A process for the preparation of 7a, 15a-dihydroxy dehydroepiandrosterone

Abstract

This invention discloses a method for preparing 7α,15α-dihydroxydehydroepiandrosterone (DHEA). The method comprises the following steps: DHEA is fed into a liquid culture medium containing *Colletotrichum lini* to convert the DHEA into 7α,15α-dihydroxydehydroepiandrosterone. The *Colletotrichum lini* liquid culture medium also contains cyclodextrin and its derivatives, and Tween. The conversion step includes: first, a low-temperature conversion, followed by a medium-temperature conversion; the low-temperature conversion conditions are: a conversion temperature of 15–20°C, preferably 16°C; the medium-temperature conversion conditions are: a conversion temperature of 28°C. Furthermore, by adding cyclodextrin and its derivatives and Tween, the feed amount of 7α,15α-dihydroxydehydroepiandrosterone prepared in this invention can be increased from 15 g / L to 30 g / L, and the relative conversion rate can reach 90%.

Description

Technical Field

[0001] This invention relates to a method for preparing 7α,15α-dihydroxydehydroepiandrosterone. Background Technology

[0002] Drospironone is a new generation of steroidal contraceptives with fewer side effects, lower toxicity, and better tolerability. It is one of the best oral contraceptives currently available. The traditional method for synthesizing drospironone requires 18 steps. This synthetic route has harsh reaction conditions, poor stereoselectivity, low product yield, and generates a large amount of contaminants.

[0003] With the in-depth research on cell transformation in steroid drugs, a new biochemical synthesis process for drospirenone has been established. By replacing a step in the traditional chemical transformation with cell transformation, the steroid biotransformation method has advantages such as higher stereoselectivity, mild transformation conditions, and environmental friendliness compared to traditional chemical synthesis.

[0004] 7α,15α-dihydroxydehydroepiandrosterone (DHEA) is a key intermediate for drospirenone. Various fungi can now perform hydroxylation reactions at the α positions of C-7 and C-15 of DHEA to form 7α,15α-dihydroxydehydroepiandrosterone. However, existing dihydroxylation processes generally suffer from problems such as low feed rate (10-15 g / L) and low conversion efficiency.

[0005] Chinese patent CN105543320B discloses a method for promoting reducing power regeneration and improving the efficiency of microbial hydroxylation of DHEA. In a 5L fermenter, through batch feeding and sugar supplementation, the temperature is controlled at 25-40℃, pH 6.5, and the tank pressure is adjusted to 0.05MPa. The aeration rate is 0.8-1.2 vvm, the rotation speed is 300-450 r / min, and the dissolved oxygen is controlled at 20-30%. After 30-48 hours of conversion, with a final DHEA concentration of 15 g / L, the yield of 7α,15α-dihydroxydehydroepiandrosterone (DHEA) is 66.6%. According to the data disclosed in this patent, the yield of 7α,15α-dihydroxydehydroepiandrosterone in the conversion product is only 66.6%, and the feed concentration of DHEA is only 15 g / L.

[0006] Chinese patent CN104561216B discloses a method for converting DHEA into 7α-hydroxy-dehydroepiandrosterone (DHEA) and 7α,15α-dihydroxydehydroepiandrosterone (DHEA) using *Colletotrichum flabellulatum* (accession number CGMCC No. 6051). The method involves adding 31.5-36 g / L of carboxymethyl oleoyl chitosan to the *Colletotrichum flabellulatum* bacterial culture and converting at 28-30°C and a rotation speed of 200-220 rpm for 36-60 hours. With a maximum DHEA concentration of 12 g / L, the conversion rate is approximately 93%. According to the data disclosed in this patent, the DHEA concentration in the conversion product is only 10 g / L.

