A preparation method of fumigatus tremorine C
By using the non-toxin species P. cypermycetes and synthetic culture medium, the culture conditions are optimized, and the problems of production safety and large-scale production of Aspergillus fumigation tremolin C in the existing technology are solved, and efficient and safe Aspergillus fumigation tremolin C are achieved. Preparation and its application in antitumor drugs.
Patent Information
- Application Number
- CN202410238395.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-04
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2044-03-04
AI Technical Summary
The use of toxic Aspergillus fumigatum strains in the prior art has a safety hazard, and the food culture medium is complex and not repetitive, so mass production and factory operations cannot be achieved.
The non-toxin species Penicillium simplicissimum is used instead of the toxic Aspergillus fumigatum, and is inoculated on synthetic culture medium for culture. The culture conditions are optimized to achieve the efficient preparation of Aspergillus fumigatum tremolin C and ensure the safety and repetition of the production process.
It has achieved safe and efficient production of Aspergillus fumigatin C, which is suitable for factory-based production, and has laid the foundation for its application in anti-tumor drugs.
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Figure CN118048412B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing fumigatus tremorine C. Background Art
[0002] Fumonisin is a type of toxic metabolite produced mainly by Aspergillus fumigatus. Aspergillus fumigatus is widely distributed in soils around the world, especially in paddy fields and vegetable fields, where it is detected most frequently. It is very likely to contaminate food, vegetables, feed, livestock and poultry houses, straw mats, etc. Fumonisin is highly toxic to the central nervous system of rats, mice, rabbits, pigs, sheep, etc., and can cause severe convulsions and even death in animals. In my country, Aspergillus fumigatus poisoning with convulsion symptoms in chickens related to this fungus has been found. Fumonisin C: CAS No. 118974-02-0, molecular formula C 22 H 25 N3O3, molecular weight 379.45. Fumonisin C is a cell cycle inhibitor that has inhibitory activity on the M phase of the mammalian cell cycle and can keep cells in the G2 / M transition phase. It is a potential candidate compound for the preparation of anti-tumor drugs.
[0003] The existing method of producing fumigatus tremorine is to collect naturally infected grains, or to inoculate Aspergillus fumigatus into grain culture medium, and then obtain fumigatus tremorine after treatment. The existing technology requires the use of the fungus Aspergillus fumigatus, which is a pathogenic bacterium that can cause pulmonary aspergillosis and other diseases in humans, livestock and poultry, and is harmful to humans and animals. The use of grain culture medium for cultivation has complex components and low repeatability, and cannot be mass-produced or factory-operated, and cannot form a complete production chain.
[0004] Therefore, the existing technology is in urgent need of improvement. It is necessary to find non-toxic strains to produce fumigatus tremorin C and optimize the culture conditions to form a complete fumigatus tremorin C production chain. Summary of the invention
[0005] The present invention aims to solve the above problems and provides a method for preparing Aspergillus fumigatus tremorine C. Non-toxic strains are selected to replace toxic Aspergillus fumigatus and inoculated into a synthetic culture medium. The operation method has strong repeatability, can realize factory production, and ensure the safety of personnel and other organisms in the production process.
[0006] Specifically, the technical solution provided by this application is as follows:
[0007] In a first aspect, the present invention provides a method for preparing fumigatus tremorine C, the method steps are as follows:
[0008] S1: Select strain: Penicillium simplicifolium Penicillium simplicissimum , the deposit number is CGMCC3.4480;
[0009] S2: Seed solution preparation: 0.1 mL of Penicillium simplex stock solution was spread on a PDA plate, placed in a constant temperature and humidity incubator at 25-28°C for 7 days, spores were scraped into a centrifuge tube under sterile conditions and suspended in sterile water to prepare seed solution;
[0010] S3: Inoculation and culture: The seed solution obtained in S2 was inoculated into the fermentation medium for culture at a temperature of 25-28°C for 240-360 hours. The spore concentration of the inoculated seed solution was 10 4 ~10 5 Pieces / ml;
[0011] S4: extracting and detecting fumigatus tremorine C from the culture solution obtained in S3 to obtain fumigatus tremorine C.
[0012] Specifically, the simple Penicillium has a preservation number of CGMCC3.4480, which was purchased from the China General Microbiological Culture Collection Center; the main biological characteristics of simple Penicillium are: the mycelium is usually white or slightly grayish white, with a velvety and flocculent texture, the conidia are oval, the conidia are green, gray-green or dark gray-green on the front, and slightly yellow on the back, lying flat without wrinkles, and easy to pick up.
[0013] Furthermore, the PDA medium formula in S2 is as follows: 200.0 g / L potato, 20.0-30 g / L glucose, 15-20.0 g / L agar, pH 7.0.
[0014] Furthermore, the fermentation medium in S3 is any one of RIChard medium, modified Czapek medium, PDB medium or malt wort medium.
[0015] Based on the above technical solution, the fermentation medium in S3 further includes one or more combinations of sodium nitrate, zinc sulfate, copper sulfate, manganese sulfate, sodium chloride, and ferric chloride. Preferably, adding sodium nitrate, manganese sulfate, copper sulfate, and zinc sulfate at the same time has a better toxin production effect.
[0016] Preferably, the formula of the fermentation medium in S3 is: glycerol 20-50 g / L, NaNO3 1.5-6 g / L, MgSO4・7H2O 0.5-1.0 g / L, KCl 0.5-1.0 g / L, FeSO4・4H2O 0.01-0.05 g / L, K2HPO4 1.0-3.0 g / L, ZnSO4・5H2O 0.1 g / L, CuSO4・5H2O 0.005 g / L, MnSO4・H2O 0.01 g / L.
[0017] Further preferably, the formula of the fermentation medium in S3 is: glycerol 30g / L, NaNO33g / L, MgSO4・7H2O 0.5g / L, KCl 0.5g / L, FeSO4・4H2O 0.01g / L, K2HPO41.0g / L, ZnSO4・5H2O 0.1g / L, CuSO4・5H2O 0.005g / L, MnSO4・H2O 0.01g / L.
[0018] Further, the spore concentration of the seed solution inoculated in S3 is 10 5 Pieces / ml.
[0019] Furthermore, the extraction method of fumigatus tremorin C in S4 is as follows: after fermentation, the culture medium of the sampled sample is taken out and combined, and an ethyl acetate solution is added according to a mass (g): volume (mL) ratio of 1:1, and the mixture is fully stirred with a stirrer at 150 rpm for 30 minutes, and the mixture is extracted overnight; after the overnight sample is separated, the upper organic phase is drawn, and the ethyl acetate solution is added to the mixture according to a mass (g): volume (mL) ratio of 1:1 for extraction, and the mixture is fully stirred with a stirrer at 150 rpm for 30 minutes, and the mixture is extracted overnight. After the overnight sample is separated, the upper organic phase is drawn, the organic phases are combined, and the obtained ethyl acetate extract is concentrated under reduced pressure at 45°C to remove the ethyl acetate until dry, to obtain an oily substance.
[0020] Furthermore, the detection method of fumigatus tremorine C in S4 is: chromatographic column: C18 column (column length 250 mm, column inner diameter 4.6 mm, filler particle size 5 μm); injection volume: 10 μL; mobile phase: acetonitrile: 0.1% formic acid water = 50:50; flow rate: 1.0 mL / min; detection wavelength: 223 nm.
[0021] In a second aspect, the present invention provides a fumigatus tremorine C prepared by any one of the methods described in the first aspect.
[0022] In the third aspect, based on the same invention, the present invention also provides the use of fumigatus tremorine C in the preparation of anti-tumor drugs.
[0023] The present invention has the following advantages:
[0024] The present invention prepares fumigatus tremorin C by selecting other non-toxic fungi Penicillium simplex to replace harmful Aspergillus fumigatus. In addition, the present invention uses synthetic culture medium to replace the food culture medium used in the prior art for cultivation, has strong repeatability, can realize artificially controlled production, is suitable for factory mass production, and can ensure the safety of personnel and other organisms in the production process. At the same time, it provides a new choice for the drug selection direction of preparing anti-tumor drugs, and lays a foundation for the application of fumigatus tremorin C components in anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.
[0026] Figure 1 : Chromatogram of the standard product of Fumonisin C of the present invention;
[0027] Figure 2 : The present invention detects the sample chromatogram;
[0028] Figure 3 : Comparative chromatograms of fumigatus tremorin C standard and test samples. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.
[0030] Experimental Materials:
[0031] Strain: Penicillium simplex, collection number: CGMCC3.4480 (purchased from China General Microbiological Culture Collection Center); the main biological characteristics of Penicillium simplex are: the mycelium is usually white or slightly grayish white, with a velvety and flocculent texture, the conidia are oval, the conidia are green, gray-green or dark gray-green on the top, and slightly yellow on the back, flat without wrinkles, and easy to pick up.
[0032] Reagents: Zinc sulfate, copper sulfate, manganese sulfate, sodium chloride, and ferric chloride were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 98%.
[0033] Experimental instruments: high performance liquid chromatograph (Shimadzu NexeraXR, Japan, photodiode array UV-visible detector model SPD-M40), SW-CJ-2D (practical vertical novel) double clean bench (Shanghai Hujing Medical Co., Ltd.), HZQ-F100 oscillating incubator (Shanghai Yuzhuo Instrument Co., Ltd.).
