Fermentation medium for converting 17alpha-hydroxyprogesterone into 11alpha, 17alpha-dihydroxyprogesterone and application of fermentation medium

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation medium for converting 17alpha-hydroxyprogesterone into 11alpha, 17alpha-dihydroxyprogesterone and application of the fermentation medium. More specifically, the invention prepares a fermentation culture medium especially suitable for fermenting and converting 17 alpha-hydroxyprogesterone by aspergillus ochraceus, and by improving the components of the culture medium, the conversion rate of producing 11 alpha, 17 alpha-dihydroxyprogesterone by converting 17 alpha-hydroxyprogesterone by aspergillus ochraceus is greatly improved, the fermentation conversion time is also greatly shortened, and the yield of 11 alpha, 17 alpha-dihydroxyprogesterone is improved. The conversion rate is improved, and the cost is reduced for industrial and efficient production of 11alpha, 17alpha-dihydroxyprogesterone.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a fermentation medium for converting 17α-hydroxyprogesterone into 11α,17α-dihydroxyprogesterone and application thereof. Background Art

[0002] Prednisone acetate is an adrenal cortex hormone drug that plays an important role in human health. It can treat diseases such as lupus erythematosus, rheumatoid arthritis, rheumatism, and bronchial asthma. 11α,17α-dihydroxyprogesterone is an important intermediate for the synthesis of prednisone acetate. The chemical synthesis of 11α,17α-dihydroxyprogesterone is cumbersome and requires a large amount of strong acid, strong base and toxic reagents. It is difficult to ensure safety and causes serious environmental pollution. The production of 11α,17α-dihydroxyprogesterone by biotransformation has a high safety factor, simple steps, and is environmentally friendly. It is a better method for synthesizing 11α,17α-dihydroxyprogesterone.

[0003] At present, the industrial bioconversion of 17α-hydroxyprogesterone takes a long time and the feed amount is low. In order to save costs and improve efficiency, a better method for converting 17α-hydroxyprogesterone to produce 11α,17α-dihydroxyprogesterone is needed to reduce costs and improve yields for the industrial production of 11α,17α-dihydroxyprogesterone. Summary of the invention

[0004] In order to solve at least one of the above problems, the present invention provides a fermentation medium in a first aspect, wherein the components of the fermentation medium include a basal medium and an additive.

[0005] in,

[0006] The additive includes at least one of sucrose, starch, sulfite and betaine,

[0007] The basic culture medium comprises corn steep liquor, glucose, peptone and ammonium sulfate.

[0008] According to a specific embodiment of the present invention, glucose is consumed quickly and a large amount of metabolic by-products are generated. The present invention adds two carbon sources, sucrose and starch, to the fermentation medium formula, which can reduce glucose consumption, eliminate the need to add glucose during the fermentation conversion process, reduce carbon source costs, maintain pH stability before and during fermentation, reduce the viscosity and surface activity of the fermentation liquid, and increase the fluidity and dissolved oxygen of the fermentation liquid.

[0009] The invention adds sulfate to the fermentation medium formula, which can enhance the P450 enzyme activity in the fermentation broth, promote the protein synthesis rate, and effectively increase the mycelium density in the fermentation broth and the conversion rate and utilization rate of 17α-hydroxyprogesterone.

[0010] Adding betaine to the fermentation medium formula of the present invention can improve the solubility of 17α-hydroxyprogesterone in the fermentation liquid, thereby further improving the conversion rate and utilization rate of 17α-hydroxyprogesterone.

[0011] The existing fermentation medium not only has a long fermentation time, but also has a low conversion rate for the substrate 17α-hydroxyprogesterone and a low feed amount. However, when 17α-hydroxyprogesterone and Aspergillus ochraceus are fermented and cultured together in the fermentation medium prepared by the present invention, more than 95% of the 17α-hydroxyprogesterone can be converted within 48 hours, which not only greatly improves the conversion rate of Aspergillus ochraceus in converting 17α-hydroxyprogesterone to produce 11α,17α-dihydroxyprogesterone, but also greatly shortens the fermentation conversion time and improves the conversion rate.

[0012] According to a specific embodiment of the present invention, the working concentration of sucrose in the fermentation medium is (5-15) g / L, preferably (8-12) g / L.

[0013] According to a specific embodiment of the present invention, the starch is soluble starch.

[0014] According to a specific embodiment of the present invention, the working concentration of the starch in the fermentation medium is (10-20) g / L, preferably (12-18) g / L.

[0015] The inventors found that adding sucrose and starch to the fermentation medium formula of the present invention can make the viscosity lower than that of glucose, enhance the fluidity of the fermentation liquid, and increase the dissolved oxygen in the fermentation liquid.

[0016] According to a specific embodiment of the present invention, the sulfite is ferrous sulfite.

[0017] According to a specific embodiment of the present invention, the working concentration of the sulfite in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

[0018] The inventors found that adding sulfate to the fermentation medium formula of the present invention can enhance the P450 enzyme activity in the fermentation broth, promote the protein synthesis rate, and effectively increase the mycelium density in the fermentation broth and the conversion rate and utilization rate of 17α-hydroxyprogesterone.

[0019] According to a specific embodiment of the present invention, the working concentration of betaine in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

[0020] According to a specific embodiment of the present invention, the working concentration of the corn steep liquor in the fermentation medium is (5-15) g / L, preferably (8-12) g / L.

[0021] According to a specific embodiment of the present invention, the glucose includes at least one of glucose and anhydrous glucose.

[0022] According to a specific embodiment of the present invention, the working concentration of glucose in the fermentation medium is (5-15) g / L, preferably (8-12) g / L.

[0023] According to a specific embodiment of the present invention, the working concentration of the peptone in the fermentation medium is (10-20) g / L, preferably (12-18) g / L.

[0024] According to a specific embodiment of the present invention, the working concentration of ammonium sulfate in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

[0025] According to a specific embodiment of the present invention, the basic culture medium further includes a surfactant and a defoaming agent.

[0026] According to a specific embodiment of the present invention, the surfactant includes at least one of Tween-80, Tween-60, Span-80 and Span-60.

[0027] According to a specific embodiment of the present invention, the working concentration of the surfactant in the fermentation medium is (1-4) g / L, preferably (1-2) g / L.

[0028] According to a specific embodiment of the present invention, the working concentration of the defoaming agent in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

[0029] The second aspect of the present invention provides use of the fermentation medium described in the first aspect in the production of 11α,17α-dihydroxyprogesterone.

[0030] The third aspect of the present invention provides a method for preparing 11α,17α-dihydroxyprogesterone, comprising:

[0031] S1, inoculating Aspergillus ochraceus into a strain culture medium for cultivation, to obtain a culture medium containing spores of Aspergillus ochraceus;

[0032] S2, washing the culture medium containing the spores of Aspergillus ochraceus with a solvent to obtain a spore suspension;

[0033] S3, inoculating the spore suspension into a seed culture medium for cultivation to obtain a seed culture solution;

[0034] S4. Inoculate the seed culture solution into the fermentation medium described in the first aspect, add 17α-hydroxyprogesterone for fermentation culture, and obtain 11α,17α-dihydroxyprogesterone.

[0035] According to a specific embodiment of the present invention, in step S1, the strain culture medium is a slant culture medium.

[0036] According to a specific embodiment of the present invention, the strain culture medium includes at least one of PDA culture medium, homemade potato culture medium, and red Bengal culture medium.

[0037] According to a specific embodiment of the present invention, in step S2, the solvent includes at least one of physiological saline and sterile water.

[0038] According to a specific embodiment of the present invention, in step S3, the components of the seed culture medium include glucose, corn steep liquor, peptone, diammonium hydrogen phosphate, and yeast extract powder.

[0039] According to a specific embodiment of the present invention, the working concentration of the corn steep liquor in the seed culture medium is (2-15) g / L, preferably (6-12) g / L.

[0040] According to a specific embodiment of the present invention, the glucose includes at least one of glucose and anhydrous glucose.

[0041] According to a specific embodiment of the present invention, the working concentration of glucose in the seed culture medium is (15-30) g / L, preferably (18-25) g / L.

[0042] According to a specific embodiment of the present invention, the working concentration of the peptone in the seed culture medium is (10-20) g / L, preferably (12-18) g / L.

[0043] According to a specific embodiment of the present invention, the working concentration of diammonium phosphate in the seed culture medium is (0.01-0.1) g / L, preferably (0.02-0.08) g / L.

[0044] According to a specific embodiment of the present invention, the working concentration of the yeast powder in the seed culture medium is (5-15) g / L, preferably (8-12) g / L.

[0045] According to a specific embodiment of the present invention, in step S4, the inoculation amount of the seed culture solution accounts for 5% of the volume of the fermentation medium.

[0046] According to a specific embodiment of the present invention, the amount of 17α-hydroxyprogesterone added is 30-60 g / L, which increases the amount of substrate converted in a single fermentation.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The existing fermentation medium not only has a long fermentation time, but also has a low conversion rate for the substrate 17α-hydroxyprogesterone. However, when 17α-hydroxyprogesterone and Aspergillus ochraceus are fermented and cultured together in the fermentation medium prepared by the present invention, more than 95% of the 17α-hydroxyprogesterone can be converted in 48 hours, which not only greatly improves the conversion rate of Aspergillus ochraceus in converting 17α-hydroxyprogesterone to produce 11α,17α-dihydroxyprogesterone, but also greatly shortens the fermentation conversion time, improves the conversion rate, and can achieve cost reduction and efficiency improvement in the industrial production of 11α,17α-dihydroxyprogesterone. DETAILED DESCRIPTION

[0049] Embodiments of the present invention are described in detail below. The embodiments are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0050] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0051] In this document, the terms “contain”, “include” or “comprise” are open expressions, that is, they include the contents specified in the present invention but do not exclude other contents.

[0052] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0054] The purchase sources and product numbers of the reagents used in this application are shown in Table 1.

[0055] Table 1

[0056]

[0057]

[0058] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0059] Example 1

[0060] S1, spore production and production slant preparation: Aspergillus ochraceus (CICC 41473) was used as the production strain, and the bacteria were placed on the PDA slant medium and cultured at 30°C for 5 days to generate yellow spores. The spore production medium was stored in a 4°C refrigerator for no more than 15 days.

[0061] S2, preparation of spore suspension: add 50 mL of physiological saline to the production slant, wash the spores to prepare a spore suspension.

[0062] S3, seed culture: inoculate 10 mL of spore suspension into the seed culture medium, and culture for 1 day in a shaker at 30°C and 220 rpm to obtain the seed culture solution. The composition of the seed culture medium is as follows: 22 g / L glucose, 8 g / L corn syrup powder, 10 g / L peptone, 0.05 g / L diammonium phosphate, and 10 g / L yeast extract powder. Adjust the pH to 6.0 and sterilize at 121°C for 25 to 30 minutes.

[0063] S4, fermentation culture: take the seed culture solution described in S3 and inoculate it into a 15L (liquid capacity 10L) fermentation tank at an inoculation rate of 5% (v / v), culture it at 28°C for 16-18h, add 17α-hydroxyprogesterone substrate to continue fermentation, and the feeding amount of 17α-hydroxyprogesterone substrate is 50g / L; the fermentation medium composition is 10g / L corn steep liquor powder, 10g / L glucose, 10g / L sucrose, 15g / L soluble starch, 15g / L peptone, 1g / L ammonium sulfate, 1g / L ferrous sulfate, 1g / L betaine, 2g / L Tween-80, and 1g / L defoaming agent.

[0064] S5, substrate conversion: fermentation until the conversion rate of the 17α-hydroxyprogesterone substrate reaches an inflection point. When the fermentation liquid level is lower than 10L, water is added to stabilize the volume of the fermentation liquid at 1L.

[0065] Example 2

[0066] S1, spore production and production slant preparation: Aspergillus ochraceus (CICC 41473) was used as the production strain, and the bacteria were placed on the PDA slant medium and cultured at 30°C for 5 days to generate yellow spores. The spore production medium was stored in a 4°C refrigerator for no more than 15 days.

[0067] S2, preparation of spore suspension: add 50 mL of physiological saline to the production slant, wash the spores to prepare a spore suspension.

[0068] S3, seed culture: inoculate 10 mL of spore suspension into the seed culture medium, and culture in a shaker at 30°C and 220 rpm for 1 day to obtain the seed culture solution.

[0069] S4, fermentation culture: take the seed culture solution described in S3 and inoculate it into a 50L (liquid capacity 30L) fermentation tank at an inoculation rate of 5% (v / v), culture it at 28°C for 18h, add 17α-hydroxyprogesterone substrate to continue fermentation, and the feeding amount of 17α-hydroxyprogesterone substrate is 50g / L; the fermentation medium composition is 10g / L corn steep liquor powder, 10g / L glucose, 10g / L sucrose, 15g / L soluble starch, 15g / L peptone, 1g / L ammonium sulfate, 1g / L ferrous sulfate, 1g / L betaine, 2g / L Tween-80, and 1g / L defoaming agent.

[0070] S5, substrate conversion: fermentation until the conversion rate of the 17α-hydroxyprogesterone substrate reaches an inflection point. When the fermentation liquid level is lower than 30 L, water is added to stabilize the volume of the fermentation liquid at 30 L.

[0071] Comparative Example 1

[0072] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10 g / L corn steep liquor powder, 35 g / L glucose, 15 g / L peptone, 1 g / L ammonium sulfate, 1 g / L ferrous sulfate, 2 g / L Tween-80, 1 g / L betaine, and 1 g / L defoaming agent.

[0073] Comparative Example 2

[0074] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10 g / L corn steep liquor powder, 25 g / L glucose, 10 g / L sucrose, 15 g / L peptone, 1 g / L ammonium sulfate, 1 g / L ferrous sulfate, 2 g / L Tween-80, 1 g / L betaine, and 1 g / L defoaming agent.

[0075] Comparative Example 3

[0076] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10 g / L corn steep liquor powder, 20 g / L glucose, 15 g / L soluble starch, 15 g / L peptone, 1 g / L ammonium sulfate, 1 g / L ferrous sulfate, 2 g / L Tween-80, 1 g / L betaine, and 1 g / L defoaming agent.

[0077] Comparative Example 4

[0078] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10g / L corn steep liquor powder, 10g / L glucose, 10g / L sucrose, 15g / L soluble starch, 15g / L peptone, 1g / L ferrous sulfate, 2g / L Tween-80, 1g / L betaine, and 1g / L defoaming agent.

[0079] Comparative Example 5

[0080] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10 g / L corn syrup powder, 10 g / L glucose, 10 g / L sucrose, 15 g / L soluble starch, 15 g / L peptone, 1 g / L ammonium sulfate, 2 g / L Tween-80, 1 g / L betaine, and 1 g / L defoaming agent.

[0081] Comparative Example 6

[0082] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 10g / L corn steep liquor powder, 10g / L glucose, 10g / L sucrose, 15g / L soluble starch, 15g / L peptone, 1g / L ammonium sulfate, 1g / L ferrous sulfate, 2g / L Tween-80, and 1g / L defoaming agent.

[0083] Comparative Example 7

[0084] Compared with Example 1, the difference is that in step S4, the fermentation medium components are: 20 g / L glucose, 10 g / L corn steep liquor powder, 10 g / L peptone, 2 g / L ammonium sulfate, 2 g / L Tween-80, and 1 g / L defoaming agent.

[0085] The high performance liquid chromatography detection method is as follows: the fermentation broth is taken once every 12 hours of conversion and detected by high performance liquid chromatography, the chromatographic column is MicroPulite Gold C18 (150mm×4.6mm); the mobile phase is water: acetonitrile = 64:36; the flow rate is 1.0mL / min; the detection wavelength is 254nm; the column temperature is 35℃; the injection volume is 5μL; the area normalization method is used for detection. The conversion rate of 17α-hydroxyprogesterone in the above examples and comparative examples is detected, and the results are shown in Table 2.

[0086] Table 217α-Hydroxyprogesterone conversion rate

[0087] Fermentation time 36h 48h 60h 72h Example 1 82.95％ 95.17％ 93.61％ - Example 2 85.20％ 95.93％ 92.10％ - Comparative Example 1 60.86％ 67.32％ 59.37％ - Comparative Example 2 72.77％ 87.13％ 84.38％ - Comparative Example 3 81.53％ 92.37％ 90.15％ - Comparative Example 4 78.67％ 93.15％ 91.94％ - Comparative Example 5 81.04％ 92.95％ 87.38％ - Comparative Example 6 81.84％ 93.55％ 91.47％ - Comparative Example 7 56.51％ 69.36％ 73.11％ 66.40％

[0088] It can be seen from the above Examples 1, 2 and Comparative Examples 1-7 that sucrose, soluble starch, ferrous sulfate and ammonium sulfate are added to the fermentation medium formula to reduce the supplementation of glucose to reduce the carbon source cost, maintain pH stability before and during fermentation, reduce the viscosity and surface activity of the fermentation liquid, increase the fluidity and dissolved oxygen of the fermentation liquid, enhance the P450 enzyme activity in the fermentation liquid, promote the protein synthesis rate, effectively increase the mycelium density in the fermentation liquid and the conversion rate and utilization rate of 17α-hydroxyprogesterone, shorten the fermentation conversion time, and increase the conversion rate, substrate concentration and substrate conversion rate, which is more beneficial to industrial production.

[0089] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fermentation medium, characterized in that The components of the fermentation medium include a basic medium and additives. in, The additive includes at least one of sucrose, starch, sulfite and betaine, The basic culture medium comprises corn steep liquor, glucose, peptone and ammonium sulfate.

2. The fermentation medium according to claim 1, characterized in that The working concentration of sucrose in the fermentation medium is (5-15) g / L, preferably (8-12) g / L; The starch is soluble starch; The working concentration of the starch in the fermentation medium is (10-20) g / L, preferably (12-18) g / L; The sulfite is ferrous sulfite; The working concentration of the sulfite in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L; The working concentration of the betaine in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

3. The fermentation medium according to claim 1, characterized in that The working concentration of the corn steep liquor in the fermentation medium is (5-15) g / L, preferably (8-12) g / L; The glucose includes at least one of glucose and anhydrous glucose; The working concentration of glucose in the fermentation medium is (5-15) g / L, preferably (8-12) g / L; The working concentration of the peptone in the fermentation medium is (10-20) g / L, preferably (12-18) g / L; The working concentration of the ammonium sulfate in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

4. The fermentation medium according to claim 1, characterized in that The basic culture medium further includes a surfactant and a defoaming agent; The surfactant includes at least one of Tween-80, Tween-60, Span-80 and Span-60; The working concentration of the surfactant in the fermentation medium is (1-4) g / L, preferably (1-2) g / L; The working concentration of the defoaming agent in the fermentation medium is (0.1-2) g / L, preferably (0.5-1.5) g / L.

5. Use of the fermentation medium according to any one of claims 1 to 4 in the production of 11α,17α-dihydroxyprogesterone.

6. A method for preparing 11α, 17α-dihydroxyprogesterone, characterized in that: include: S1. inoculating Aspergillus ochraceus into a strain culture medium for cultivation to obtain a culture medium containing spores of Aspergillus ochraceus; S2, washing the culture medium containing the spores of Aspergillus ochraceus with a solvent to obtain a spore suspension; S3, inoculating the spore suspension into a seed culture medium for cultivation to obtain a seed culture solution; S4. Inoculate the seed culture solution into the fermentation medium according to any one of claims 1 to 4, add 17α-hydroxyprogesterone for fermentation culture, and obtain 11α,17α-dihydroxyprogesterone.

7. The method according to claim 6, characterized in that In step S1, the strain culture medium is a slant culture medium; The strain culture medium comprises at least one of a PDA culture medium, a homemade potato culture medium and a red Bengal culture medium.

8. The method according to claim 6, characterized in that In step S2, the solvent includes at least one of physiological saline and sterile water.

9. The method according to claim 6, characterized in that In step S3, the components of the seed culture medium include glucose, corn steep liquor, peptone, diammonium phosphate, and yeast extract powder; The working concentration of the corn steep liquor in the seed culture medium is (2-15) g / L, preferably (6-12) g / L; The glucose includes at least one of glucose and anhydrous glucose; The working concentration of glucose in the seed culture medium is (15-30) g / L, preferably (18-25) g / L; The working concentration of the peptone in the seed culture medium is (10-20) g / L, preferably (12-18) g / L; The working concentration of diammonium phosphate in the seed culture medium is (0.01-0.1) g / L, preferably (0.02-0.08) g / L; The working concentration of the yeast powder in the seed culture medium is (5-15) g / L, preferably (8-12) g / L.

10. The method according to claim 6, characterized in that In step S4, the inoculation amount of the seed culture solution accounts for 5% of the volume percentage of the fermentation medium; The added amount of the 17α-hydroxyprogesterone is 30-80 g / L.

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