[0007] Therefore, in the process of converting DHEA to 7α,15α-dihydroxydehydroepiandrosterone using *Colletotrichum flaxensis*, improving the feed concentration and substrate conversion rate is an urgent problem to be solved in the existing technology. Summary of the Invention

[0008] To address the aforementioned technical problems, the present invention aims to provide an improved method for synthesizing 7α,15α-dihydroxydehydroepiandrosterone (7α,15α-dihydroxydehydroepiandrosterone) using *Colletotrichum flaxensis* with high feed concentration and high conversion rate. By adding cyclodextrin and its derivatives, as well as Tween, the conversion temperature is lowered, achieving the desired high substrate concentration and high substrate conversion rate.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A method for preparing 7α,15α-dihydroxydehydroepiandrosterone (DHEA) is provided, comprising the following steps: in a liquid culture medium system containing Colletotrichum lini, DHEA is fed and converted to obtain 7α,15α-dihydroxydehydroepiandrosterone; wherein the Colletotrichum lini liquid culture medium system further contains cyclodextrin and its derivatives and Tween.

[0011] In this invention, the feeding and conversion steps include: first performing low-temperature conversion, and then performing medium-temperature conversion;

[0012] The conditions for the low-temperature conversion are: conversion temperature of 15-20℃, preferably 16℃;

[0013] The intermediate-temperature conversion condition is a conversion temperature of 28°C;

[0014] Preferably, the conversion time for the low-temperature conversion is 20-48 hours, for example, 24 hours; and the conversion time for the medium-temperature conversion is 24-72 hours.

[0015] Preferably, the temperature conditions for the low-temperature conversion and the medium-temperature conversion are such that the target temperature is reached within 0.5-1 hour.

[0016] In this invention, the cyclodextrin and its derivatives and Tween are conventional in the art, and the cyclodextrin and its derivatives are one or more of β-cyclodextrin, α-cyclodextrin, γ-cyclodextrin, carboxymethyl β-cyclodextrin, methyl β-cyclodextrin and hydroxypropyl β-cyclodextrin, preferably β-cyclodextrin;

[0017] The Tween is Tween 60 and / or Tween 80, preferably Tween 80.

[0018] In this invention, the mass fraction of dehydroepiandrosterone (DHEA) is 2-3%, the mass fraction of cyclodextrin and its derivatives is 3-5%, the volume fraction of Tween is 1-2%, and the wet weight of Colletotrichum lini before feeding is 40-50 g / L.

[0019] More preferably, the dehydroepiandrosterone (DHEA) has a mass fraction of 3%, the cyclodextrin and its derivatives are β-cyclodextrin with a mass fraction of 3%, and the Tween is Tween 80 with a volume fraction of 1.5%.

[0020] The concentrations of dehydroepiandrosterone (DHEA) and β-cyclodextrin are 30 g / L, and the concentrations of Tween 80 are 15 g / L.

[0021] In this invention, the dissolved oxygen during the feeding and conversion process is controlled at 30±2%, the rotation speed during the feeding and conversion process is 200-500 rpm, for example, 300-500 rpm or 200-350 rpm, and the aeration rate during the feeding and conversion process is 1.5-2 vvm, preferably 1.5 vvm.

[0022] The *Colletotrichum lini* strain mentioned is a commercially available strain, CGMCC, NO. 3.4486.

[0023] In this invention, the liquid culture medium comprises: glucose 30-50 g / L, corn steep liquor 10-30 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-0.3 g / L, sodium nitrate 1-5 g / L, and pH 5.5-5.8.

[0024] The liquid culture medium containing Colletotrichum lini was prepared by the following process: the Colletotrichum lini strain was successively subjected to slant culture, seed culture and fermentation culture.

[0025] The slant culture includes culturing a commercially available strain of Colletotrichum lini on a PDA tube slant.

[0026] The seed culture is a primary seed culture, or a primary seed culture followed by a secondary seed culture.