[0034] Example 1
[0035] 1.1 Preparation of seed solution
[0036] PDA culture medium formula: potato 200g / L, glucose 20-30g / L, agar 15-20g / L, pH 7.0; strain: Penicillium simplex, preservation number: CGMCC3.4480.
[0037] Take 0.1mL of Penicillium simplex culture stock solution and spread it on the PDA plate, place it in a constant temperature and humidity incubator at 25-28℃ for static culture, and after 7 days of culture, scrape the spores into a centrifuge tube under sterile conditions and suspend them in sterile water. The spore concentration under microscopic examination is 10 4 ~10 5 / ml, to prepare a seed solution, wherein the spore concentration of the seed solution is 10 4 ~10 5 Pieces / ml.
[0038] 1.2 Preparation and cultivation of Aspergillus fumigatus tremorine C fermentation medium
[0039] Fermentation medium types and preparation:
[0040] RIChard medium: glycerol 50 g, KNO3 10 g, KH2PO4 5 g, MgSO4·7H2O 2.5 g, FeCl3 0.02 g, distilled water 1000 mL
[0041] Malt extract medium: 20.0 g malt extract, 20.0 g glycerol, 1.0 g peptone, 1000 mL distilled water
[0042] PDB medium: 200 g potato, 20 g glycerol, 1000 mL distilled water, pH 7.0.
[0043] Modified Czapek medium: glycerol 30.0 g, NaNO3 3.0 g, MgSO4・7H2O 0.5 g, KCl 0.5 g, FeSO4・4H2O 0.01 g, K2HPO4 1.0 g, distilled water 1000 mL, pH 6.0~6.5.
[0044] Inoculation: The seed solution was inoculated with a final spore concentration of 10 4The inoculum of 1000 μg / ml was added into the fermentation medium, the culture temperature was 28°C, the rotation speed was 150 rpm, and the culture was carried out for 240 hours. The production of fumigatus tremorine C was detected by HPLC.
[0045] 1.3 Extraction of fumigatus tremorin C
[0046] After fermentation, the culture medium of the sample was taken out and combined, and ethyl acetate solution was added according to the mass (g): volume (mL) ratio of 1:1, and the mixture was fully stirred with a stirrer at 150 rpm for 30 min, and the mixture was extracted overnight; after the overnight sample was separated, the upper organic phase was drawn, and the ethyl acetate solution was added according to the mass (g): volume (mL) ratio of 1:1 for extraction, and the mixture was fully stirred with a stirrer at 150 rpm for 30 min, and the mixture was extracted overnight. After the overnight sample was separated, the upper organic phase was drawn, the organic phases were combined, and the obtained ethyl acetate extract was concentrated under reduced pressure at 45°C to remove ethyl acetate until dry, and fumigatus tremorine C (oily substance) was obtained.
[0047] Liquid phase (HPLC) detection method of fumigatus tremorin C:
[0048] Chromatographic column: C18 column (column length 250 mm, column inner diameter 4.6 mm, filler particle size 5 μm); injection volume: 10 μL; mobile phase: acetonitrile: 0.1% formic acid water = 50:50; flow rate: 1.0 mL / min; detection wavelength: 223 nm.
[0049] 1.4 Results and Analysis
[0050] The test results are shown in Table 1. Among the different fermentation media, the highest yield of fumigatus tremorine C prepared by the modified Czapek medium was 7.115 mg / L.
[0051] Table 1 Effects of different fermentation media on the production of Aspergillus fumigatus tremorine C
[0052]
[0053] Example 2
[0054] Based on the improved Czapek medium obtained in Example 1, the effect of the inoculation spore concentration on the yield of Aspergillus fumigatus tremorine C (the higher the concentration, the higher the yield, and the concentration and yield in the text refer to the same meaning) was verified and optimized. The method of this example is different from that of Example 1 in that the inoculation spore concentration is 10 5 The experimental method was the same as in Example 1.
[0055] The test results are shown in Table 2. The final yield of Aspergillus fumigatus tremorin C prepared at different inoculation spore concentrations is also different. 5When the concentration of fumonisin C was 1.0447 W / ml, the production of fumonisin C was higher, reaching 8.563 mg / L.
[0056] Table 2 Effects of different inoculation spore concentrations on the production of Aspergillus fumigatus tremorine C
[0057]
[0058] Example 3
[0059] The fermentation medium obtained by verification in Example 1 is a modified Czapek medium, and the inoculation spore concentration obtained by verification in Example 2 is 10 5 On the basis of 100 μg / ml, the formula of the fermentation medium was further optimized.
[0060] This embodiment is different from embodiment 2 in that a mixed solution of trace elements is added to the improved Czapek medium: ZnSO4·5H2O 0.1 g / L, CuSO4·5H2O 0.005 g / L, and MnSO4·H2O 0.01 g / L.
