CN118345131A - Method for producing penicillic acid by penicillium fermentation and application thereof - Google Patents
Method for producing penicillic acid by penicillium fermentation and application thereof Download PDFInfo
- Publication number
- CN118345131A CN118345131A CN202410508497.1A CN202410508497A CN118345131A CN 118345131 A CN118345131 A CN 118345131A CN 202410508497 A CN202410508497 A CN 202410508497A CN 118345131 A CN118345131 A CN 118345131A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- penicillium
- penicillic acid
- producing
- acid
- 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
- WNPVZANXRCPJPW-UHFFFAOYSA-N 5-[isocyano-(4-methylphenyl)sulfonylmethyl]-1,2,3-trimethoxybenzene Chemical compound COC1=C(OC)C(OC)=CC(C([N+]#[C-])S(=O)(=O)C=2C=CC(C)=CC=2)=C1 WNPVZANXRCPJPW-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 238000000855 fermentation Methods 0.000 title claims abstract description 52
- 230000004151 fermentation Effects 0.000 title claims abstract description 52
- YCOFRPYSZKIPBQ-UHFFFAOYSA-N penicillic acid Natural products COC1=CC(=O)OC1(O)C(C)=C YCOFRPYSZKIPBQ-UHFFFAOYSA-N 0.000 title claims abstract description 52
- VOUGEZYPVGAPBB-UHFFFAOYSA-N penicillin acid Natural products OC(=O)C=C(OC)C(=O)C(C)=C VOUGEZYPVGAPBB-UHFFFAOYSA-N 0.000 title claims abstract description 52
- 241000228143 Penicillium Species 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims abstract description 30
- 241000233866 Fungi Species 0.000 claims abstract description 20
- 239000000287 crude extract Substances 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 241000207199 Citrus Species 0.000 claims abstract description 6
- 235000020971 citrus fruits Nutrition 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 239000000047 product Substances 0.000 claims abstract description 5
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- 239000000178 monomer Substances 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000000605 extraction Methods 0.000 claims description 10
- 238000004821 distillation Methods 0.000 claims description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 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 5
- 239000012153 distilled water Substances 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 230000000813 microbial effect Effects 0.000 claims description 5
- 239000012046 mixed solvent Substances 0.000 claims description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 4
- 229930195725 Mannitol Natural products 0.000 claims description 4
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- 240000008042 Zea mays Species 0.000 claims description 4
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 4
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 4
- 239000002253 acid Substances 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
- 235000005822 corn Nutrition 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- 239000000594 mannitol Substances 0.000 claims description 4
- 235000010355 mannitol Nutrition 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 4
- 239000011782 vitamin Substances 0.000 claims description 4
- 229930003231 vitamin Natural products 0.000 claims description 4
- 229940088594 vitamin Drugs 0.000 claims description 4
- 235000013343 vitamin Nutrition 0.000 claims description 4
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 4
- 239000012138 yeast extract Substances 0.000 claims description 4
- 244000052616 bacterial pathogen Species 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 230000006837 decompression Effects 0.000 claims description 3
- 208000025865 Ulcer Diseases 0.000 claims description 2
- 231100000397 ulcer Toxicity 0.000 claims description 2
- 238000003810 ethyl acetate extraction Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 2
- 244000005700 microbiome Species 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 10
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 238000002386 leaching Methods 0.000 description 2
- 238000009630 liquid culture Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004611 spectroscopical analysis Methods 0.000 description 2
- 238000010183 spectrum analysis Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- SZUVOHPGDMWXRD-UHFFFAOYSA-N 1-(3,4-dimethoxyfuran-2-yl)ethanone Chemical compound COC1=COC(C(C)=O)=C1OC SZUVOHPGDMWXRD-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- 206010064458 Penicilliosis Diseases 0.000 description 1
- 241001496939 Penicillium pulvillorum Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- PVTHJAPFENJVNC-MHRBZPPQSA-N kasugamycin Chemical compound N[C@H]1C[C@H](NC(=N)C(O)=O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O PVTHJAPFENJVNC-MHRBZPPQSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 150000002960 penicillins Chemical class 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
Abstract
The invention discloses a method for producing penicillic acid by penicillium fermentation and application thereof, belonging to the technical field of microorganism development and application, wherein the method for producing penicillium fermentation comprises the following steps of S1, inoculating fungi with the preservation number of CGMCC No.41147 into a liquid fermentation medium, and carrying out shaking fermentation culture for 10d at 28 ℃ to obtain a crude fermentation product; and S2, extracting fermentation supernatant by ethyl acetate, decompressing and distilling to obtain a crude extract, separating and purifying by utilizing a chromatography means to obtain a penicillic acid monomer, wherein compared with the conventional penicillic acid-producing fungi, the penicillic acid-producing fungi has the advantages of about 2-3 times of output, effectively improving the output and the production efficiency of penicillic acid when being applied to the mass production of penicillic acid, expanding the application of penicillic acid in resisting citrus canker, and being convenient for mass production, high in content and wide in application range.