[0027] The primary seed culture step includes: culturing the strain obtained from the slant culture in a liquid shake flask medium;

[0028] The secondary seed culture step includes: inoculating the strain obtained from the primary seed culture into a secondary seed shake flask or seed tank for culture;

[0029] The fermentation culture involves inoculating the bacterial strain obtained from the seed culture medium into the fermentation culture medium for further culture.

[0030] The conditions for the slant culture were: streak slant culture at 28℃ for 6-7 days;

[0031] The conditions for primary seed culture were: 28℃ for 40-48 hours, shaking speed of 200 rpm, inoculum size of 1%, and fungal spore concentration of 10-1. 8 pcs / ml;

[0032] The conditions for the secondary seed culture are: culture at 28℃ for 20-24 hours, rotation speed of 200-500 rpm, for example, 300-500 rpm; inoculum size of 5%-10%, bacterial concentration of 20-30 g / L based on wet bacterial weight, and aeration rate of 1.5 vvm.

[0033] The fermentation conditions are as follows: culture at 28℃ for 16-24 hours, dissolved oxygen linked to rotation speed, dissolved oxygen 30%, rotation speed 200-500 rpm, for example, 300-500 rpm or 200-350 rpm; inoculum size 5%-10%, bacterial concentration 20-30 g / L based on wet bacterial weight, and aeration rate 1.5 vvm.

[0034] In this invention, the PDA solid culture medium consists of 200 g / L potato, 20 g / L glucose, and 15-20 g / L agar, with a pH of 6-8.

[0035] The liquid culture medium used for seed culture and fermentation culture is the liquid culture medium as described above.

[0036] In this invention, the process after material conversion also includes a post-processing step;

[0037] The post-processing steps include (1) filtering the conversion liquid obtained from the feed conversion to obtain bacterial residue and fermentation supernatant; (2) extracting the bacterial residue, combining the extracts and concentrating until no liquid residue remains; (3) adding methyl isobutyl ketone, pulping and concentrating, filtering, repeating twice to obtain a solid, and collecting the filtrate; (4) adding the solid to pure water, pulping, filtering, and obtaining the target product; (5) combining the filtrate from (3), concentrating, cooling to 10°C to precipitate, filtering, and obtaining mother liquor; (6) adding the mother liquor to pure water, pulping, filtering, obtaining a solid, drying, and using it for refeeding; repeating steps (1)-(4) to obtain the target product after refeeding.

[0038] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0039] The reagents and raw materials used in this invention are all commercially available.

[0040] The positive and progressive effects of this invention are as follows: by simultaneously adding cyclodextrin and its derivatives and Tween, the feed amount can be increased from 15 g / L to 30 g / L. At the same time, this invention lowers the conversion temperature to 15-20°C after feeding and then raises the temperature to 28°C for conversion, which is more conducive to the conversion of substrate to product, and the relative conversion rate can reach 90%. Detailed Implementation

[0041] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0042] Example 1

[0043] (1) Slant culture: Take the glycerol tube containing Colletotrichumlini (commercially available product number CGMCC NO.3.4486) from the -80℃ freezer to thaw, dip the tube in the glycerol, and streak it onto the slant of the PDA tube. Incubate at 28℃ for 6-7 days. (PDA solid medium: potato 200g / L, glucose 20g / L, agar 15-20g / L, pH 6-8)

[0044] (2) Primary seed shake flask culture: Take the slant of a PDA test tube, add 20 ml of sterile water, scrape off the spores with an inoculation needle to prepare a spore suspension, transfer it to a 20 ml liquid shake flask culture medium and culture for 40-48 h at a temperature of 28℃ and a shaking speed of 200 rpm. The inoculation amount is 1%, and the fungal spore concentration is 10^6. 8Cells / ml. (Primary and secondary seed culture and fermentation medium: glucose 30-50g / L, corn steep liquor 10-30g / L, potassium dihydrogen phosphate 1-5g / L, dipotassium hydrogen phosphate 1-5g / L, magnesium sulfate 0.1-0.3g / L, sodium nitrate 1-5g / L, pH 5.5-5.8)

[0045] (3) Fermentation shake flask culture: Take the seed from the first-stage shake flask and inoculate it into a 200ml fermentation shake flask and culture for 20-24h at a temperature of 28℃ and 200rpm. The inoculation amount is 10%, and the bacterial concentration is 20-30g / L based on the wet bacterial weight.

