CN114438158A - Method for preparing cycloastragenol by fermenting and converting astragaloside IV by using Fusarium proliferatum - Google Patents
Method for preparing cycloastragenol by fermenting and converting astragaloside IV by using Fusarium proliferatum Download PDFInfo
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- CN114438158A CN114438158A CN202011184825.5A CN202011184825A CN114438158A CN 114438158 A CN114438158 A CN 114438158A CN 202011184825 A CN202011184825 A CN 202011184825A CN 114438158 A CN114438158 A CN 114438158A
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- WENNXORDXYGDTP-UOUCMYEWSA-N cycloastragenol Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O)C4(C)C)[C@H]4[C@@H](O)C[C@H]3[C@]2(C)C[C@@H]1O WENNXORDXYGDTP-UOUCMYEWSA-N 0.000 title claims abstract description 32
- WENNXORDXYGDTP-UHFFFAOYSA-N cyclosiversigenin Natural products O1C(C(C)(O)C)CCC1(C)C1C2(C)CCC34CC4(CCC(O)C4(C)C)C4C(O)CC3C2(C)CC1O WENNXORDXYGDTP-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 31
- 241000690372 Fusarium proliferatum Species 0.000 title claims abstract description 13
- QMNWISYXSJWHRY-YLNUDOOFSA-N astragaloside IV Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)[C@H]4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)C[C@H]3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-YLNUDOOFSA-N 0.000 title abstract description 9
- QMNWISYXSJWHRY-BCBPIKMJSA-N astragaloside IV Natural products CC(C)(O)[C@@H]1CC[C@@](C)(O1)[C@H]2[C@@H](O)C[C@@]3(C)[C@@H]4C[C@H](O[C@@H]5O[C@H](CO)[C@H](O)[C@@H](O)[C@H]5O)[C@H]6C(C)(C)[C@H](CC[C@@]67C[C@@]47CC[C@]23C)O[C@@H]8OC[C@@H](O)[C@H](O)[C@H]8O QMNWISYXSJWHRY-BCBPIKMJSA-N 0.000 title abstract description 9
- PFKIBRPYVNVMRU-UHFFFAOYSA-N cyclosieversioside F Natural products CC(C)(O)C1COC(C)(C1)C2C(O)CC3(C)C4CC(OC5OC(CO)C(O)C(O)C5O)C6C(C)(C)C(CCC67CC47CCC23C)OC8OCC(O)C(O)C8O PFKIBRPYVNVMRU-UHFFFAOYSA-N 0.000 title abstract description 9
- 238000000855 fermentation Methods 0.000 claims abstract description 32
- 230000004151 fermentation Effects 0.000 claims abstract description 32
- SMDOOINVMJSDPS-UHFFFAOYSA-N Astragaloside Natural products C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)=C1 SMDOOINVMJSDPS-UHFFFAOYSA-N 0.000 claims abstract description 20
- QMNWISYXSJWHRY-XWJCTJPOSA-N astragaloside Chemical compound O1[C@H](C(C)(O)C)CC[C@]1(C)[C@@H]1[C@@]2(C)CC[C@]34C[C@]4(CC[C@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)CO4)O)C4(C)C)C4[C@@H](O[C@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O4)O)CC3[C@]2(C)C[C@@H]1O QMNWISYXSJWHRY-XWJCTJPOSA-N 0.000 claims abstract description 20
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims description 25
- 239000002609 medium Substances 0.000 claims description 15
- 241001061264 Astragalus Species 0.000 claims description 11
- 235000006533 astragalus Nutrition 0.000 claims description 11
- 210000004233 talus Anatomy 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 10
- 238000010898 silica gel chromatography Methods 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 230000008020 evaporation Effects 0.000 claims description 5
- 238000011081 inoculation Methods 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000001953 recrystallisation Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 abstract description 10
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 24
- 238000000605 extraction Methods 0.000 description 11
- 230000000813 microbial effect Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 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 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 239000000945 filler Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- TWCMVXMQHSVIOJ-UHFFFAOYSA-N Aglycone of yadanzioside D Natural products COC(=O)C12OCC34C(CC5C(=CC(O)C(O)C5(C)C3C(O)C1O)C)OC(=O)C(OC(=O)C)C24 TWCMVXMQHSVIOJ-UHFFFAOYSA-N 0.000 description 2
- PLMKQQMDOMTZGG-UHFFFAOYSA-N Astrantiagenin E-methylester Natural products CC12CCC(O)C(C)(CO)C1CCC1(C)C2CC=C2C3CC(C)(C)CCC3(C(=O)OC)CCC21C PLMKQQMDOMTZGG-UHFFFAOYSA-N 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- PFOARMALXZGCHY-UHFFFAOYSA-N homoegonol Natural products C1=C(OC)C(OC)=CC=C1C1=CC2=CC(CCCO)=CC(OC)=C2O1 PFOARMALXZGCHY-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000003301 hydrolyzing effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 1
- 241000235389 Absidia Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 241000223218 Fusarium Species 0.000 description 1
- 239000009636 Huang Qi Substances 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 241000544020 Stratiotes Species 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940107666 astragalus root Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010047754 beta-Glucosidase Proteins 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- -1 oleanase Proteins 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 235000017709 saponins Nutrition 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J53/00—Steroids in which the cyclopenta(a)hydrophenanthrene skeleton has been modified by condensation with a carbocyclic rings or by formation of an additional ring by means of a direct link between two ring carbon atoms, including carboxyclic rings fused to the cyclopenta(a)hydrophenanthrene skeleton are included in this class
- C07J53/002—Carbocyclic rings fused
- C07J53/004—3 membered carbocyclic rings
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
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- General Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for preparing cycloastragenol by fermenting and converting astragaloside IV (Fusarium proliferatum 0821 CGMCC No. 16482). The invention utilizes the screened Fusarium proliferatum and astragaloside IV as a substrate to prepare the cycloastragenol through fermentation. The invention utilizes Fusarium proliferatum fermentation technology to convert astragaloside to obtain high-purity cycloastragenol, the whole process is carried out at normal temperature and normal pressure, the required equipment condition is low, the method is suitable for mass production, and finally the conversion rate of the cycloastragenol reaches more than 80 percent and the purity reaches more than 95 percent.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemicals, and particularly relates to a method for preparing cycloastragenol by fermenting and converting astragaloside IV by using fusarium stratiotes.
Background
Astragalus root is one of the most common medicines for clinical treatment in traditional Chinese medicine. The effective components of radix astragali include flavonoids, saponins, polysaccharides and amino acids. Astragaloside IV is a representative compound of astragaloside, and has effects of improving immunity, scavenging free radicals, lowering blood pressure, and resisting bacteria. The cycloastragenol is aglycone of astragaloside, has a molecular formula of C30H5OO5 and a relative molecular mass of 490.71, is a main product of astragaloside metabolism in intestinal tracts, and has the effects of promoting wound healing, promoting nerve cell proliferation, resisting aging and the like. And researches find that the cycloastragenol can play a role in activating telomerase and effectively inhibit the reduction of telomeres, so that the immunity of the organism is enhanced and the aging is resisted.
Cycloastragenol is aglycone of astragaloside, is mainly obtained by hydrolyzing astragaloside IV, and is easily soluble in methanol, ethyl acetate, etc. At present, the production process for preparing cycloastragenol by hydrolyzing astragaloside is mainly a chemical method, an enzymatic method and a microbial fermentation method.
The chemical method is to break off the xylosidic bond at the C3 position and the glucosidic bond at the C6 position of the astragaloside by hydrolysis with a strong oxidant to obtain the cycloastragaloside. The Chinese invention patent CN104817610A uses sulfuric acid to hydrolyze astragaloside to prepare cycloastragenol, the reactor is a high-pressure reaction kettle, and the hydrolysis temperature is 130 ℃. The sulfuric acid hydrolysis reaction condition is severe, which causes serious damage to the three-membered ring of the astragaloside and generates a large amount of byproducts.
The enzyme method is to adopt a compound form of multiple hydrolases to hydrolyze astragaloside to prepare the cycloastragenol. The Chinese patent CN105734109A uses the complex enzyme combination of beta-glucosidase, oleanase, cellulase and helicase, and the defect of using the complex enzyme is that the cost of the enzyme is high and the large-scale production is not easy.
The microbial fermentation method is a method for preparing cycloastragenol by using Absidia molderiana to ferment and hydrolyze astragaloside IV in the Chinese invention patent CN 106011213A. Compared with the method, the method has lower conversion rate of astragaloside IV.
Disclosure of Invention
The invention utilizes the screened strain Fusarium proliferatum 0821 (CGMCC No.16482 China general microbiological culture Collection center Beijing city North Chen Xilu No.1 institute No. 3 preservation date: 20180911) to select proper culture medium and ferment and transform astragaloside under proper culture conditions to prepare the cycloastragenol. The invention aims to overcome the defects of more byproducts, high cost, environmental pollution and the like in the prior art for preparing the cycloastragenol by a chemical method and an enzymatic method by adopting a microbial conversion method, and has higher conversion rate than the prior art for preparing the cycloastragenol by microbial fermentation.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
(1) seed culture
Inoculating the preserved strain Fusarium proliferatum into 100mL conical flask filled with wort culture medium, and culturing at 25-30 deg.C and 150-.
(2) Transformation culture
Inoculating the seed solution cultured in the step (1) into a 100mL conical flask filled with a fermentation medium according to the inoculation amount of 10%, and culturing for 6 days at the temperature of 25-30 ℃ and the temperature of 150-200r/min to obtain a fermentation liquid.
(3) Separating and purifying
And (3) extracting the fermentation liquor obtained in the step (2) for three times by using water saturated n-butanol in an isovolumetric manner, then carrying out vacuum concentration and evaporation to obtain an extracted product, further carrying out separation and purification by using silica gel column chromatography, and finally improving the purity of the product to more than 95% by ethanol recrystallization to obtain the cycloastragenol product.
The fermentation medium in the step (2) comprises the following components: 2g/L of astragalus powder, 5g/L of peptone, 2g/L of yeast powder, K2HPO 41 g/L, 0.5g/L of MgSO4 & 7H2O 0.5, and 0.2g/L of astragaloside substrate.
The astragalus powder in the fermentation medium formula in the step (2) is 80-mesh astragalus powder, and is prepared by directly crushing cleaned astragalus roots and stems, and the purity of the astragaloside substrate is 50-100%.
The silica gel column chromatography packing in the step (3) is 100-200-mesh silica gel powder, and the eluent is lower layer solution of methanol-chloroform-water with the volume ratio of 13: 6: 2 respectively.
The invention uses Fusarium proliferatum fermentation technology to convert astragaloside to obtain high-purity cycloastragenol. Compared with the prior art, the invention has the following remarkable beneficial effects:
compared with the chemical method for preparing the cycloastragenol, the microbial fermentation method solves the problems of by-products and environmental pollution in the chemical method preparation process, and has mild fermentation reaction conditions and lower requirements on equipment.
Compared with the method for preparing the cycloastragenol by the enzyme method, the microbial fermentation method has low cost and is easy for industrial production.
Compared with the prior method for preparing the cycloastragenol by the microbial fermentation method, the method has the advantages that the conversion rate of the cycloastragenol is higher and reaches more than 80 percent, and the purity of the cycloastragenol reaches more than 95 percent.
Detailed Description
The present invention is further described with reference to specific examples, which are not intended to limit the scope of the present invention.
Example 1
This example prepares cycloastragenol as follows:
(1) seed culture
The preserved strain Fusarium proliferatum is inoculated into a 100mL conical flask filled with a wort culture medium, and cultured for 48h under the conditions of 25 ℃ and 180r/min to obtain a seed solution. The wort medium consisted of: 10g/L of glucose, 4g/L of peptone, 3g/L of yeast extract powder and 10g/L of malt powder.
(2) Transformation culture
Inoculating the seed solution cultured in the step (2) into a 100mL conical flask filled with a fermentation medium according to the inoculation amount of 10%, and culturing for 6 days at 25 ℃ and 180r/min to obtain a fermentation liquid. The fermentation medium comprises the following components: 2g/L of astragalus powder, 5g/L of peptone, 2g/L of yeast powder, K2HPO 41 g/L, 0.5g/L of MgSO4 & 7H2O 0.5, and 0.2g/L of astragaloside substrate with the mass percent of 50%.
(3) Separating and purifying
And (3) adding 100mL of water saturated n-butanol into the fermentation liquor obtained in the step (2) for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain an extraction product. Separating and purifying 1g of the extraction product by silica gel column chromatography, wherein the filler is 100-mesh silica gel powder of 200 meshes, and the extraction product is loaded by a dry method, and the eluent is a lower layer solution of methanol-chloroform-water with the volume ratio of 13: 6: 2. The fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected, concentrated to dryness in vacuo and weighed dry, and calculated to yield 0.8092g of product with 86.38% purity.
Recrystallizing the product after silica gel column chromatography with ethanol to obtain cycloastragenol product with purity of 95.47% and conversion rate of 80.92%.
Example 2
This example prepares cycloastragenol as follows:
(1) seed culture
Inoculating Fusarium proliferatum into 100mL conical flask filled with wort culture medium, and culturing at 30 deg.C and 180r/min for 36h to obtain seed solution. The wort medium consisted of: 10g/L of glucose, 4g/L of peptone, 3g/L of yeast extract powder and 10g/L of malt powder.
(2) Transformation culture
Inoculating the seed solution cultured in the step (2) into a 100mL conical flask filled with a fermentation medium according to the inoculation amount of 10%, and culturing for 6 days at the temperature of 30 ℃ and at the speed of 180r/min to obtain a fermentation liquid. The fermentation medium comprises the following components: 2g/L of astragalus powder, 5g/L of peptone, 2g/L of yeast powder, K2HPO 41 g/L, 0.5g/L of MgSO4 & 7H2O 0.5, and 0.2g/L of astragaloside substrate with the mass percent of 50%.
(3) Separating and purifying
And (3) adding 100mL of water saturated n-butanol into the fermentation liquor obtained in the step (2) for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain an extraction product. Separating and purifying 1g of the extraction product by silica gel column chromatography, wherein the filler is 100-mesh silica gel powder of 200 meshes, and the extraction product is loaded by a dry method, and the eluent is a lower layer solution of methanol-chloroform-water with the volume ratio of 13: 6: 2. The fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected, concentrated to dryness in vacuo and weighed dry, and calculated to yield 0.7584g of product with 81.57% purity.
Recrystallizing the product after silica gel column chromatography with ethanol to obtain cycloastragenol product with purity of 94.81% and conversion rate of 75.84%.
Example 3
This example prepares cycloastragenol as follows:
(1) seed culture
Inoculating the strain Fusarium proliferatum into a 100mL conical flask filled with a wort culture medium, and culturing at 30 ℃ for 36h at 180r/min to obtain a seed solution. The wort medium consisted of: 10g/L of glucose, 4g/L of peptone, 3g/L of yeast extract powder and 10g/L of malt powder.
(2) Transformation culture
Inoculating the seed solution cultured in the step (2) into a 100mL conical flask filled with a fermentation medium according to the inoculation amount of 10%, and culturing for 6 days under the conditions of 30 ℃ and 180r/min to obtain a fermentation liquid. The fermentation medium comprises the following components: 2g/L of astragalus powder, 5g/L of peptone, 2g/L of yeast powder, K2HPO 41 g/L, 0.5g/L of MgSO4 & 7H2O 0.5 and 0.2g/L of 100 percent of astragaloside substrate by mass percentage.
(3) Separating and purifying
And (3) adding 100mL of water saturated n-butanol into the fermentation liquor obtained in the step (2) for extraction, continuously extracting for three times, and then carrying out vacuum concentration and evaporation drying on the obtained extract liquor to obtain an extraction product. Separating and purifying 1g of the extraction product by silica gel column chromatography, wherein the filler is 100-200 mesh silica gel powder, dry loading is adopted, and the eluent is a lower layer solution of methanol-chloroform-water with the volume ratio of 13: 6: 2. The fractions containing the product were collected and tested for concentration in the liquid phase, after which all fractions were collected, concentrated to dryness in vacuo and weighed dry, and calculated to yield 0.7885g of product with 83.48% purity.
Recrystallizing the product after silica gel column chromatography with ethanol to obtain cycloastragenol product with purity of 95.21% and conversion rate of 78.85%.
Claims (5)
1. The screened strain Fusarium proliferatum 0821 CGMCC No.16482 is utilized, and astragaloside is used as a substrate to prepare the cycloastragenol through fermentation.
2. The method according to claim 1, characterized by comprising the steps of:
(1) seed culture
Inoculating the preserved strain Fusarium proliferatum into 100mL conical flask filled with wort culture medium, and culturing at 25-30 deg.C and 150-.
(2) Transformation culture
Inoculating the seed solution cultured in the step (1) into a 100mL conical flask filled with a fermentation medium according to the inoculation amount of 10%, and culturing for 6 days at the temperature of 25-30 ℃ and under the condition of 150-.
(3) Separating and purifying
And (3) extracting the fermentation liquor obtained in the step (2) for three times by using water saturated n-butanol in an isovolumetric manner, then carrying out vacuum concentration and evaporation to obtain an extracted product, further carrying out separation and purification by using silica gel column chromatography, and finally improving the purity of the product to more than 95% by ethanol recrystallization to obtain the cycloastragenol product.
3. The method of claim 2, wherein: the fermentation medium in the step (2) comprises the following components: 2g/L of astragalus powder, 5g/L of peptone, 2g/L of yeast powder, K2HPO 41 g/L, 0.5g/L of MgSO4 & 7H2O 0.5 and 0.2g/L of astragaloside substrate.
4. The method of claim 2, wherein: the astragalus powder in the fermentation medium formula is 80-mesh astragalus powder, and is prepared by directly crushing cleaned astragalus roots, and the purity of the astragaloside substrate is 50-100%.
5. The method of claim 2, wherein: the silica gel column chromatography packing in the step (3) is 100-200-mesh silica gel powder, and the eluent is lower layer solution of methanol-chloroform-water with the volume ratio of 13: 6: 2 respectively.
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