Description
Technical Field
The invention relates to the technical field of microbial development and application, in particular to a method for producing penicillic acid by fermenting penicillium and application thereof.
Background
Penicillic acid is colorless needle crystal, with melting point of 83 deg.C, molecular formula of C8H10O4, relative molecular weight of 170.16, and is very soluble in solvents such as hot water, ethanol, diethyl ether and chloroform, and insoluble in pentane and hexane. Penicillic acid is a major component of secondary metabolites of a few penicillium strains and exhibits a wide range of biological activities, and at the same time, it has a large biotoxicity to various animals, mainly causes damage to organs such as heart, liver and kidney, and is mainly present in animal spoilage feeds.
At present, the toxicology research of penicillic acid is an effective method for preventing and treating animal penicilliosis, but the research must require high-purity compounds as standard substances, and the penicillins produced by the microbial fermentation method at present have the problems of low yield and complex components, often depend on the subsequent purification process, easily cause the condition of insufficient penicillic acid supply, and simultaneously increase the production cost of penicillic acid.
Therefore, there is a need to provide a method for producing penicillic acid by penicillium fermentation and application thereof, which aims to solve the above problems.
Disclosure of Invention
Aiming at the defects existing in the prior art, the embodiment of the invention aims to provide a method for producing penicillic acid by fermenting penicillium and application thereof, so as to solve the problems in the background art.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for producing penicillic acid by penicillium fermentation, comprising the following steps:
Step S1, inoculating fungi with the preservation number of CGMCC No.41147 into a liquid fermentation medium, and carrying out shaking fermentation culture for 10d at the temperature of 28 ℃ by a shaking table to obtain a fermentation crude product;
And S2, extracting the fermentation supernatant by using ethyl acetate, distilling under reduced pressure to obtain a crude extract, and separating and purifying by using a chromatographic method to obtain the penicillic acid monomer.
As a further scheme of the invention, in the step S1, before the fungi are inoculated to a fermentation culture medium, the fungi are subjected to expansion culture, wherein PDA solid culture medium is adopted in the expansion culture, the temperature of the expansion culture is 28 ℃, the environment of the expansion culture is a microbial biochemical incubator, and the time of the expansion culture is 3-5d.
As a further scheme of the invention, the fermentation liquid culture medium in the step S1 comprises 200mL of distilled water, 4g of glucose, 4g of mannitol, 2g of peptone, 0.1g of monopotassium phosphate, 0.06g of magnesium sulfate heptahydrate, 1g of yeast extract powder, 0.2g of corn steep liquor and 0.1mL of vitamin mixed solution.
As a further scheme of the invention, the extraction time of the ethyl acetate in the step S2 is 2-5h, and the extraction temperature is 22-27 ℃.
As a further embodiment of the present invention, the extraction temperature is 25 ℃.
As a further scheme of the invention, the pressure of reduced pressure distillation in the step S2 is-0.1 MPa, and the temperature of a reduced pressure distillation water bath is 24 ℃.
As a further aspect of the present invention, the separation and purification in the step S2 includes the steps of:
Step a, separating the crude extract by a decompression ODS reversed phase column, and collecting fractions;
And b, purifying the fraction by high performance liquid chromatography to obtain a pure product of the penicillic acid.
As a further aspect of the present invention, in the step a, the mobile phase is a mixed solvent of methanol and water, wherein the volume ratio of methanol to water is 3:7.
As a further scheme of the invention, in the step b, the high performance liquid chromatography separates the methanol aqueous solution with 21% of mobile phase and the retention time is 15min.
Use of penicillium fermentation to produce penicillium acid for controlling pathogenic bacteria from citrus ulcers.
In summary, compared with the prior art, the embodiment of the invention has the following beneficial effects:
the invention provides fungus producing group effect inhibitor and application thereof, the yield of penicillic acid produced by the fungus is about 0.35g of penicillic acid produced and extracted by each 1L of fermentation liquor, and the content of penicillic acid in an extract is more than 90 percent.
In order to more clearly illustrate the structural features and efficacy of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is an HPLC fingerprint of a crude extract obtained by shaking fermentation of a fungus JW-13-16 in the example of the invention.
FIG. 2 is an MS of an inventive example of a target compound.
FIG. 3 is a 1 H-NMR chart of a target compound according to an example of the invention.
FIG. 4 is a diagram showing 13 C-NMR of a target compound according to an embodiment of the invention.
FIG. 5 is a graph showing the activity of pure penicillic acid against pathogenic bacteria of citrus canker in the inventive example.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
In one embodiment of the present invention, a method for producing penicillic acid by fermentation of penicillium comprises the steps of:
Step S1, inoculating fungi with the preservation number of CGMCC No.41147 into a liquid fermentation medium, and carrying out shaking fermentation culture for 10d at the temperature of 28 ℃ by a shaking table to obtain a fermentation crude product;
And S2, extracting the fermentation supernatant by using ethyl acetate, distilling under reduced pressure to obtain a crude extract, and separating and purifying by using a chromatographic method to obtain the penicillic acid monomer.
In this embodiment, the fungus is Penicillium pad (Penicillium pulvillorum) JW-13-16, and the preservation number is CGMCC No. 41147, and the fungus is preserved in China general microbiological culture Collection center (CGMCC) at the address of 04/07: the dynasty district North Star, department 1, hospital 3 in Beijing;
1) In the step S1, before the fungi are inoculated to a fermentation culture medium, the fungi are subjected to expansion culture, wherein the expansion culture adopts a PDA solid culture medium, the temperature of the expansion culture is 28 ℃, the environment of the expansion culture is a microbial biochemical incubator, and the time of the expansion culture is 3-5d; the preparation method of the PDA solid culture medium comprises the following steps: 200g of peeled potatoes are diced, added with distilled water and boiled for 20min, filtered by gauze to obtain a leaching solution, 20g of glucose and 18g of agar are added into the leaching solution, water is used for constant volume to 1L, and steam sterilization is carried out for 30min at the high temperature and the high pressure of 121 ℃;
The fermentation liquid culture medium in the step S1 comprises 200mL of distilled water, 4g of glucose, 4g of mannitol, 2g of peptone, 0.1g of monopotassium phosphate, 0.06g of magnesium sulfate heptahydrate, 1g of yeast extract powder, 0.2g of corn steep liquor and 0.1mL of vitamin mixed solution; the liquid fermentation medium is preferably contained in 500mL conical flasks;
In the step 1, the temperature of the fermentation culture is preferably 28 ℃, and the environment of the fermentation culture is preferably a constant temperature shaking incubator for fermentation culture for 10d, which is more beneficial to maintaining a constant temperature;
4) The extraction time of ethyl acetate in the step S2 is 2-5h, preferably 5h, the extraction is favorable for dissolving the penicillic acid in the fermentation supernatant in the ethyl acetate, the extraction temperature is 22-27 ℃, preferably 25 ℃, and the stirring extraction is favorable for fully dissolving the penicillic acid in the ethyl acetate;
5) The pressure of reduced pressure distillation in the step S2 is-0.1 MPa, the temperature of reduced pressure distillation water bath is 24 ℃, the method of reduced pressure distillation is not particularly limited, and the method of reduced pressure distillation well known in the art can be adopted;
The crude penicillic acid extract is obtained by extraction with ethyl acetate, and is subjected to fingerprint spectrum analysis, and the gradient elution method comprises the following steps:
0 to 5 minutes: 10% by volume of aqueous methanol solution;
5-45 minutes: 10-100% methanol aqueous solution by volume concentration;
45-55 minutes: a pure methanol solution;
The compound represented by the chromatographic peak is penicillic acid after the fingerprint analysis at 18-19 minutes;
6) The separation and purification in the step S2 comprises the following steps: step a, separating the crude extract by a decompression ODS reversed phase column, and collecting fractions; purifying the fraction by high performance liquid chromatography to obtain a pure product of the penicillic acid; wherein the stationary phase of the reduced pressure ODS reversed phase column in the step a is preferably C 18, and the mobile phase of the reduced pressure reversed phase column is preferably a methanol-water mixed solvent; in the methanol-water mixed solvent, the volume ratio of methanol to water is 3:7, in the step b, separating the methanol aqueous solution with 21% of mobile phase by high performance liquid chromatography, wherein the retention time is 15min;
in order to clearly separate pure components, the compounds which show peaks when separated by high performance liquid chromatography for 15 minutes are respectively subjected to mass spectrometry, nuclear magnetic resonance hydrogen spectrometry and carbon spectrometry, and the result shows that the compounds are penicillic acid.