[0046] (4) Feeding and conversion: Accurately weigh 0.6g DHEA, 0.6g β-CD, and 0.3g Tween 80 into a conical flask, then add them to the 20mL bacterial solution. Place the flask in a shaker at 200rpm for conversion. After feeding, lower the temperature to 16℃ and convert for 24h. Then raise the temperature to 28℃ and convert for 24-72h. The conversion temperature should be achieved within 0.5-1h. Before feeding, the wet weight of Colletotrichum lini should be 40-50g / L. The relative conversion rate of 7α,15α-dihydroxydehydroepiandrosterone is 90.541%. No post-treatment is required.

[0047] Repeating the above process steps (1) to (4), the relative conversion rate of 7α,15α-dihydroxydehydroepiandrosterone can reach 91.235% or 90.562%.

[0048] Example 2

[0049] (1) Slant culture: Take out the glycerol tube containing Colletotrichumlini with commercial product number CGMCC NO.3.4486 from the -80℃ freezer to thaw, dip the tube with an inoculation needle and streak it on the slant of the PDA tube, and incubate at 28℃ for 6-7 days.

[0050] (2) Primary seed shake flask culture: Take the slant of a PDA test tube, add 20 ml of sterile water, scrape off the spores with an inoculation needle to prepare a spore suspension, transfer it to a 60 ml liquid shake flask culture medium and culture for 40-48 h at a culture temperature of 28℃ and a shaker speed of 200 rpm. The inoculation amount is 1%, and the fungal spore concentration is 10^6. 8 per ml.

[0051] (3) Secondary seed shake flask culture: Take the seeds from the primary shake flask and inoculate them into a 600ml secondary seed shake flask and culture for 20-24h at a temperature of 28℃ and a rotation speed of 200rpm. The inoculation amount is 10%, and the bacterial concentration is 20-30g / L based on the wet bacterial weight.

[0052] (4) 20L fermenter culture: Take the second-grade seed shake flask, inoculate it into the 20L fermenter and culture for 20-24h. The culture temperature is 28℃, dissolved oxygen is linked to the rotation speed, dissolved oxygen is 30%, rotation speed is 300-500rpm, inoculation amount is 5%, bacterial concentration is 20-30g / L based on wet bacterial weight, and aeration rate is 1.5vvm.

[0053] (5) Feeding and conversion: Accurately weigh 360g of DHEA and 360g of β-CD into 1L of sterile water and put them into the fermenter. Then add 180ml of Tween 80. After feeding, lower the temperature to 16℃ and convert for 24h. Then raise the temperature to 28℃ for 24-72h. The conversion temperature should be achieved within 0.5-1h. Dissolved oxygen is linked to the rotation speed. Dissolved oxygen is 30% and the rotation speed is 300-500rpm. Before feeding, the wet weight of Colletotrichum lini should be 40-50g / L and the aeration rate should be 1.5vvm.

[0054] (6) Product extraction:

[0055] 6.1 After confirming the reaction endpoint, filter to obtain bacterial residue (1W) and fermentation supernatant (discarded);

[0056] 6.2 The bacterial residue was extracted three times with 10V acetone at 50℃, each time for 1 hour;

[0057] 6.3 Combine the extracts and concentrate until no liquid residue remains;

[0058] 6.4 Add 30V of methyl isobutyl ketone and beat at 70℃ for 1 hour. Concentrate until 3V of methyl isobutyl ketone remains. Filter. Repeat the solid test twice. Collect the filtrate.

[0059] 6.5 After refining three times, the solid is added to 10V pure water at 30℃ and pulped for 1 hour. The mixture is then filtered to obtain the finished product, and the filtrate is discarded.

[0060] 6.6 Combine the filtrates from 6.4, concentrate until no more liquid is effluent, cool to 10℃ for 1 hour to allow crystals to crystallize, filter to obtain the mother liquor, and discard the filtrate.

[0061] 6.7 Add 10V pure water to the mother liquor and beat at 30℃ for 1 hour. Filter to obtain solid. After drying, it can be used for recycling. Discard the filtrate.

[0062] 6.8 Return to base: Repeat steps (1)-(6);

[0063] 6.9 Refined purity: 98.530%, Refined purity of crude product after reprocessing: 98.674%, Average yield: 80.164%.

[0064] Example 3

[0065] (1) Slant culture: Take out the glycerol tube containing Colletotrichumlini with commercial product number CGMCC NO.3.4486 from the -80℃ freezer to thaw, dip the tube with an inoculation needle and streak it on the slant of the PDA tube, and incubate at 28℃ for 6-7 days.

[0066] (2) Primary seed shake flask culture: Take the slant of a PDA test tube, add 20 ml of sterile water, scrape off the spores with an inoculation needle to make a spore suspension, and transfer it to a 600 ml liquid shake flask culture medium for 40-48 h at a culture temperature of 28℃ and a shaker speed of 200 rpm. The inoculation amount is 1%, and the fungal spore concentration is 10^6. 8 per ml.

[0067] (3) 20L seed tank culture: Take a Grade 1 seed shake flask, inoculate it into a 20L seed tank and culture for 20-24h. The culture temperature is 28℃, dissolved oxygen is linked to the rotation speed, dissolved oxygen is 30%, rotation speed is 300-500rpm, inoculation amount is 5%, bacterial concentration is 20-30g / L based on wet bacterial weight, and aeration rate is 1.5vvm.

[0068] (4) 200L fermenter culture: Before transplanting, if the bacteria in the 20L seed tank are free of contaminants and the bacteria are growing well, they can be transplanted to the 200L fermenter. The culture temperature is 28℃, dissolved oxygen is linked to the rotation speed, dissolved oxygen is 30%, rotation speed is 200-350rpm, inoculum size is 10%, bacterial concentration is 20-30g / L based on wet bacterial weight, aeration rate is 1.5vvm, and culture time is 16-22h.

[0069] (5) Feeding and conversion: Accurately weigh 3.6 kg DHEA and 3.6 kg β-CD into 10 L of sterile water and put them into the fermenter. Then add 1.8 L of Tween 80. After feeding, lower the temperature to 16℃ and convert for 24 h. Then raise the temperature to 28℃ for 24-72 h. The conversion temperature should be achieved within 0.5-1 h. Dissolved oxygen and rotation speed are linked. Dissolved oxygen is 30% and rotation speed is 200-350 rpm. Before feeding, the wet weight of Colletotrichumlini should be 40-50 g / L and the aeration rate is 1.5 vvm.

[0070] (6) Product extraction:

[0071] 6.1 After confirming the reaction endpoint, filter to obtain bacterial residue (1W) and fermentation supernatant (discarded);

[0072] 6.2 The bacterial residue was extracted three times with 10V acetone at 50℃, each time for 1 hour;

[0073] 6.3 Combine the extracts and concentrate until no liquid residue remains;

[0074] 6.4 Add 30V of methyl isobutyl ketone and beat at 70℃ for 1 hour. Concentrate until 3V of methyl isobutyl ketone remains. Filter. Repeat the solid test twice. Collect the filtrate.

[0075] 6.5 After the third refining, the solid is added to 10V pure water at 30℃ and pulped for 1 hour. After filtration, the finished product is obtained, and the filtrate is discarded.

[0076] 6.6 Combine the filtrates from 6.4, concentrate until no more liquid is effluent, cool to 10℃ for 1 hour to allow crystals to crystallize, filter to obtain the mother liquor, and discard the filtrate.

[0077] 6.7 Add 10V pure water to the mother liquor and beat at 30℃ for 1 hour. Filter to obtain solid. After drying, it can be used for recycling. Discard the filtrate.

[0078] 6.8 Return to base: Repeat steps (1)-(6);

[0079] 6.9 Refined purity: 98.665%, Refined purity of crude product after reprocessing: 98.545%, Average yield: 81.254%.

[0080] Comparative Example 1

[0081] This comparative example replicates the technical solution of Example 1, with the only change being that the temperature after feeding is maintained at 28°C throughout the conversion process, and the relative conversion rate of 7α,15α-dihydroxydehydroepiandrosterone is 82.507%, without any post-treatment.

[0082] Comparative Example 2

[0083] This comparative example replicates the technical solution of Example 1, with the only modification being that the temperature was lowered to 16°C throughout the conversion process after feeding, and the relative conversion rate of 7α,15α-dihydroxydehydroepiandrosterone was 75.403%, without post-treatment.

[0084] Comparative Example 3

[0085] This comparative example replicates the technical solution of Example 1, with the only modification being that β-CD,7α,15α-dihydroxydehydroepiandrosterone was not added during the conversion process, resulting in a relative conversion rate of 69.343%, and no post-treatment was performed.

[0086] Comparative Example 4

[0087] This comparative example replicates the technical solution of Example 1, with the only modification being that Tween 80,7α,15α-dihydroxydehydroepiandrosterone was not added during the feeding and conversion process, resulting in a relative conversion rate of 60.693%, and no post-processing was performed.

[0088] Comparative Example 5

[0089] This comparative example replicates the technical solution of Example 1, with the only modification being that no β-CD or Tween 80, 7α,15α-dihydroxydehydroepiandrosterone was added during the feed conversion, resulting in a relative conversion rate of 38.403%, and no post-treatment was performed.

Claims

1. A method for preparing 7α,15α-dihydroxydehydroepiandrosterone, characterized in that, The steps include: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] Colletotrichum lini In a liquid culture medium system, dehydroepiandrosterone (DHEA) was converted to 7α,15α-dihydroxydehydroepiandrosterone; wherein, the *Colletotrichum flaxensis*... Colletotrichum lini The liquid culture medium system also contains cyclodextrin and its derivatives, as well as Tween; The feeding and conversion steps include: first performing low-temperature conversion, and then performing medium-temperature conversion; the conditions for low-temperature conversion are: conversion temperature 15~16℃; the conditions for medium-temperature conversion are: conversion temperature 28℃; wherein, the conversion time for low-temperature conversion is 20h-48h; and the conversion time for medium-temperature conversion is 24-72h. The cyclodextrin and its derivatives are β-cyclodextrin; the Tween is Tween 80. The mass fraction of dehydroepiandrosterone (DHEA) is 2-3%, the mass fraction of cyclodextrin and its derivatives is 3-5%, the volume fraction of Tween is 1-2%, and the DHEA is added before feeding with *Colletotrichum flaxensis*. Colletotrichum lini The wet bacterial count is 40-50 g / L.

2. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The conditions for the low-temperature conversion are: a conversion temperature of 16°C.

3. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The conversion time for the low-temperature conversion is 24 hours.

4. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The temperature conditions for the low-temperature conversion and the medium-temperature conversion are that the target temperature can be reached within 0.5-1 hour.

5. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The dehydroepiandrosterone (DHEA) has a mass fraction of 3%, the cyclodextrin and its derivatives are β-cyclodextrin with a mass fraction of 3%, and the Tween is Tween 80 with a volume fraction of 1.5%.

6. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The dissolved oxygen level during the feeding and conversion process is controlled at 30±2%, the rotation speed during the feeding and conversion process is 200-500 rpm, and the aeration rate during the feeding and conversion process is 1.5-2 vvm.

7. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The rotation speed of the feeding and conversion is 300-500 rpm; the aeration rate of the feeding and conversion is 1.5 vvm.

8. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The rotation speed for feeding and conversion is 200-350 rpm.

9. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The linthellae Colletotrichum lini It is a commercially available strain, CGMCC NO.3.4486.

10. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The liquid culture medium comprises: glucose 30-50 g / L, corn steep liquor 10-30 g / L, potassium dihydrogen phosphate 1-5 g / L, dipotassium hydrogen phosphate 1-5 g / L, magnesium sulfate 0.1-0.3 g / L, sodium nitrate 1-5 g / L, pH 5.5-5.

8.

11. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The one containing lindendra Colletotrichum lini The liquid culture medium is prepared by the following process: *Colletotrichum flaxensis* Colletotrichum lini The strain was obtained by sequentially performing slant culture, seed culture, and fermentation culture.

12. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 11, characterized in that, The slant culture includes the culture of *Colletotrichum flaxensis*. Colletotrichum lini Commercially available bacterial strains were cultured on PDA test tube slant agar. The seed culture is a primary seed culture, or a primary seed culture and a secondary seed culture are performed sequentially; wherein, the primary seed culture step includes: culturing the strain obtained from the slant culture in a liquid shake flask medium; wherein, the secondary seed culture step includes: inoculating the strain obtained from the primary seed culture into a secondary seed shake flask or seed tank for culture; the fermentation culture involves inoculating the strain obtained from the seed culture medium into a fermentation medium for culture.

13. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 12, characterized in that, The conditions for the slant culture were streak culture at 28℃ for 6-7 days; the conditions for the primary seed culture were culture at 28℃ for 40-48 hours, shaking speed of 200 rpm, inoculum size of 1%, and fungal spore concentration of 10^6. 8 The conditions for the secondary seed culture are: culture at 28℃ for 20-24 hours, rotation speed of 200-500 rpm, inoculum size of 5%-10%, bacterial concentration of 20-30 g / L (wet bacterial weight), and aeration rate of 1.5 vvm; the conditions for the fermentation culture are: culture at 28℃ for 16-24 hours, dissolved oxygen-linked rotation speed of 30%, rotation speed of 200-500 rpm, inoculum size of 5%-10%, bacterial concentration of 20-30 g / L (wet bacterial weight), and aeration rate of 1.5 vvm; the liquid culture medium for both the seed culture and fermentation culture is the liquid culture medium as described in claim 10.

14. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 12, characterized in that, The conditions for the secondary seed culture are a rotation speed of 300-500 rpm; The fermentation conditions are as follows: 28℃ for 20-24h, dissolved oxygen linked to rotation speed, dissolved oxygen 30%, rotation speed 300-500rpm; PDA solid medium consists of 200 g / L potato, 20 g / L glucose, and 15-20 g / L agar, with a pH of 6-8.

15. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 12, characterized in that, The fermentation conditions are as follows: culture at 28℃ for 16-22 hours, dissolved oxygen linked to rotation speed, dissolved oxygen 30%, and rotation speed 200-350 rpm.

16. The method for preparing 7α,15α-dihydroxydehydroepiandrosterone as described in claim 1, characterized in that, The feeding conversion process also includes a post-processing step; the post-processing step includes (1) filtering the conversion liquid obtained from the feeding conversion to obtain bacterial residue and fermentation supernatant; (2) extracting the bacterial residue, combining the extract and concentrating until there is no liquid residue; (3) adding methyl isobutyl ketone, pulping and concentrating, filtering, repeating twice to obtain a solid, and collecting the filtrate; (4) adding the solid to pure water, pulping, filtering, and obtaining the target product; (5) combining the filtrate in (3), concentrating, cooling to 10°C to precipitate, filtering, and obtaining the mother liquor; (6) adding the mother liquor to pure water, pulping, filtering, obtaining a solid, drying for refeeding; repeating steps (1)-(4) to obtain the target product after refeeding.