CN103923966A - Method of preparing 4-androstenedione by fermentatively degrading phytosterol by virtue of single water phase system - Google Patents
Method of preparing 4-androstenedione by fermentatively degrading phytosterol by virtue of single water phase system Download PDFInfo
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- CN103923966A CN103923966A CN201410113381.4A CN201410113381A CN103923966A CN 103923966 A CN103923966 A CN 103923966A CN 201410113381 A CN201410113381 A CN 201410113381A CN 103923966 A CN103923966 A CN 103923966A
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Abstract
The invention provides a method of preparing 4-androstenedione by fermentatively degrading phytosterol by virtue of a single water phase system. The method comprises the following step: 1, strain cultivation, namely inoculating a mycobacterium smegmatis stain into a seed medium and cultivating; 2, sterol emulsification, namely emulsifying the phytosterol as a substrate under the action of a surfactant; 3, fermentation cultivation, namely inoculating the mycobacterium smegmatis stain obtained in the first step in a fermentation medium, adding the phytosterol obtained in the second step and carrying out fermentation cultivation; 4, finished product extraction. According to the method of preparing the 4-androstenedione by fermentatively degrading the phytosterol by virtue of the single water phase system, an emulsifier is added, so that the yield of 4-androstenedione is increased, and the extraction technology of the 4-androstenedione is also simplified; the cost is low, the pollution is little, and the environmental protection pressure is weak.
Description
Technical field
The present invention relates to the microbiological transformation technology field of steroid drugs, refer to that especially a kind of single water system fermentative degradation plant sterol prepares the method for 4-AD.
Background technology
4-AD, English name: androst-4-ene-3,17-dione, referred to as 4-AD, be the irreplaceable intermediate of steroid hormone class medicine, current most of steroid hormone series products is all produced as raw material using 4-AD.
The production technique that 4-AD is taked at present mainly contains two kinds: one is to extract dioscin from the plants such as wild Chinese medicinal materials Dioscorea nipponica Mak. Ningpo Yam Rhizome, yellow ginger, then prepares 4-AD through chemosynthesis; Another kind is to utilize microbial transformation phytosterin to produce 4-AD.The former process costs is high, pollution is heavy, the production cycle is long; The latter is simple to operate, the cycle is short, is the focus of present stage research.
At present, mainly take-profit of the method biphasic fermentation system that domestic existing microbial transformation phytosterin is produced 4-AD is carried out fermentation culture.But biphasic fermentation system ubiquity complex process, plant factor are low, target product 4-AD extracts and the problem such as oil treatment difficulty, and the organic solvent use kind in diphasic system is many simultaneously, and consumption is large, and environmental pollution is very large; Although superpolymer/superpolymer double water-phase fermentation system substrate plant sterol consistency in oil phase is good, be beneficial to the conversion of bacterial classification, the solubilising of superpolymer is limited and expensive, has limited its application in industry.
Summary of the invention
The present invention proposes a kind of single water system fermentative degradation plant sterol and prepare the method for 4-AD, the method for preparing 4-AD for solving existing microbial transformation phytosterin exists product to extract difficulty and the serious problem of environmental pollution.
Technical scheme of the present invention is achieved in that
Single water system fermentative degradation plant sterol is prepared a method for 4-AD, comprises the steps: the first step, culture propagation: will in M. smegmatics bacterial classification access seed culture medium, cultivate; Second step, sterol emulsification: substrate plant sterol is carried out under the effect of emulsifying agent to emulsification; The 3rd step, fermentation culture: in the M. smegmatics bacterial classification access fermention medium that the first step is obtained and add the plant sterol emulsion that second step obtains and carry out fermentation culture; The 4th step, finished product extracts.
Preferably, in second step, emulsifying agent comprises: the one in polysorbate60, tween 80, sorbester p18 or sorbester p17.
Preferably, in fermention medium, the massfraction of plant sterol is: 1.5%-3.0%; In fermention medium, the massfraction of emulsifying agent is: polysorbate60 0.1-0.5%, tween 80 0.5-2.0%, sorbester p18 0.5-1.0%, sorbester p17 0.5-2.0%.
Preferably, the temperature of second step plant sterol emulsification is 50-70 DEG C.
Preferably, the concrete operations of the first step culture propagation are: by concentration 10
7-10
9the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with the inoculum size of 5%-10%.
Preferably, the concrete operations of the 3rd step fermentation culture are: treat the bacterium culture medium OD in the first step
600during for 15-20, with the inoculum size of 20%-50%, its access is equipped with in the fermentor tank of fermention medium and cultivates.