Specific:
(1): peat moss collected from the rock wall of Jian Jinggang mountain area is used for subsequent separation, a sample is ground and then inoculated on a PDA culture medium for separation culture at 28 ℃, and fungus obtained by screening is JW-13-16 after morphological identification;
(2): inoculating the fungi obtained in the step (1) to a PDA strain culture medium for culturing to obtain fermentation strains, preparing a culture medium containing the following components as the fermentation culture medium, adding 4g of glucose, 4g of mannitol, 2g of peptone, 0.1g of monopotassium phosphate, 0.06g of magnesium sulfate heptahydrate, 1g of yeast extract powder, 0.2g of corn steep liquor, 0.1mL of vitamin mixed solution and 200mL of distilled water into a 500mL conical flask, and carrying out high-pressure sterilization. Inoculating the prepared strain agar blocks with the specification of 0.5cm multiplied by 0.5cm into a conical flask, and placing the inoculated conical flask in a constant-temperature shaking culture at 28 ℃ for 9 days to obtain a fermented product.
And (3) carrying out solid-liquid separation on the fermentation liquid, extracting the fermentation supernatant by adopting ethyl acetate, and carrying out reduced pressure distillation (the water bath temperature of a rotary evaporator is 24 ℃ C., and the pump pressure of a circulating water type vacuum pump is-0.1 MPa) on the extract to obtain a fermentation crude extract. Fingerprint analysis is carried out on the crude extract, and gradient elution conditions are as follows:
0 to 5 minutes: 10% by volume of aqueous methanol solution; 5-45 minutes: 10-100% methanol aqueous solution by volume concentration; 45-55 minutes: pure methanol solution.
The fingerprint of the fermented crude extract is shown in figure 1, and the compound with retention time of 18-19 min is penicillic acid.
(3): And (3) passing the crude extract in the step (2) through an ODS reversed phase column, wherein a stationary phase is C 18, a mobile phase is a methanol-water mixed solvent, the volume ratio of methanol to water is 3:7, obtaining a fraction, purifying the obtained fraction through high performance liquid chromatography, and the mobile phase is a 21% methanol aqueous solution, and the retention time is 15 minutes, thus obtaining a pure compound. The compound is subjected to nuclear magnetic resonance hydrogen spectrum/carbon spectrum and mass spectrum analysis. The nuclear magnetic data contrast identification shows that the penicillic acid is obtained, the mass spectrum of the penicillic acid is shown in figure 2, the hydrogen spectrum is shown in figure 3, and the carbon spectrum is shown in figure 4.
Through detection, the purity of the pure penicillic acid extracted by the invention is more than 99.5 percent.
(4): And (3) verifying the activity of the pure penicillic acid obtained in the step (3) against citrus canker, wherein kasugamycin is used as a positive control drug, and the antibacterial activity result shows that the effective antibacterial concentration of penicillic acid is less than 2mg/mL, and the activity of the penicillic acid against citrus canker is shown in figure 5.
The invention provides fungus producing group effect inhibitor and application thereof, the yield of penicillic acid produced by the fungus is about 0.35g of penicillic acid produced and extracted by each 1L of fermentation liquor, and the content of penicillic acid in an extract is more than 90 percent.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method for producing penicillic acid by penicillium fermentation, comprising the steps of:
Step S1, inoculating fungi with the preservation number of CGMCC No.41147 into a liquid fermentation medium, and carrying out shaking fermentation culture for 10d at the temperature of 28 ℃ by a shaking table to obtain a fermentation crude product;
And S2, extracting the fermentation supernatant by using ethyl acetate, distilling under reduced pressure to obtain a crude extract, and separating and purifying by using a chromatographic method to obtain the penicillic acid monomer.
2. The method for producing penicillium fermentation to penicillium acid according to claim 1, wherein in the step S1, the fungi is subjected to expansion culture before being inoculated into a fermentation medium, wherein PDA solid medium is adopted for the expansion culture, the temperature of the expansion culture is 28 ℃, the environment of the expansion culture is a microbial biochemical incubator, and the time of the expansion culture is 3-5d.
