with distilled water, packaging, and sterilizing to obtain liquid fermentation culture medium;
(2) subpackaging the liquid fermentation culture medium in the step (1) in conical flasks, beating the preserved beauveria bassiana into fungus cakes by using a puncher, culturing in the conical flasks, centrifuging the cultured fermentation liquor, taking supernate, and filtering by using a microporous filter membrane to obtain beauveria bassiana BB-7 fermentation liquor;
(3) and (3) freeze-drying the Beauveria bassiana BB-7 fermentation liquor obtained in the step (2), dispersing the fermentation liquor in distilled water, sequentially extracting the fermentation liquor with petroleum ether, chloroform and ethyl acetate, combining the extraction liquor with the same solvent, and performing vacuum concentration and drying on the extraction liquor to respectively obtain a petroleum ether extract, a chloroform extract, an ethyl acetate extract and a water extract.
4. A pharmaceutical composition characterized by: comprising the beauveria bassiana BB-7 fermentation broth of claim 2 and a pharmaceutically acceptable carrier.
5. Use of the beauveria bassiana BB-7 fermentation broth of claim 2 or the pharmaceutical composition of claim 2 in the preparation of a medicament for the treatment or prevention of fungal infection.
6. Use of the beauveria bassiana BB-7 fermentation broth of claim 2 or the pharmaceutical composition of claim 4 in the preparation of a medicament for preventing and treating skin diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110085335.8A CN112680363A (en) | 2021-01-22 | 2021-01-22 | Preparation and application of beauveria bassiana BB-7 metabolite |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110085335.8A CN112680363A (en) | 2021-01-22 | 2021-01-22 | Preparation and application of beauveria bassiana BB-7 metabolite |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112680363A true CN112680363A (en) | 2021-04-20 |
Family
ID=75458920
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110085335.8A Pending CN112680363A (en) | 2021-01-22 | 2021-01-22 | Preparation and application of beauveria bassiana BB-7 metabolite |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112680363A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114456949A (en) * | 2022-01-13 | 2022-05-10 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
-
2021
- 2021-01-22 CN CN202110085335.8A patent/CN112680363A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114456949A (en) * | 2022-01-13 | 2022-05-10 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
| CN114456949B (en) * | 2022-01-13 | 2023-06-16 | 贵州民族大学 | Beauveria bassiana JSHA-MD912 and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fraser et al. | Aphanomyces species associated with red spot disease: an ulcerative disease of estuarine fish from eastern Australia | |
| CN111662880B (en) | Clostridium perfringens bacteriophage, bacteriostatic agent containing bacteriophage, preparation method and application | |
| CN104789483A (en) | Method for isolated culture of endoclyta signifer walker beauveria bassiana (bals.) vuill strain, and prevention and control of endoclyta signifer walkers | |
| CN114774287B (en) | Endophytic chaetomium globosum MG2 and application thereof in preventing and treating apple tree rot | |
| WO2005029958A1 (en) | Use of anthraquinone derivatives as pestcides for controlling plant diseases | |
| CN112680363A (en) | Preparation and application of beauveria bassiana BB-7 metabolite | |
| CN102660467A (en) | Liquorice endophytic fungus for producing glycyrrhetinic acids | |
| CN103371988B (en) | Pterostilbene is used for the application of antimycotic biofilm medicine | |
| CN115500355B (en) | Control of downy mildew of litchi by Podophyllotoxin and Gentisic acid | |
| GB2025222A (en) | Vaccine for treatment of trichophytosis caused by trichophyton mentagrophytes | |
| CN108635490A (en) | Pale reddish brown jade hairpin endogenous fungus metabolite, Medical bioactive and its application | |
| CN111905018B (en) | Preparation method and application of zanthoxylum bungeanum maxim extract | |
| CN103392746B (en) | Application of polygonum multiflorum extract | |
| CN108837029B (en) | Pharmaceutical composition for preventing and treating saprolegniasis of aquatic animals and preparation method thereof | |
| AU2020103248A4 (en) | The preparation and application of antifungal extracts from Oxytropis falcate Bunge | |
| CN110840865A (en) | Application of nerolidol in preparation of medicine for treating fungal keratitis | |
| Adetumbi et al. | Inhibition of in vitro germination and spherulation of Coccidioides immitis by Allium sativum | |
| CN109700858A (en) | Rhizoma Polygoni suffulti is preparing the application in antifungal drug and its synergist | |
| CN112159766B (en) | Aphyllophora fuscopsis new strain, fermentation method thereof and application thereof in bacteriostasis | |
| CN104306477B (en) | Coptis water extract and monomer are preparing the purposes in suppressing Streptococcus suis or intervening streptococcus suis biofilm medicine | |
| CN114404566B (en) | Application of trichoderma | |
| CN116473060B (en) | Medicine for preventing and treating rice blast | |
| CN110964644B (en) | Penicillium and fermentation method and application thereof | |
| Kitz et al. | Radiomyces a genus in the Mucorales pathogenic for mice | |
| Nehmatullah et al. | Antimicrobial potential of some plant extracts against Candida species Isolated from infants oral thrush |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |