Method for preparing steroid intermediate by microbial fermentation of sterol

Abstract

The invention belongs to the technical field of steroid compound preparation, and particularly relates to a method for preparing a steroid intermediate by microbial fermentation of sterol. According to the method for preparing the steroid intermediate through microbial fermentation of sterol, sterol serves as a carbon source, mycobacteria serves as a fermentation strain, polyglycerol fatty acid ester is added in the initial stage of fermentation to serve as a fermentation aid, hydroxytyrosol or sodium dehydroacetate is added in the middle stage of fermentation to serve as a fermentation aid, and AD, ADD, 1, 3-butanediol and 1, 3-butanediol are prepared through fermentation culture. The invention relates to four steroid intermediates, namely 4-HBC and 4-HBC. Compared with conventional auxiliary-free fermentation culture, the preparation method has the advantages that the yield of a steroid intermediate can be remarkably increased, and the yield of the intermediate can reach 160 mg / L or above. Therefore, a novel preparation strategy can be provided for synthesis of the steroid intermediates, more raw materials can be provided for synthesis of hormone drugs, and meanwhile, the preparation method has important significance in promoting development and application of the steroid intermediates.

Description

Technical Field

[0001] The invention belongs to the technical field of steroid compound preparation, and in particular relates to a method for preparing steroid intermediates by fermenting sterols through microorganisms. Background Art

[0002] Steroid compounds are widely found in plants, animals, yeast and fungi. They are a class of terpene lipids containing a special structure of sterane nucleus and are also important raw materials for the preparation of steroid hormone drugs. Although steroid compounds are widely found in nature, their production is still obviously insufficient to meet the huge medical market demand.

[0003] Currently, steroidal compounds are mainly derived from various steroidal intermediates. Steroidal intermediates mainly include 4-androstene-3,17-dione (AD), 1,4-androstadiene-3,17-dione (ADD), 9-hydroxy-4-androstene-3,17-dione (9-OH-AD), 22-hydroxy-23,24-dicholesterol-4-ene-3-one (4-HBC), 22-hydroxy-23,24-dicholesterol-1,4-diene-3-one (1,4-HBC) and 9,22-dihydroxy-23,24-dicholesterol-4-ene-3-one (9-OH-4-HBC). Steroidal intermediates can be further produced into steroid hormone drugs for clinical use by synthetic means.

[0004] At present, the methods for preparing steroid intermediates mainly include chemical methods and microbial fermentation methods. As a traditional synthesis method, the chemical method has a relatively mature process flow, and the synthesis route is "Dioscoreaceae plants → diosgenin → diene intermediates → steroid drugs", but this method relies on wild Chinese medicinal materials such as "Dioscorea chinensis". As diosgenin resources are gradually depleted, the cost of chemical synthesis is gradually increasing. In addition, the chemical synthesis process is complicated. The conversion process from Dioscorea chinensis to androstenedione contains 8 main links, which is complex and energy-intensive. It will also produce a large amount of acid hydrolyzate, which is unfavorable to the environment and cannot meet the needs of the sustainable development of the steroid intermediate industry.

[0005] Microbial transformation can avoid many shortcomings of chemical methods. It has the characteristics of mild reaction conditions, short cycle, high efficiency, and environmental friendliness. It is a very promising and potential method for preparing steroid intermediates. Among them, plant sterols are natural substrates for preparing steroid drug intermediates. Studies have shown that mycobacteria and other microorganisms can convert sterols into various steroid intermediates such as ADD and AD. At present, although significant progress has been made in the use of microorganisms to transform sterols to produce steroid intermediates, there are still problems such as long fermentation cycle, low sterol conversion rate, and low intermediate yield.

[0006] In the patent application with publication number CN105861612A, attapulgite is added to promote the solubility of sterols in the aqueous phase, thereby preparing steroid intermediate AD, the fermentation cycle is 120-168h, and the final AD yield is 52.81±0.88%. In the patent application with publication number CN112301083A, plant sterols, dichloromethane and tetramethylpiperidinium nitrogen oxide mixture are used as raw material systems, and the AD yield reaches 42.3%. In the patent application with publication number CN103343155A, Mycobacteriumfortuitum ATCC35855 is used as the transformation strain, and the sterol conversion time is shortened to 72-120h, but the 9-OH-AD yield is only about 55%. Xue Kai et al. (Xue Kai, Wang Rong, Wang Youfu, et al. Optimization of plant sterol conversion to prepare 9-hydroxy-androstenedione by response surface methodology [J]. Food Industry, 2017(9):5) induced mutations in mycobacteria and, through preliminary fermentation tank amplification and conversion, the accumulation of 9-OH-AD reached 50.41 mg / L.

[0007] In summary, the current process of microbial conversion of sterols to produce steroidal intermediates is relatively mature, but the low yield of the target product is still a thorny problem. Therefore, how to increase the yield of sterols converted into steroidal intermediates has become a problem that technicians in this field need to solve urgently. Summary of the invention

[0008] To overcome the deficiencies of the prior art, the present invention aims to provide a method for preparing steroid intermediates by microbial fermentation of sterols, which has a simple process and can effectively increase the yield of steroid intermediates prepared by microbial fermentation of sterols.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for preparing a steroid intermediate by fermenting sterols with microorganisms comprises the following steps:

[0011] Activating mycobacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for a fermentation culture; the fermentation medium includes the following components at the following concentrations: 0.5-2 g / L sterol, 0.8-1.2 g / L polyglycerol fatty acid ester;

[0012] Adding hydroxytyrosol or sodium dehydroacetate to the system after the first fermentation culture to carry out a second fermentation culture; the amount of hydroxytyrosol or sodium dehydroacetate added to the system is 100-200 mg / L;

[0013] After the secondary fermentation culture is completed, the fermentation broth is separated and purified to obtain a steroid intermediate.

[0014] As a preferred embodiment, the mycobacterium is Mycobacterium sp. 191574.

[0015] As a preferred embodiment, the activation is to inoculate the mycobacterium into a nutrient broth medium for activation culture.

[0016] As a further preferred solution, the inoculation amount of mycobacteria in the nutrient broth medium is 0.08% to 0.15% v / v; the activation culture is to culture the inoculated mycobacteria in a shaker at 28 to 32°C and a rotation speed of 150 to 200 r / min until OD 600nm OD is 0.8~1.0. 600nm It is the absorbance value at a wavelength of 600nm. Through activation culture, the biological activity of mycobacteria is improved and the strains are enriched.

[0017] As a preferred solution, the fermentation medium further includes the following components at the following concentrations: 0.8-3 g / L citric acid, 0.5-2 g / L MgSO4·7H2O, 0.5-2 g / L K2HPO4, 3-5 g / L (NH4)2HPO4, and the solvent is water. By adding the above components, the pH value and stability of the fermentation medium are maintained.

[0018] As a preferred solution, the sterol is a combination of one or more of stigmasterol, campesterol, and β-sitosterol; the polyglycerol fatty acid ester is one or more of triglycerol dioleate, hexaglycerol monooleate, and decaglycerol monooleate. As a further preferred solution, the sterol is a mixed sterol composed of stigmasterol, campesterol, and β-sitosterol; and the polyglycerol fatty acid ester is triglycerol dioleate.

[0019] As a preferred solution, the inoculation amount of the seed liquid in the fermentation medium is 0.8% to 1.5% v / v, more preferably 1.0% v / v.

[0020] As a preferred solution, the time of the primary fermentation culture is 42 to 54 hours, more preferably 48 hours; the time of the secondary fermentation culture is 64 to 80 hours, more preferably 72 hours.

[0021] As a preferred solution, the process conditions of the primary fermentation culture and the secondary fermentation culture are: shaking culture under the conditions of temperature of 28-32°C and rotation speed of 150-200 r / min.

[0022] As a preferred solution, the separation is centrifugal separation; the purification process is: first extracting and concentrating the fermentation broth, then extracting the obtained concentrated product, and separating the supernatant, which is the steroid intermediate.

[0023] The technical solution of the present invention has the following advantages and beneficial effects:

[0024] The invention provides a method for preparing steroid intermediates by fermenting sterols with microorganisms. Sterols are used as carbon sources, mycobacteria are used as fermentation strains, polyglycerol fatty acid esters are added as fermentation aids in the early stage of fermentation, hydroxytyrosol or sodium dehydroacetate is added as fermentation aids in the middle stage of fermentation, and androstenedione (AD), androstenedione (ADD), 22-hydroxy-23,24-dicholesterol-1,4-diene-3-one (1,4-HBC) and 22-hydroxy-23,24-dicholesterol-4-ene-3-one (4-HBC) are prepared through fermentation and culture.

[0025] The preparation method of the present invention adopts a one-step fermentation method to prepare four steroid drug intermediates, namely ADD, AD, 1,4-HBC and 4-HBC. Compared with the traditional chemical synthesis method, the method has the characteristics of cheap and easy-to-obtain fermentation aids, natural intracellular synthesis products, environmental friendliness, few by-products, simple separation and purification, and convenient industrial production. In particular, experiments have confirmed that compared with conventional fermentation culture without adjuvants, the present invention can significantly increase the yield of steroid intermediates by introducing specific fermentation aids in the early and middle fermentation stages, and the yield of intermediates can reach more than 160 mg / L.

[0026] Therefore, the present invention can provide a novel preparation strategy for the synthesis of steroid intermediates, and can also provide more raw material supply for the synthesis of hormone drugs, and at the same time has important significance for promoting the development and application of steroid intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a liquid chromatogram of the steroid intermediate prepared in Example 1 of the present invention. DETAILED DESCRIPTION

[0028] The technical scheme of the present invention is clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in 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. The test methods used in the following embodiments are conventional methods unless otherwise specified; the raw materials used are materials commonly used in the field and available through commercial channels unless otherwise specified.

[0029] The present invention provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, comprising the following steps:

[0030] Activating mycobacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for primary fermentation; the fermentation medium includes the following components at the following concentrations: 0.5-2 g / L sterol, 0.8-1.2 g / L polyglycerol fatty acid ester;

[0031] Adding hydroxytyrosol or sodium dehydroacetate to the system after the first fermentation culture to carry out a second fermentation culture; the amount of hydroxytyrosol or sodium dehydroacetate added to the system is 100-200 mg / L;

[0032] After the secondary fermentation culture is completed, the fermentation broth is separated and purified to obtain a steroid intermediate.

[0033] In some embodiments of the present invention, the mycobacterium is Mycobacterium sp. 191574.

[0034] In some embodiments of the present invention, preferably, the activation is performed by inoculating the mycobacterium into a nutrient broth medium for activation culture.

[0035] In some embodiments of the present invention, further preferably, the inoculation amount of mycobacteria in the nutrient broth medium is 0.08% to 0.15% v / v; the activation culture is to culture the inoculated mycobacteria in a shaker at 28 to 32°C and a rotation speed of 150 to 200 r / min until OD 600nm The activation culture can enhance the biological activity of mycobacteria and enrich the strains.

[0036] In some embodiments of the present invention, the fermentation medium further comprises the following components at the following concentrations: 0.8-3 g / L citric acid, 0.5-2 g / L MgSO4·7H2O, 0.5-2 g / L K2HPO4, 3-5 g / L (NH4)2HPO4, and the solvent is water. The above components are added to maintain the pH value and stability of the fermentation medium.

[0037] In some embodiments of the present invention, preferably, the sterol is a combination of one or more of stigmasterol, campesterol, and β-sitosterol; the polyglycerol fatty acid ester is one or more of triglycerol dioleate, hexaglycerol monooleate, and decaglycerol monooleate. Further preferably, the sterol is a mixed sterol composed of stigmasterol, campesterol, and β-sitosterol; and the polyglycerol fatty acid ester is triglycerol dioleate.

[0038] In some embodiments of the present invention, the inoculation amount of the seed liquid in the fermentation medium is 0.8% to 1.5% v / v, more preferably 1.0% v / v.

[0039] In some embodiments of the present invention, the time of the first fermentation culture is 42 to 54 hours, more preferably 48 hours; the time of the second fermentation culture is 64 to 80 hours, more preferably 72 hours.

[0040] In some embodiments of the present invention, the process conditions of the primary fermentation culture and the secondary fermentation culture are: shaking culture at a temperature of 28 to 32° C. and a rotation speed of 150 to 200 r / min.

[0041] In some embodiments of the present invention, separation is centrifugal separation; the purification process is: first extracting and concentrating the fermentation broth, then extracting the obtained concentrated product, and separating the supernatant, which is the steroid intermediate.

[0042] In some embodiments of the present invention, it is further preferred that the extraction is to extract the fermentation broth with an extraction solvent; the extraction solvent is ethyl acetate or ether; the concentration is reduced pressure concentration; the extraction solvent used for extraction is acetonitrile or methanol. It is further preferred that the number of extractions is 2 to 5 times; the volume ratio of the fermentation broth to the extraction solvent is (1.8 to 2.2): (0.8 to 1.2), more preferably 2: 1. The number of extractions is 2 to 5 times; the volume ratio of the concentrated product to the extraction solvent is (1 to 2): (4 to 6).

[0043] In the following embodiments and comparative examples of the present invention, the mycobacterium strain used is specifically mycobacterium (Mycobacterium sp.) 191574, which is purchased from Beijing Beina Chuanglian Biotechnology Research Institute. The polyglycerol fatty acid ester used is specifically triglycerol dioleate, which is a commercial product of Shandong Binzhou Jinsheng New Material Technology Co., Ltd. Sodium dehydroacetate and hydroxytyrosol are both conventional commercial products. The sterol used is a mixed sterol product, including stigmasterol 22.41±0.05%, campesterol 16.79±0.16%, and β-sitosterol 60.70±1.37%.

[0044] In the following embodiments and comparative examples of the present invention, the nutrient broth medium adopts the conventional composition in the art, and is prepared with water as the matrix according to the following raw material concentrations: 3.0 g / L beef extract powder, 10.0 g / L peptone, 5.0 g / L sodium chloride, and sterilized at 121° C. for 20 min.

[0045] Example 1

[0046] This embodiment provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0047] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD600nm is 0.80, and the seed solution is obtained;

[0048] (2) Inoculate the seed liquid into 100 mL fermentation medium at an inoculum volume of 1% (v / v) and ferment for 48 hours, then add sodium dehydroacetate to the fermentation system at a concentration of 100 mg / L, and continue fermentation for 72 hours; wherein the composition of the fermentation medium is: sterol 1g / L, polyglycerol fatty acid ester 1g / L, citric acid 1g / L, MgSO4·7H2O 0.5g / L, K2HPO4 0.5g / L, (NH4)2HPO4 3.5g / L, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160r / min;

[0049] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0050] Example 2

[0051] This embodiment provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0052] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0053] (2) Inoculate the seed liquid into 100 mL fermentation medium at an inoculum volume of 1% (v / v) and ferment for 48 hours, then add hydroxytyrosol to the fermentation system at a concentration of 200 mg / L, and continue fermentation for 72 hours; wherein the composition of the fermentation medium is: sterol 1g / L, polyglycerol fatty acid ester 1g / L, citric acid 1g / L, MgSO4·7H2O 0.5g / L, K2HPO4 0.5g / L, (NH4)2HPO4 3.5g / L, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160r / min;

[0054] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0055] Comparative Example 1

[0056] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0057] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0058] (2) Inoculating the seed liquid into 100 mL of fermentation medium at an inoculum rate of 1% (v / v) and fermenting for 120 h; wherein the fermentation medium comprises: 1 g / L sterol, 1 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 3.5 g / L (NH4)2HPO4, and the solvent is water. The fermentation is carried out in a shaking incubator at a temperature of 30°C and a rotation speed of 160 r / min;

[0059] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0060] Comparative Example 2

[0061] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0062] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0063] (2) Inoculating the seed liquid into 100 mL of fermentation medium at an inoculum volume of 1% (v / v) and fermenting for 120 h; wherein the fermentation medium comprises: 1 g / L sterol, 1 g / L polyglycerol fatty acid ester, 1 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 3.5 g / L (NH4)2HPO4, and the solvent is water. The fermentation is carried out in a shaking incubator at a temperature of 30°C and a rotation speed of 160 r / min;

[0064] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0065] Comparative Example 3

[0066] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0067] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0068] (2) Inoculate the seed liquid into 100 mL fermentation medium at an inoculation rate of 1% (v / v) and ferment for 48 hours, then add sodium dehydroacetate to the fermentation system at concentrations of 50, 100, 250, and 300 mg / L, respectively, and continue fermentation for 72 hours; wherein the composition of the fermentation medium is: sterol 1g / L, citric acid 1g / L, MgSO4·7H2O 0.5g / L, K2HPO4 0.5g / L, (NH4)2HPO4 3.5g / L, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160r / min;

[0069] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0070] Comparative Example 4

[0071] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0072] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0073] (2) Inoculate the seed liquid into 100 mL fermentation medium at an inoculum volume of 1% (v / v) and ferment for 48 hours, then add hydroxytyrosol to the fermentation system at concentrations of 20, 100, 200, and 500 mg / L, and continue fermentation for 72 hours; wherein the composition of the fermentation medium is: sterol 1g / L, citric acid 1g / L, MgSO4·7H2O 0.5g / L, K2HPO40.5g / L, (NH4)2HPO4 3.5g / L, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160r / min;

[0074] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0075] Comparative Example 5

[0076] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0077] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0078] (2) Inoculate the seed liquid into 100 mL of fermentation medium at an inoculum volume of 1% (v / v) and ferment for 120 h; wherein the composition of the fermentation medium is: 1 g / L sterol, 1 g / L polyglycerol fatty acid ester, 100 mg / L sodium dehydroacetate, 1 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 3.5 g / L (NH4)2HPO4, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160 r / min;

[0079] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0080] Comparative Example 6

[0081] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0082] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0083] (2) Inoculating the seed liquid into 100 mL of fermentation medium at an inoculum volume of 1% (v / v) and fermenting for 120 hours; wherein the composition of the fermentation medium is: 1 g / L sterol, 1 g / L polyglycerol fatty acid ester, 200 mg / L hydroxytyrosol, 1 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 3.5 g / L (NH4)2HPO4, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160 r / min;

[0084] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0085] Comparative Example 7

[0086] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0087] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0088] (2) Inoculating the seed liquid into 100 mL of fermentation medium at an inoculum volume of 1% (v / v) and fermenting for 120 hours; wherein the composition of the fermentation medium is: 1 g / L sterol, 0.5 g / L polyglycerol fatty acid ester, 50 mg / L sodium dehydroacetate, 100 mg / L hydroxytyrosol, 1 g / L citric acid, 0.5 g / L MgSO4·7H2O, 0.5 g / L K2HPO4, 3.5 g / L (NH4)2HPO4, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160 r / min;

[0089] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0090] Comparative Example 8

[0091] This comparative example provides a method for preparing a steroid intermediate by fermenting sterols by microorganisms, which specifically comprises the following steps:

[0092] (1) Mycobacterium strains were inoculated into 100 mL of nutrient broth at a concentration of 0.1% (v / v) and cultured in a shaker at 30°C and 160 rpm until OD 600nm is 0.80, and the seed solution is obtained;

[0093] (2) Inoculate the seed liquid into 100 mL fermentation medium at an inoculation rate of 1% (v / v) and ferment for 48 hours, then add sodium dehydroacetate to the fermentation system at a concentration of 50 mg / L, and add hydroxytyrosol to the fermentation system at a concentration of 100 mg / L, and continue fermentation for 72 hours; wherein the composition of the fermentation medium is: sterol 1g / L, polyglycerol fatty acid ester 1g / L, citric acid 1g / L, MgSO4·7H2O 0.5g / L, K2HPO4 0.5g / L, (NH4)2HPO4 3.5g / L, and the solvent is water. The fermentation is carried out in a shaking table at a temperature of 30°C and a rotation speed of 160r / min;

[0094] (3) After the fermentation culture is completed, the bacteria are removed by centrifugation to obtain a fermentation broth; the fermentation broth is extracted with ethyl acetate (the single amount of ethyl acetate is 50 mL, and the number of extractions is 3 times), and then distilled under reduced pressure to obtain a concentrated product, and then the concentrated product is extracted with acetonitrile (the single amount of acetonitrile is 5 mL, and the number of extractions is 5 times) to obtain a steroid intermediate.

[0095] Test example

[0096] Liquid chromatography was used to determine the contents of ADD, AD, 1,4-HBC, and 4-HBC in the steroid intermediates prepared in Examples 1 to 2 and Comparative Examples 1 to 8, and the total yield of the steroid intermediates was calculated. The conditions of liquid chromatography were as follows: chromatographic column: ZORBAX Eclipse Plus Phenyl-Hexyl; detector: UV detector (254 nm); mobile phase: methanol, water, high pressure binary gradient elution: A: water; B: methanol. 1 to 4 min: 70% B; 4 to 20 min: 70%-100% B; 20 to 25 min: 100% B; 25 to 26 min: 100%-70% B; 26 to 32 min: 70% B. Flow rate 1 mL / min, injection volume 10 μL. According to the retention time and peak area, the yield of each intermediate was quantitatively calculated using an external standard, and parallel tests were performed.

[0097] in, Figure 1 This is a liquid chromatogram of the steroid intermediates prepared in Example 1 of the present invention, which shows the peak positions and peak area sizes of four steroid intermediates ADD, AD, 1,4-HBC, and 4-HBC. The liquid chromatograms of other embodiments and comparative examples are similar thereto, and the present invention will not list them one by one.

[0098] According to the results of liquid chromatography, the yield of each steroid intermediate can be obtained, and then the total yield of the steroid intermediate can be calculated. Table 1 lists the yield and total yield of each steroid intermediate in Examples 1 to 2 and Comparative Examples 1 to 8 determined by liquid chromatography. Note: In the column of total yield, different lowercase letters indicate significant differences between the groups (P<0.05).

[0099] Table 1. Yield and total yield of steroid intermediates prepared in Examples and Comparative Examples

[0100]

[0101] As shown in Table 1, the test results of steroidal intermediates are as follows: compared to the test group (Comparative Example 1) without adding a fermentation aid, other test groups with added fermentation aids can significantly improve the total amount of steroidal intermediates. In Comparative Example 2, after adding 1g / L of polyglycerol fatty acid ester, intermediate yield is significantly improved, which is 12.2 times higher than without adding a fermentation aid. In Comparative Example 3, after adding 100mg / L of sodium dehydroacetate, the best intermediate yield can be obtained compared to other additions (50, 250, 300mg / L), which is 11.5 times higher than without adding a fermentation aid. In Comparative Example 4, after adding 200mg / L of hydroxytyrosol, intermediate yield is significantly improved, which is 10.8 times higher than without adding a fermentation aid.

[0102] Comparative Examples 5 to 7 explore the effect of polyglycerol fatty acid esters, hydroxytyrosol and / or sodium dehydroacetate on sterol conversion when added together in the early stage of fermentation. It can be seen from Comparative Examples 5 to 7 that when polyglycerol fatty acid esters and hydroxytyrosol and / or sodium dehydroacetate are added simultaneously, the total amount of steroid drug intermediates is 143.12, 124.82 and 131.65 mg / L, respectively, which is not much different from Comparative Example 2 (131.84 mg / L), but less than Examples 1 and 2 of the present invention.

[0103] The present invention changes the hydroxytyrosol / sodium dehydroacetate input time, that is, adds hydroxytyrosol / sodium dehydroacetate after fermentation and culture for 48 hours. As shown in Example 1 and Example 2, after changing the feeding time, the total amount of steroidal intermediates is significantly increased compared with Comparative Example 2, increasing by 33.11 mg / L and 33.87 mg / L respectively, indicating that sodium dehydroacetate / hydroxytyrosol and polyglycerol fatty acid ester have a synergistic effect on sterol conversion, and the feeding time is very critical. At this time, the output of the steroidal intermediates of Example 1 and 2 is increased by 15.5 times and 15.6 times respectively compared with Comparative Example 1.

[0104] In Comparative Example 8, after adding polyglycerol fatty acid esters and fermenting for 48 hours, a certain amount of sodium dehydroacetate and hydroxytyrosol were added, and the total amount of steroidal intermediates was significantly reduced compared with Examples 1 and 2, indicating that when the two were added together, the fermentation process of mycobacteria was adversely affected, but it still had a promoting effect compared with Comparative Example 1, which was increased by 7.1 times.

[0105] In summary, the method for preparing steroidal intermediates by microbial fermentation of sterols provided by the present invention uses sterols as carbon source, mycobacteria as fermentation strains, polyglycerol fatty acid esters as fermentation aids are added in the early stage of fermentation, hydroxytyrosol or sodium dehydroacetate is added as fermentation aids in the middle stage of fermentation, and four steroidal intermediates AD, ADD, 1,4-HBC and 4-HBC are prepared by fermentation culture. Compared with conventional fermentation culture without auxiliary agents, the above-mentioned preparation method of the present invention can significantly improve the yield of steroidal intermediates, and the yield of intermediates can reach more than 160mg / L. Therefore, the present invention can provide a novel preparation strategy for the synthesis of steroidal intermediates, and can also provide more raw material supply for the synthesis of hormone drugs, and at the same time, it is of great significance to promote the development and application of steroidal intermediates.

[0106] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing steroid intermediates by microbial fermentation of sterols, characterized in that: The following steps are involved: Activating mycobacteria to obtain seed liquid, inoculating the seed liquid into a fermentation medium for a fermentation culture; the fermentation medium includes the following components at the following concentrations: 0.5-2 g / L sterol, 0.8-1.2 g / L polyglycerol fatty acid ester; Adding hydroxytyrosol or sodium dehydroacetate to the system after the first fermentation culture to carry out a second fermentation culture; the amount of hydroxytyrosol or sodium dehydroacetate added to the system is 100-200 mg / L; After the secondary fermentation culture is completed, the fermentation broth is separated and purified to obtain a steroid intermediate.

2. The method for preparing steroid intermediates by microbial fermentation of sterols according to claim 1, characterized in that: The mycobacterium is Mycobacterium sp. 191574.

3. The method for preparing steroid intermediates by microbial fermentation of sterols according to claim 1, characterized in that: The activation is to inoculate the mycobacterium into a nutrient broth medium for activation culture.

4. The method for preparing steroid intermediates by microbial fermentation of sterols according to claim 3, characterized in that: The inoculation amount of mycobacteria in the nutrient broth medium is 0.08% to 0.15% v / v; the activation culture is to culture the inoculated mycobacteria in a shaking incubator at 28 to 32°C and a rotation speed of 150 to 200 r / min until OD 600nm It is 0.8~1.

0.

5. The method for preparing steroid intermediates by microbial fermentation of sterols according to claim 1, characterized in that: The fermentation medium also includes components with the following concentrations: 0.8-3 g / L of citric acid, 0.5-2 g / L of MgSO4·7H2O, 0.5-2 g / L of K2HPO4, 3-5 g / L of (NH4)2HPO4, and the solvent is water.

6. The method for preparing steroid intermediates by microbial fermentation of sterols according to claim 1, characterized in that: The sterol is a combination of one or more of stigmasterol, campesterol, and β-sitosterol; the polyglycerol fatty acid ester is one or more of triglycerol dioleate, hexaglycerol monooleate, and decaglycerol monooleate.

7. The method for preparing steroid intermediates by fermenting sterols by microorganisms according to any one of claims 1 to 6, characterized in that: The inoculation amount of the seed liquid in the fermentation medium is 0.8% to 1.5% v / v.

8. The method for preparing steroid intermediates by fermenting sterols by microorganisms according to any one of claims 1 to 6, characterized in that: The time of the primary fermentation culture is 42 to 54 hours; the time of the secondary fermentation culture is 64 to 80 hours.

9. The method for preparing steroid intermediates by fermenting sterols by microorganisms according to any one of claims 1 to 6, characterized in that: The process conditions of the primary fermentation culture and the secondary fermentation culture are: shaking culture under the conditions of temperature of 28-32°C and rotation speed of 150-200r / min.

10. The method for preparing steroid intermediates by fermenting sterols by microorganisms according to any one of claims 1 to 6, characterized in that: The separation is centrifugal separation; the purification process is: first extracting and concentrating the fermentation broth, then extracting the concentrated product, and separating the supernatant, which is the steroid intermediate.

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