3. The method for producing penicillic acid by fermentation of penicillium according to claim 1, wherein the fermentation broth in the step S1 comprises distilled water 200mL, glucose 4g, mannitol 4g, peptone 2g, potassium dihydrogen phosphate 0.1g, magnesium sulfate heptahydrate 0.06g, yeast extract 1g, corn steep liquor 0.2g and vitamin mixture solution 0.1mL.
4. The method for producing penicillic acid by fermentation of penicillium according to claim 1, wherein the ethyl acetate extraction time in the step S2 is 2-5h, and the extraction temperature is 22-27 ℃.
5. The method for producing penicillic acid by fermentation of penicillium according to claim 4, wherein the extraction temperature is 25 ℃.
6. The method for producing penicillic acid according to claim 1, wherein the reduced pressure distillation in the step S2 is performed under a pressure of-0.1 MPa, and the reduced pressure distillation water bath temperature is 24 ℃.
7. The method for producing penicillic acid by fermentation of penicillium according to claim 1, wherein the separation and purification in the step S2 comprises the steps of:
Step a, separating the crude extract by a decompression ODS reversed phase column, and collecting fractions;
And b, purifying the fraction by high performance liquid chromatography to obtain a pure product of the penicillic acid.
8. The method for producing penicillic acid by fermentation of penicillium according to claim 7, wherein in the step a, the mobile phase is a mixed solvent of methanol and water, wherein the volume ratio of methanol and water is 3:7.
9. The method for producing penicillic acid according to claim 8, wherein in the step b, the mobile phase of the high performance liquid chromatography is 21% methanol aqueous solution, and the retention time is 15min.
10. The application of penicillium fermentation to produce penicillium acid is characterized in that the penicillium acid is used for preventing and controlling pathogenic bacteria of citrus ulcers.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118345131A true CN118345131A (en) | 2024-07-16 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114478565B (en) | Monascin A with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof | |
| CN111154658B (en) | Marine fungus, novel skeleton heteroterpene derivative prepared from marine fungus, and preparation method and application of novel skeleton heteroterpene derivative | |
| CN113151080A (en) | Lactobacillus plantarum and application thereof in producing phenyllactic acid | |
| CN114350722B (en) | Method for preparing genistein | |
| CN114276395A (en) | N having pancreatic lipase inhibitory activity6-hydroxyethyl-5' -acetyl-beta-ribose adenosine and preparation method thereof | |
| CN104894173B (en) | A kind of preparation method of curcumin derivate | |
| CN118345131A (en) | Method for producing penicillic acid by penicillium fermentation and application thereof | |
| CN114478564B (en) | Monascin C with glycosidase activity inhibiting and immunoregulatory activity and preparation method thereof | |
| CN114292280A (en) | Monascus B with glycosidase inhibiting activity and immunoregulation activity and preparation method thereof | |
| CN114395010B (en) | Tripterine derivative and application thereof in tumor resistance | |
| CN109456899A (en) | A kind of method of Penicillium notatum and its fermenting and producing penicillic acid | |
| CN112725205B (en) | Saccharomyces strain and screening method and application thereof | |
| CN118146958A (en) | Fungus for producing group effect inhibitor | |
| CN106399397A (en) | Method for increasing content of tyrosol in rhodiola rosea by microorganism fermentation | |
| CN108374029B (en) | Method for promoting antrodia camphorata liquid fermentation to produce Antrodin C | |
| CN109022512B (en) | Method for biologically synthesizing Hispidin | |
| CN108998480B (en) | Method for preparing phenol compounds by using microorganisms | |
| CN113004237A (en) | Spiro compound and preparation method and application thereof | |
| CN113173969A (en) | Marine fungus-derived heteroterpene compound and application thereof in preparation of anti-hepatic fibrosis drugs | |
| CN115385878B (en) | Natural active compound moenofuran and preparation method and application thereof | |
| CN109810906B (en) | Endophytic fungi and application thereof in fermentation preparation of phenolic acid compounds | |
| CN115611914A (en) | Monascus A, B and C with glycosidase inhibiting activity, immunoregulation activity and anti-aging activity and preparation method thereof | |
| CN110092794A (en) | The method and application of chaetomium globosum QY-1 separation and Extraction chaetomium globosum A | |
| CN113072605B (en) | Fumosoroseside B with antibacterial, free radical scavenging and tyrosinase activity inhibiting effects and its preparation method | |
| CN113004357B (en) | Fumosoroseside A with antibacterial, free radical scavenging and tyrosinase activity inhibiting effects and its preparation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication |