CN110592169A - Method for preparing ADD by phytosterol microbial transformation - Google Patents
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Abstract
The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing ADD by phytosterol microbial transformation. The invention comprises the following steps: (1) fermenting and converting phytosterol by using mycobacterium B-NRRL 3683, and standing and layering fermentation liquor to obtain an upper oil layer; (2) and (3) carrying out conversion reaction by taking the oil layer as a substrate and a bacterial solution of the nocardia simplicifolia as an enzyme source, and extracting and refining reactants to obtain the ADD. The invention adopts a two-step conversion method, firstly converts the phytosterol into a mixture of 4-AD and ADD, then converts the 4-AD enriched in an oil layer into ADD, adopts different strains in the two steps, omits the step of independently extracting the 4-AD in the whole reaction, has high conversion rate, strong reaction specificity, relatively quick conversion time, low degradation rate of products, high product quality, and high application prospect, and the purity of the obtained final product ADD can reach more than 99.5 percent.
Description
Technical Field
The invention relates to a production method of a steroid drug intermediate, in particular to a method for preparing ADD by phytosterol microbial transformation.
Background
Androst-4-ene-3,17-dione (androst-4-ene-3,17-dione, abbreviated as androstenedione, AD or 4-AD) and androst-1, 4-diene-3,17-dione (androsta-1,4-diene-3,17-dione, abbreviated as androstenedione, ADD) are important steroid intermediate compounds, androstenedione (4-AD) can be used as precursor of hormone substances such as male hormone, anabolin, etc., and can also be used for synthesizing drugs such as spironolactone, hydrocortisone, prednisone oxide, dexamethasone, etc.; the Androstenedione (ADD) can be used for synthesizing female hormone, oral contraceptive and other medicines.
Currently, there are two main methods for synthesizing androstenedione: firstly, dioscin is used as a starting material and is obtained through 11 steps of chemical synthesis reaction. And secondly, directly obtaining ADD from phytosterol through microbial transformation. The microbial conversion has the advantages of mild reaction conditions, energy conservation, environmental protection and the like, and whether the microbial conversion has economy including reaction substrate concentration, conversion rate and conversion specificity is determined, wherein the conversion specificity refers to the ratio of ADD to byproduct 4-AD in the conversion process, and the higher the ratio is, the better the conversion specificity is. Us patent USP4345029 describes the transformation of Mycobacterium species (Mycobacterium phlei) to AD and ADD; U.S. Pat. No. 3,7259005 describes the transformation of phytosterol into AD and ADD using another Mycobacterium (Mycobacterium fortuitum), but the transformation efficiency and specificity are not high, the transformation rate is only 64% at the maximum, and the industrial value is low.
Disclosure of Invention
In view of the above technical problems, an object of the present invention is to provide a method for preparing ADD by microbial transformation of phytosterol, which can solve the problems of low conversion rate and high degradation rate in the current methods for preparing androstenedione, and can rapidly and highly purify phytosterol to be converted into ADD.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for preparing ADD by phytosterol microbial transformation comprises the following steps:
(1) carrying out slant culture and shake flask seed culture on mycobacteria (Mycobacterium sp.) B-NRRL 3683, then carrying out fermentation conversion on phytosterol, heating and cooling fermentation liquor after the conversion is finished, standing for layering, and discarding a lower water phase to obtain an upper oil layer;
(2) and (2) taking the oil layer obtained in the step (1) as a substrate, taking a bacterial liquid obtained by performing slant culture and shake flask seed culture on the Nocardia simplex as an enzyme source, adding the bacterial liquid into a conversion system, performing conversion reaction at 28-32 ℃, completely reacting, and extracting and refining reactants to obtain the ADD.
Further, the formula of the slant culture medium of the Mycobacterium (Mycobacterium sp.) B-NRRL 3683 in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, agar 20g/L, pH 7.5-8.0;
the formula of the shake flask seed culture medium in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.
Further, the formula (w/v) of the transformation medium in the step (1) is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 1-10% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
Preferably, the conversion conditions in step (1) are: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.
Further, heating the fermentation liquor in the step (1) to 80 ℃, preserving heat, standing for 2 hours, layering, and treating the lower-layer water phase as wastewater.
Further, the formula (w/v) of the slant culture medium of the nocardia simplex in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract, 2% of agar powder and 7.0-7.2% of PH;
the formula (w/v) of the primary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract and pH value of 7.0-7.2;
the formula (w/v) of the secondary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.
Further, the formula (w/v) of the transformation medium in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.
Preferably, the conversion conditions in step (2) are: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.
Further, the extraction method in the step (2) is as follows: heating and standing the conversion product, and removing a water layer and leaving an oil layer; adding methanol into the oil layer, stirring and standing at normal temperature, separating out the upper layer of methanol, concentrating under reduced pressure to dry, adding a small amount of water to carry out suction filtration on residual methanol in the dry oil, cooling and discharging; the separated oil layer is extracted twice with methanol to obtain crude ADD product.
Further, the refining method in the step (2) is as follows: adding a sodium hydroxide solution with the mass concentration of 2% into the ADD crude product, heating for saponification, cooling, performing suction filtration, and leaching to be neutral to obtain a white-like solid; and drying and weighing the white-like solid, adding methanol, heating, stirring, dissolving, adding activated carbon, stirring, decoloring, performing suction filtration and leaching while the white-like solid is hot, concentrating the filtrate under reduced pressure, cooling, growing crystals, performing suction filtration, and drying a filter cake to obtain an ADD fine product.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts a two-step conversion method, firstly converts the phytosterol into a mixture of 4-AD and ADD, then converts the 4-AD enriched in an oil layer into ADD, adopts different strains in the two steps, omits the step of independently extracting the 4-AD in the whole reaction, has high conversion rate, strong reaction specificity, relatively quick conversion time, low degradation rate of products and high product quality, and the purity of the obtained final product ADD can reach more than 99.5 percent, thereby having great application prospect.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The materials used in the following examples are all commercially available from conventional sources.
Example 1
1. Conversion of phytosterols to 4-AD
1.1 seed culture
The strain name is as follows: mycobacterium sp.B-NRRL 3683
1.1.1 slant culture Medium
The formula is as follows: 0.1-1.0g/L of peptone, 0.1-1.0g/L of yeast extract, 0.1-1.0g/L of glucose, 0.1-1.0g/L of disodium hydrogen phosphate, 20g/L of agar and 7.5-8.0 of pH.
Sterilizing at 121 deg.C for 30 min. After coagulation and forming, inoculation is carried out under aseptic conditions.
After inoculation, the culture is carried out for 4 days at 30 ℃, and the culture is stored in a refrigerator at 4 ℃ for no more than 1 month.
1.1.2 Shake flask seed Medium
The formula is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.
Sterilizing at 121 deg.C for 30 min. And cooling to room temperature.
1.1.2.1 first stage culture
Inoculation under sterile conditions, inoculum size: scrape 1 ring per 100 ml. After inoculation, the cells were incubated with shaking at 200rpm for 48 hours at 30 ℃.
1.1.2.2 Secondary culture
Inoculation under sterile conditions, inoculum size: 10 percent. After inoculation, the cells were incubated with shaking at 200rpm for 48 hours at 30 ℃.
1.210 liter tank fermentation conversion
The conversion was carried out in a 10 liter tank. Measuring volume: 6 liters. Post-inoculation volume: 6 liters.
1.2.1 transformation Medium
The formula is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 3% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, and inoculating under aseptic conditions, wherein the inoculation amount is as follows: 20 percent.
1.2.2 transformation conditions
28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 88 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
1.3 treatment of the materials before dehydrogenation
Heating the fermented liquid to 80 deg.C, maintaining the temperature, standing for 2 hr, layering, and treating the lower water phase with wastewater to obtain upper oil layer.
Conversion of 4-AD to ADD
2.1 seed culture
The strain name is as follows: nocardia simplex
2.1.1 slant culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of potassium dihydrogen phosphate, 0.01-0.05% of yeast extract, 2% of agar powder and 7.0-7.2% of PH.
The culture conditions are as follows: 30 plus or minus 1 ℃ for 3 to 5 days.
2.1.2 first order seed culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of potassium dihydrogen phosphate and 0.01-0.05% of yeast extract, and the PH value is 7.0-7.2.
The culture conditions are as follows: 500 ml of shake flask is filled with 100 ml of culture medium, and shake culture is carried out at the rotation speed of 200rpm, the temperature of 30 +/-1 ℃ and the culture time of 24 hours.
2.1.3 Secondary seed culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.
The culture conditions are as follows: 500 ml of shake flask is filled with 100 ml of culture medium, and shake culture is carried out at the rotating speed of 200rpm and the temperature of 30 +/-1 ℃ for 16-24 hours.
2.1.410L tank seed transformation culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2. Sterilizing at 121 deg.C for 30 min, cooling to 30 deg.C, inoculating with 10% of inoculum size.
Adding the oil layer obtained in the step 1.3, and culturing conditions are as follows: 28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 64 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
3. Extracting and refining
3.1 extraction
(1) Heating the transformed fermentation liquor to 80 ℃, standing for 2 hours, removing a water layer, and leaving an oil layer;
(2) adding 3 liters of methanol into an oil layer, stirring for 20 minutes at normal temperature, standing for 2 hours, separating out upper-layer methanol, concentrating under reduced pressure at 50 ℃ to dry, adding a small amount of water to bring residual methanol in the dry oil, cooling to below 30 ℃, and performing suction filtration and discharging;
(3) extracting the oil layer separated in the step (2) twice by using methanol, using 3 liters of methanol each time, combining the methanol separated in the step (2) and the methanol separated in the step (3), heating and concentrating under reduced pressure;
(4) the resulting mixture is concentrated and filtered to obtain a solid, i.e., crude ADD.
3.2 refining
(1) Adding 500 ml of 2% sodium hydroxide solution into the obtained ADD crude product, heating to 60 ℃, and saponifying for 2 hours;
(2) cooling to 10-20 ℃, carrying out suction filtration and leaching to be neutral to obtain a white solid;
(3) drying the obtained solid at 50 ℃, weighing, adding 4V methanol, heating to 60 ℃, stirring for dissolving, adding 0.1W activated carbon, stirring for decoloring for 2 hours, carrying out suction filtration and leaching while the solid is hot, concentrating the filtrate under reduced pressure to 2V volume, cooling to 4 ℃, growing crystals for 1 hour, and carrying out suction filtration;
(4) and drying a filter cake, and recovering 55.2 g of ADD with the purity of 99.5%.
The above reaction process is as follows:
example 2
1. Conversion of phytosterols to 4-AD
1.1 seed culture according to the method of example 1
1.210 liter tank fermentation conversion
The conversion was carried out in a 10 liter tank. Measuring volume: 6 liters. Post-inoculation volume: 6 liters.
1.2.1 transformation Medium
The formula is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 5% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, and inoculating under aseptic conditions, wherein the inoculation amount is as follows: 20 percent.
1.2.2 transformation conditions
28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 102 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
1.3 treatment of the materials before dehydrogenation
Heating the fermented liquid to 80 deg.C, maintaining the temperature, standing for 2 hr, layering, and treating the lower water phase with wastewater to obtain upper oil layer.
Conversion of 4-AD to ADD
2.1 seed culture according to example 1
2.1.410L tank seed transformation culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2. Sterilizing at 121 deg.C for 30 min, cooling to 30 deg.C, inoculating with 10% of inoculum size.
Adding the oil layer obtained in the step 1.3, and culturing conditions are as follows: 28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 72 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
3. Extracting and refining
The extraction and refining steps were carried out as in example 1, the filter cake was dried, 94.5 g of ADD was recovered, the purity was 99.6%.
Example 3
1. Conversion of phytosterols to 4-AD
1.1 seed culture according to the method of example 1
1.210 liter tank fermentation conversion
The conversion was carried out in a 10 liter tank. Measuring volume: 6 liters. Post-inoculation volume: 6 liters.
1.2.1 transformation Medium
The formula is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 1% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, and inoculating under aseptic conditions, wherein the inoculation amount is as follows: 20 percent.
1.2.2 transformation conditions
28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 60 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
1.3 treatment of the materials before dehydrogenation
Heating the fermented liquid to 80 deg.C, maintaining the temperature, standing for 2 hr, layering, and treating the lower water phase with wastewater to obtain upper oil layer.
Conversion of 4-AD to ADD
2.1 seed culture according to example 1
2.1.410L tank seed transformation culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2. Sterilizing at 121 deg.C for 30 min, cooling to 30 deg.C, inoculating with 10% of inoculum size.
Adding the oil layer obtained in the step 1.3, and culturing conditions are as follows: 28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 48 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
3. Extracting and refining
The extraction and refining steps were carried out as in example 1, the filter cake was dried, 18.8 g of ADD was recovered, the purity was 99.8%.
Example 4
1. Conversion of phytosterols to 4-AD
1.1 seed culture according to the method of example 1
1.210 liter tank fermentation conversion
The conversion was carried out in a 10 liter tank. Measuring volume: 6 liters. Post-inoculation volume: 6 liters.
1.2.1 transformation Medium
The formula is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 10% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
Sterilizing at 121 ℃ for 30 minutes, cooling to room temperature, and inoculating under aseptic conditions, wherein the inoculation amount is as follows: 20 percent.
1.2.2 transformation conditions
28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 180 hours, TLC spot plate monitoring conversion condition, and waiting for conversion completion.
1.3 treatment of the materials before dehydrogenation
Heating the fermented liquid to 80 deg.C, maintaining the temperature, standing for 2 hr, layering, and treating the lower water phase with wastewater to obtain upper oil layer.
Conversion of 4-AD to ADD
2.1 seed culture according to example 1
2.1.410L tank seed transformation culture
The formula of the culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2. Sterilizing at 121 deg.C for 30 min, cooling to 30 deg.C, inoculating with 10% of inoculum size.
Adding the oil layer obtained in the step 1.3, and culturing conditions are as follows: 28-32 ℃, 200rpm, air flow of 0.1-1.0vvm, pot pressure of 0.01-0.1MPa, conversion time of 120 hours, TLC point plate monitoring conversion condition, and waiting for conversion completion.
3. Extracting and refining
The extraction and refining steps were carried out as in example 1, the filter cake was dried, ADD 186.4 g was recovered, purity 99.5%.
Claims (10)
1. The method for preparing ADD by phytosterol microbial transformation is characterized by comprising the following steps:
(1) carrying out slant culture and shake flask seed culture on mycobacteria (Mycobacterium sp.) B-NRRL 3683, then carrying out fermentation conversion on phytosterol, heating and cooling fermentation liquor after the conversion is finished, standing for layering, and discarding a lower water phase to obtain an upper oil layer;
(2) and (2) taking the oil layer obtained in the step (1) as a substrate, taking a bacterial liquid obtained by performing slant culture and shake flask seed culture on the Nocardia simplex as an enzyme source, adding the bacterial liquid into a conversion system, performing conversion reaction at 28-32 ℃, completely reacting, and extracting and refining reactants to obtain the ADD.
2. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula of the slant culture medium of Mycobacterium (Mycobacterium sp.) B-NRRL 3683 in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, agar 20g/L, pH 7.5-8.0;
the formula of the shake flask seed culture medium in the step (1) is as follows: peptone 0.1-1.0g/L, yeast extract 0.1-1.0g/L, glucose 0.1-1.0g/L, disodium hydrogen phosphate 0.1-1.0g/L, and pH 7.5-8.0.
3. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the transformation medium in the step (1) is as follows: 1-10% of corn steep liquor, 0.1-1% of sodium nitrate, 0.01-0.1% of diammonium phosphate, 0.1-2% of PPE, 1-10% of phytosterol and 16% of soybean oil, wherein the pH value is 7.5-8.0.
4. The method for preparing ADD by microbial transformation of phytosterol according to claim 1 or 3, wherein the transformation conditions in the step (1) are as follows: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.
5. The method for preparing ADD through phytosterol microbial conversion as claimed in claim 1, wherein the fermentation broth in step (1) is heated to 80 ℃, kept at the temperature for standing for 2 hours, layered, and the lower aqueous phase is treated with wastewater.
6. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the slant culture medium of Nocardia simplex in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract, 2% of agar powder and 7.0-7.2% of PH;
the formula (w/v) of the primary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.15% of monopotassium phosphate, 0.01-0.05% of yeast extract and pH value of 7.0-7.2;
the formula (w/v) of the secondary seed culture medium is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.
7. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the formula (w/v) of the transformation medium in the step (2) is as follows: 0.1-1% of glucose, 0.1-1% of corn steep liquor, 0.1-0.5% of peptone, 0.01-0.25% of monopotassium phosphate, 0.05% of 4-AD and 0.02% of natural killer, and the PH value is 7.0-7.2.
8. The method for preparing ADD by microbial transformation of phytosterol according to claim 1 or 7, wherein the transformation conditions in the step (2) are as follows: at 28-32 deg.C, 200rpm, air flow rate of 0.1-1.0vvm, and tank pressure of 0.01-0.1 MPa.
9. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the extraction method in the step (2) is as follows: heating and standing the conversion product, and removing a water layer and leaving an oil layer; adding methanol into the oil layer, stirring and standing at normal temperature, separating out the upper layer of methanol, concentrating under reduced pressure to dry, adding a small amount of water to carry out suction filtration on residual methanol in the dry oil, cooling and discharging; the separated oil layer is extracted twice with methanol to obtain crude ADD product.
10. The method for preparing ADD by microbial transformation of phytosterol according to claim 1, wherein the refining method in the step (2) is as follows: adding a sodium hydroxide solution with the mass concentration of 2% into the ADD crude product, heating for saponification, cooling, performing suction filtration, and leaching to be neutral to obtain a white-like solid; and drying and weighing the white-like solid, adding methanol, heating, stirring, dissolving, adding activated carbon, stirring, decoloring, performing suction filtration and leaching while the white-like solid is hot, concentrating the filtrate under reduced pressure, cooling, growing crystals, performing suction filtration, and drying a filter cake to obtain an ADD fine product.
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| CN112175033A (en) * | 2020-10-30 | 2021-01-05 | 湖南新合新生物医药有限公司 | Method for refining steroid intermediate mother liquor material |
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