[0061] The test results are shown in Table 3. Compared with the original modified Czapek medium, the addition of the trace element mixture helps to increase the production of Aspergillus fumigatus tremorine C to 9.643 mg / L.
[0062] Table 3 Effects of trace element mixture on the production of tremorin C in Aspergillus fumigatus
[0063]
[0064] Example 4
[0065] The method of this example is different from that of Example 2 in terms of the culture temperature, and the effect of culturing at 25°C and 28°C on the yield of fumigatus tremorine C was verified.
[0066] The test results are shown in Table 4. When the culture temperature is 25°C, the yield of Aspergillus fumigatus tremorine C is better increased, reaching 11.425 mg / L.
[0067] Table 4 Effect of culture temperature on the production of tremorin C in Aspergillus fumigatus
[0068]
[0069] Example 5
[0070] The method of this embodiment is different from that of embodiment 2 in that: the culture time is 360 hours; and the concentrations of sodium nitrate in the culture medium are 1.5 g / ml, 3 g / ml, and 6 g / ml, respectively.
[0071] The experimental settings and test results are shown in Table 5. When the culture time was 360 hours and the sodium nitrate concentration was 3 g / ml, the optimal yield of fumigatus tremorine C reached 13.672 mg / L, which was 60.7% higher than that of the treatment with a culture time of 240 hours.
[0072] Table 5 Effects of culture time and sodium nitrate dosage on the yield of Aspergillus fumigatus tremorine C
[0073]
[0074] Example 6
[0075] Based on the optimization of culture conditions in Examples 1-5, the fermentation medium formula used in this example is as follows: 30 g / L glycerol, 33 g / L NaNO, 0.5 g / L MgSO4・7H2O, 0.5 g / L KCl, 0.01 g / L FeSO4・4H2O, 1.0 g / L K2HPO4, 0.1 g / L ZnSO4・5H2O, 0.005 g / L CuSO4・5H2O, 0.01 g / L MnSO4・H2O. The spore inoculation concentration is 10 5 The culture temperature was 25°C and the culture time was 360 hours. The experimental method was the same as the above embodiment.
[0076] The test results show that the concentration of Aspergillus fumigatus tremorine C fermented under the culture medium and culture conditions of this embodiment can reach 30.406 mg / L. Compared with the concentration of 7.115 mg / L obtained under the traditional culture medium and culture conditions in Example 1, the concentration of Aspergillus fumigatus tremorine C increased by 23.291 mg / L, which is 4.27 times the original concentration.
[0077] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.
Claims
1. A method for preparing fumigatus tremorine C, characterized in that: The steps are as follows: S1: Select strain: Penicillium simplicifolium Penicillium simplicissimum , the deposit number is CGMCC3.4480; S2: Seed solution preparation: 0.1 mL of Penicillium simplex stock solution was spread on a PDA plate, placed in a constant temperature and humidity incubator at 25-28°C for 7 days, spores were scraped into a centrifuge tube under sterile conditions and suspended in sterile water to prepare seed solution; S3: Inoculation and culture: The seed solution obtained in S2 was inoculated into the fermentation medium for culture at a temperature of 25°C for 360 hours. The spore concentration of the inoculated seed solution was Pieces / ml; S4: extracting and detecting fumigatus tremorine C from the culture solution obtained in S3 to obtain fumigatus tremorine C.
2. The method for preparing fumigatus tremorine C according to claim 1, characterized in that: The PDA medium formula in S2 is as follows: 200.0 g / L potato, 20.0-30 g / L glucose, 15-20.0 g / L agar, pH 7.
0.
3. The method for preparing fumigatus tremorine C according to claim 1, characterized in that: The extraction method of fumigatus tremorine C in S4 is as follows: 1) After fermentation, the culture medium of the sample was taken out and combined, and ethyl acetate solution was added according to the mass (g): volume (mL) ratio of 1:1, and the mixture was fully stirred with a stirrer at 150 rpm for 30 min and extracted overnight; 2) After the overnight sample is separated, the upper organic phase is drawn and extracted with ethyl acetate solution at a mass (g): volume (mL) ratio of 1:
1. The mixture is stirred thoroughly with a stirrer at 150 rpm for 30 min and the extraction is carried out overnight. 3) After the overnight sample is separated, the upper organic phase is aspirated, the organic phases are combined, and the resulting ethyl acetate extract is concentrated under reduced pressure at 45°C to remove the ethyl acetate until dryness, thereby obtaining fumigatus tremorine C.
4. The method for preparing fumigatus tremorine C according to claim 1, characterized in that: The detection method of fumigatus tremorine C in the S4 is as follows: chromatographic column: C18 column; injection volume: 10 μL; mobile phase: acetonitrile: 0.1% formic acid water = 50:50; flow rate: 1.0 mL / min; detection wavelength: 223 nm.