Preferably, the condition of the 3rd step fermentation culture is: temperature 25-30 DEG C, and the initial pH6.0-8.0 that ferments, dissolved oxygen amount 20-40%, cultivates rotating speed 150-200rpm, culture cycle 80-120h.
Preferably, the seed culture medium of the first step comprises the component of following concentration: glucose 10-12g/L, K
2hPO
40.5-0.6g/L, MgSO
47H
2o0.5-0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.3g/L and citric acid 2.0-2.5g/L, the pH of seed culture medium is 6.0-8.0.
Preferably, in the 3rd step, fermention medium comprises the component of following concentration: glucose 20-25g/L, peptone 20-25g/L, soybean cake powder 15-20g/L, K
2hPO
40.7-0.8g/L, MgSO
47H
2o0.6-0.7g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.5g/L, citric acid 2.0-2.5g/L and NH
4nO
30.9-1.0g/L, the pH of described fermention medium is 6.0-8.0.
Preferably, the concrete technology that the 4th step finished product extracts is: filtering fermentation liquor is obtained to filter cake, use the ethanol of at least 2 times of amounts or acetone that the distillation that extracts successively, filters, decolours after described filter cake dissolving is obtained to crude product; Crude product extracts successively, decolours, filters, distills after dissolving with the methylene dichloride of at least 4 times of amounts or chloroform, then adds at least 2 times to measure and distill crystallization after hexanes or toluene and obtain highly finished product.
Single water system fermentative degradation plant sterol that the present invention proposes is prepared the method for 4-AD, has following beneficial effect:
1, improved the utilization ratio of plant sterol, fermentation substrate transformation efficiency reaches more than 95%;
2, fermentation in adopt single aqueous phase system and extract operation in solvent kind few, consumption is little, low in the pollution of the environment, production cost is low;
3, reduce the difficulty that finished product extracts, extracted simple to operate, efficient, environmental protection.
Brief description of the drawings
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, to the accompanying drawing of required use in embodiment or description of the Prior Art be briefly described below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, do not paying under the prerequisite of creative work, can also obtain according to these accompanying drawings other accompanying drawing.
Fig. 1 is the schematic flow sheet that a kind of single water system fermentative degradation plant sterol of the present invention is prepared the method for 4-AD.
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtaining under creative work prerequisite, belong to the scope of protection of the invention.
As shown in Figure 1: a kind of single water system fermentative degradation plant sterol is prepared the method for 4-AD, comprises the steps: the first step, culture propagation: will in M. smegmatics bacterial classification access seed culture medium, cultivate; Second step, sterol emulsification: substrate plant sterol is carried out under the effect of emulsifying agent to emulsification; The 3rd step, fermentation culture: in the M. smegmatics bacterial classification access fermention medium that the first step is obtained and add the plant sterol emulsion that second step obtains and carry out fermentation culture; The 4th step, finished product extracts.
As the preferred technical scheme of one, in second step, emulsifying agent comprises: the one in polysorbate60, tween 80, sorbester p18 or sorbester p17.
As the preferred technical scheme of one, in fermention medium, the massfraction of plant sterol is: 1.5%-3.0%; In fermention medium, the massfraction of emulsifying agent is: polysorbate60 0.1-0.5%, tween 80 0.5-2.0%, sorbester p18 0.5-1.0%, sorbester p17 0.5-2.0%.
As the preferred technical scheme of one, the temperature of second step plant sterol emulsification is 50-70 DEG C.
Kind, consumption and the emulsifying temperature of emulsifying agent, the absorbancy of the emulsion that can be mixed with by test plants sterol and emulsifying agent is determined.Concrete testing method is: by plant sterol and selected certain density emulsifying agent according to certain proportioning constant temperature emulsification 30min after, after centrifugal 15min, get its absorbancy at 678nm place of clear liquid dilution test, absorbancy height illustrates plant sterol emulsify well in the emulsifying agent of this concentration, after repetition test, determine polysorbate60, tween 80, sorbester p18 and sorbester p17 are preferred emulsifying agent, their preferred concentration (massfraction of emulsifying agent in fermention medium) is respectively: polysorbate60 0.1-0.5%, tween 80 0.5-2.0%, sorbester p18 0.5-1.0%, sorbester p17 0.5-2.0%, preferred emulsifying temperature is 50-70 DEG C.
As the preferred technical scheme of one, the concrete operations of the first step culture propagation are: by concentration 10
7-10
9the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with the inoculum size of 5%-10%.
As the preferred technical scheme of one, the concrete operations of the 3rd step fermentation culture are: treat the bacterium culture medium OD in the first step
600during for 15-20, with the inoculum size of 20%-50%, its access is equipped with in the fermentor tank of fermention medium and cultivates.
As the preferred technical scheme of one, the condition of the 3rd step fermentation culture is: temperature 25-30 DEG C, and the initial pH6.0-8.0 that ferments, dissolved oxygen amount 20-40%, cultivates rotating speed 150-200rpm, culture cycle 80-120h.
As the preferred technical scheme of one, the seed culture medium of the first step comprises the component of following concentration: glucose 10-12g/L, K
2hPO
40.5-0.6g/L, MgSO
47H
2o0.5-0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.3g/L and citric acid 2.0-2.5g/L, the pH of seed culture medium is 6.0-8.0.
As the preferred technical scheme of one, in the 3rd step, fermention medium comprises the component of following concentration: glucose 20-25g/L, peptone 20-25g/L, soybean cake powder 15-20g/L, K
2hPO
40.7-0.8g/L, MgSO
47H
2o0.6-0.7g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.5g/L, citric acid 2.0-2.5g/L and NH
4nO
30.9-1.0g/L, the pH of fermention medium is 6.0-8.0.
As the preferred technical scheme of one, the concrete technology that the 4th step finished product extracts is: filtering fermentation liquor is obtained to filter cake, use the ethanol of at least 2 times of amounts or acetone that the distillation that extracts successively, filters, decolours after described filter cake dissolving is obtained to crude product; Crude product extracts successively, decolours, filters, distills after dissolving with the methylene dichloride of at least 4 times of amounts or chloroform, then adds to distill crystallization after the hexane of at least 2 times of amounts or toluene and obtain highly finished product.
Compared with prior art, the method plant sterol transformation efficiency that single water system fermentative degradation plant sterol that the present invention proposes is prepared 4-AD is high, more than the concentration of fermentation culture after product 4-AD reaches 9.0g/L; Later stage finished product extracts simple, of reduced contamination, and the solvent quantity of its use is few, crude product is repeated to extract after twice, more than the purity to 99% of 4-AD with above-mentioned extracting method.
Embodiment 1
Single water system fermentative degradation plant sterol is prepared the method for 4-AD, comprises the steps:
The first step, culture propagation: by concentration 10
7the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with 10% inoculum size, and seed culture medium comprises the component of following concentration: glucose 10g/L, K
2hPO
40.6g/L, MgSO
47H
2o0.5g/L, (NH
4)
2fe(SO
4)
26H
2o0.05g/L, CaCO
35.0g/L and citric acid 2.5g/L, the pH of seed culture medium is 6.0;
Second step, sterol emulsification:
Substrate plant sterol is carried out under the effect of emulsifier tween 80 to emulsification, 60 DEG C of emulsifying temperatures;
The 3rd step, fermentation culture:
Treat the bacterium culture medium OD in the first step
600be 20 o'clock, the inoculum size with 50% is equipped with its access in the fermentor tank of fermention medium and adds the plant sterol emulsion that second step obtains and carry out fermentation culture;
The condition of fermentation culture is: 25 DEG C of temperature, and the initial pH6 that ferments, dissolved oxygen amount 40%, cultivates rotating speed 200rpm, culture cycle 120h;
Fermention medium comprises the component of following concentration: glucose 20g/L, peptone 25g/L, soybean cake powder 20g/L, K
2hPO
40.8g/L, MgSO
47H
2o0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.05g/L, CaCO
35.0g/L, citric acid 2.5g/L and NH
4nO
30.9g/L, the pH of fermention medium is 6.0, the massfraction 0.5% of tween 80, the massfraction of plant sterol is 3.0%;
The 4th step, finished product extracts:
The filtering fermentation liquor obtaining in the 3rd step is obtained to filter cake, after with the ethanol of 4 times of amounts, described filter cake being dissolved, extract successively, decolour, filter, distill and obtain crude product; Crude product decolours successively, filters, distills after dissolving with the methylene dichloride of 8 times of amounts, then adds to distill crystallization after the toluene of 4 times of amounts and obtain highly finished product.
Test result: the transformation efficiency of plant sterol is 96.0%, the 14.4g/L that finally tires, the purity of the 4-AD obtaining is greater than 99.0%.
Embodiment 2
Emulsifying temperature in embodiment 1 is changed to 50 DEG C, and all the other operations are identical with embodiment 2.
Test result: the transformation efficiency of plant sterol is 89.6%, the 13.44g/L that finally tires, the purity of the 4-AD obtaining is greater than 99.0%.
Embodiment 3
Emulsifying temperature in embodiment 1 is changed to 70 DEG C, and all the other operations are identical with embodiment 2.
Test result: the transformation efficiency of plant sterol is 84.3%, the 12.65g/L that finally tires, the purity of the 4-AD obtaining is greater than 99.0%.Comprehensively analyze embodiment 1-3 and learn, highly preferred emulsifying temperature is 60 DEG C.
Embodiment 4
The massfraction of tween 80 in fermention medium in embodiment 1 is changed to 2.0%, and all the other operations are identical with embodiment 2.
Test result: the transformation efficiency of plant sterol is 76.4%, the 11.46g/L that finally tires, the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 5
The massfraction of tween 80 in fermention medium in embodiment 1 is changed to 1%, and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 80.3%, the 12.04g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 6
Dissolved oxygen amount in fermentation culture conditions in embodiment 1 is changed to 20%, and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 80.6%, the 12.09g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 7
Dissolved oxygen amount in fermentation culture conditions in embodiment 1 is changed to 30%, and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 82.6%, the 12.39g/L that finally tires, and the purity of the 4-AD obtaining is greater than 99.0%.
Embodiment 8
Emulsifier tween in embodiment 1 80 is changed to polysorbate60, and addition and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 76.9%, the 11.54g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 9
The addition of the polysorbate60 in embodiment 8 is changed to 0.1%, and all the other operations are identical with embodiment 8.
Test result is: the transformation efficiency of plant sterol is 81.2%, the 12.18g/L that finally tires, and the purity of the 4-AD obtaining is greater than 99.0%.
Embodiment 10
The addition of the polysorbate60 in embodiment 8 is changed to 0.3%, and all the other operations are identical with embodiment 8.
Test result is: the transformation efficiency of plant sterol is 79.9%, the 11.98g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 11
Emulsifier tween in embodiment 1 80 is changed to sorbester p18, and addition and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 75.0%, the 11.25g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 12
Emulsifier tween in embodiment 1 80 is changed to sorbester p17, and addition and all the other operations are identical with embodiment 2.
Test result is: the transformation efficiency of plant sterol is 74.1%, the 11.12g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.Comprehensive embodiment 1, embodiment 8-12 are known, and in above-mentioned four kinds of emulsifying agents, tween 80 is most preferred emulsifying agent, and comprehensive embodiment 1,4,5 is known, and the preferred massfraction of tween 80 is 0.5%.
Embodiment 13
The operation that in embodiment 1, the 4th step finished product extracts is changed to: obtain crude product by filtering successively after described filter cake dissolving, extract, distilling with the acetone of 2 times of amounts; Crude product decolours successively, extracts, distills after dissolving with the chloroform of 4 times of amounts, then adds to distill crystallization after the toluene of 2 times of amounts and obtain highly finished product.
Test result is: the transformation efficiency of plant sterol is 95.7%, the 14.36g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 14
The initial pH of fermentation culture in the 3rd step in embodiment 1 is changed to 8, and all the other operations are identical with embodiment 1.
Test result is: the transformation efficiency of plant sterol is 69.9%, the 10.48g/L that finally tires, and the purity of the 4-AD obtaining is greater than 98.5%.
Embodiment 15
The initial pH of fermentation culture in the 3rd step in embodiment 1 is changed to 7, and all the other operations are identical with embodiment 1.
Test result is: the transformation efficiency of plant sterol is 95.9%, the 14.38g/L that finally tires, and the purity of the 4-AD obtaining is greater than 99%.Comprehensive embodiment 1, the embodiment 14 and embodiment 15 of analyzing, the suitable pH of visible fermentation culture is slightly acidic or neutral environment.
Embodiment 16
Single water system fermentative degradation plant sterol is prepared the method for 4-AD, comprises the steps:
The first step, culture propagation: by concentration 10
9the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with 5% inoculum size, and seed culture medium comprises the component of following concentration: glucose 12g/L, K
2hPO
40.5g/L, MgSO
47H
2o0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.04g/L, CaCO
35.3g/L and citric acid 2.0g/L, the pH of seed culture medium is 8.0;
Second step, sterol emulsification:
Substrate plant sterol is carried out under the effect of emulsifier tween 80 to emulsification, 60 DEG C of emulsifying temperatures;
The 3rd step, fermentation culture:
Treat the bacterium culture medium OD in the first step
600be 15 o'clock, the inoculum size with 30% is equipped with its access in the fermentor tank of fermention medium and adds the plant sterol emulsion that second step obtains and carry out fermentation culture;
The condition of fermentation culture is: 25 DEG C of temperature, and the initial pH that ferments is 7.0, dissolved oxygen amount 30% is cultivated rotating speed 180rpm, culture cycle 100h;
Fermention medium comprises the component of following concentration: glucose 25g/L, peptone 20g/L, soybean cake powder 15g/L, K
2hPO
40.7g/L, MgSO
47H
2o0.7g/L, (NH
4)
2fe(SO
4)
26H
2o0.04g/L, CaCO
35.5g/L, citric acid 2.0g/L and NH
4nO
31.0g/L, the pH of fermention medium is 8, the massfraction 0.5% of tween 80, the massfraction of plant sterol is 2.0%;
The 4th step, finished product extracts:
The filtering fermentation liquor obtaining in the 3rd step is obtained to filter cake, after with the acetone of 4 times of amounts, described filter cake being dissolved, extract successively, decolour, filter, distill and obtain crude product; Crude product decolours successively, filters, distills after dissolving with the chloroform of 6 times of amounts, then adds to distill crystallization after the hexane of 4 times of amounts and obtain highly finished product.
Test result: the transformation efficiency of plant sterol is 93.18%, the 9.32g/L that finally tires, the purity of the 4-AD obtaining is greater than 99%.
Embodiment 17
Single water system fermentative degradation plant sterol is prepared the method for 4-AD, comprises the steps:
The first step, culture propagation: by concentration 10
9the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with 5% inoculum size, and seed culture medium comprises the component of following concentration: glucose 12g/L, K
2hPO
40.5g/L, MgSO
47H
2o0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.04g/L, CaCO
35.3g/L and citric acid 2.0g/L, the pH of seed culture medium is 8.0;
Second step, sterol emulsification:
Substrate plant sterol is carried out under the effect of emulsifier tween 80 to emulsification, 60 DEG C of emulsifying temperatures;
The 3rd step, fermentation culture:
Treat the bacterium culture medium OD in the first step
600be 15 o'clock, the inoculum size with 20% is equipped with its access in the fermentor tank of fermention medium and adds the plant sterol emulsion that second step obtains and carry out fermentation culture;
The condition of fermentation culture is: 25 DEG C of temperature, and the initial pH that ferments is 8.0, dissolved oxygen amount 20% is cultivated rotating speed 150rpm, culture cycle 80h;
Fermention medium comprises the component of following concentration: glucose 25g/L, peptone 20g/L, soybean cake powder 15g/L, K
2hPO
40.7g/L, MgSO
47H
2o0.7g/L, (NH
4)
2fe(SO
4)
26H
2o0.04g/L, CaCO
35.5g/L, citric acid 2.0g/L and NH
4nO
31.0g/L, the pH of fermention medium is 8, the massfraction 0.5% of tween 80, the massfraction of plant sterol is 1.5%;
The 4th step, finished product extracts:
The filtering fermentation liquor obtaining in the 3rd step is obtained to filter cake, after with the acetone of 2 times of amounts, described filter cake being dissolved, extract successively, decolour, filter, distill and obtain crude product; Crude product decolours successively, filters, distills after dissolving with the chloroform of 4 times of amounts, then adds to distill crystallization after the hexane of 2 times of amounts and obtain highly finished product.
Test result: the transformation efficiency of plant sterol is 73.3%, the 5.50g/L that finally tires, the purity of the 4-AD obtaining is greater than 98.0%.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. single water system fermentative degradation plant sterol is prepared a method for 4-AD, it is characterized in that: comprise the steps:
The first step, culture propagation:
To in M. smegmatics bacterial classification access seed culture medium, cultivate;
Second step, sterol emulsification:
Substrate plant sterol is carried out under the effect of emulsifying agent to emulsification;
The 3rd step, fermentation culture:
In the M. smegmatics bacterial classification access fermention medium that the first step is obtained and add the plant sterol emulsion that second step obtains and carry out fermentation culture;
The 4th step, finished product extracts.
2. single water system fermentative degradation plant sterol according to claim 1 is prepared the method for 4-AD, it is characterized in that: in described second step, emulsifying agent comprises: the one in polysorbate60, tween 80, sorbester p18 or sorbester p17.
3. single water system fermentative degradation plant sterol according to claim 2 is prepared the method for 4-AD, it is characterized in that:
In described fermention medium, the massfraction of plant sterol is: 1.5%-3.0%;
In described fermention medium, the massfraction of emulsifying agent is: polysorbate60 0.1-0.5%, tween 80 0.5-2.0%, sorbester p18 0.5-1.0%, sorbester p17 0.5-2.0%.
4. single water system fermentative degradation plant sterol according to claim 3 is prepared the method for 4-AD, it is characterized in that: the temperature of described second step plant sterol emulsification is 50-70 DEG C.
5. single water system fermentative degradation plant sterol according to claim 1 is prepared the method for 4-AD, it is characterized in that: the concrete operations of described the first step culture propagation are: by concentration 10
7-10
9the M. smegmatics bacterial classification of individual/ml accesses in the culture tank that seed culture medium is housed and cultivates with the inoculum size of 5%-10%.
6. single water system fermentative degradation plant sterol according to claim 5 is prepared the method for 4-AD, it is characterized in that: the concrete operations of described the 3rd step fermentation culture are: treat the bacterium culture medium OD in the described the first step
600during for 15-20, with the inoculum size of 20%-50%, its access is equipped with in the fermentor tank of fermention medium and cultivates.
7. single water system fermentative degradation plant sterol according to claim 6 is prepared the method for 4-AD, it is characterized in that: the condition of described the 3rd step fermentation culture is: temperature 25-30 DEG C, initial pH6.0-8.0 ferments, dissolved oxygen amount 20-40%, cultivate rotating speed 150-200rpm, culture cycle 80-120h.
8. the method for preparing 4-AD according to the single water system fermentative degradation plant sterol described in claim 1-7 any one, is characterized in that: the seed culture medium of the described the first step comprises the component of following concentration: glucose 10-12g/L, K
2hPO
40.5-0.6g/L, MgSO
47H
2o0.5-0.6g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.3g/L and citric acid 2.0-2.5g/L, the pH of described seed culture medium is 6.0-8.0.
9. prepare the method for 4-AD according to the single water system fermentative degradation plant sterol described in claim 1-7 any one, it is characterized in that: in described the 3rd step, fermention medium comprises the component of following concentration: glucose 20-25g/L, peptone 20-25g/L, soybean cake powder 15-20g/L, K
2hPO
40.7-0.8g/L, MgSO
47H
2o0.6-0.7g/L, (NH
4)
2fe(SO
4)
26H
2o0.04-0.05g/L, CaCO
35.0-5.5g/L, citric acid 2.0-2.5g/L and NH
4nO
30.9-1.0g/L, the pH of described fermention medium is 6.0-8.0.
10. single water system fermentative degradation plant sterol according to claim 1 is prepared the method for 4-AD, it is characterized in that: the concrete technology that described the 4th step finished product extracts is: filtering fermentation liquor is obtained to filter cake, use the ethanol of at least 2 times of amounts or acetone that the distillation that extracts successively, filters, decolours after described filter cake dissolving is obtained to crude product; Crude product extracts successively, decolours, filters, distills after dissolving with the methylene dichloride of at least 4 times of amounts or chloroform, then adds at least 2 times to measure and distill crystallization after hexanes or toluene and obtain highly finished product.
Priority Applications (1)
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| CN104195209A (en) * | 2014-09-02 | 2014-12-10 | 常州大学 | Method for preparing 4-androstenedione through phytosterin |
| CN104232673A (en) * | 2014-08-21 | 2014-12-24 | 宋浩雷 | Method for producing dehydroepiandrosterone through microbial fermentation |
| CN107119100A (en) * | 2016-05-27 | 2017-09-01 | 保定北瑞甾体生物有限公司 | A kind of method for preparing 4-AD |
| CN112359087A (en) * | 2020-11-12 | 2021-02-12 | 湖南新合新生物医药有限公司 | Preparation method of 9-hydroxy androstenedione |
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| CN107119100B (en) * | 2016-05-27 | 2020-05-08 | 保定北瑞甾体生物有限公司 | Method for preparing 4-androstenedione |
| CN112359087A (en) * | 2020-11-12 | 2021-02-12 | 湖南新合新生物医药有限公司 | Preparation method of 9-hydroxy androstenedione |
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