CN112725238B - Streptomyces toxytricini strain for producing lipstatin through fermentation and application thereof - Google Patents
Streptomyces toxytricini strain for producing lipstatin through fermentation and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of microorganisms, and particularly relates to a streptomycin toxytricin strain for producing lipstatin by fermentation, and further discloses application of the streptomycin toxytricin strain for producing lipstatin by fermentation. The Streptomyces toxytricini FIM-28-93 (Streptomyces toxytricini) capable of producing lipstatin with high yield is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, and can ferment the lipstatin with high yield, in a fermentation experiment, the titer of the lipstatin produced by fermenting the Streptomyces toxytricini FIM-28-93 is as high as 13539 mu g/mL, the yield of the lipstatin is greatly improved, the fermentation period is shortened to 108 hours, and the method is more suitable for industrial fermentation production.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a streptomycin toxytricin strain for producing lipstatin by fermentation, and further discloses application of the streptomycin toxytricin strain for producing lipstatin by fermentation.
Background
Lipstatin (lipstatin), a metabolite of Streptomyces toxytricini (Streptomyces toxytricini), is an endogenous lipase inhibitor that selectively inhibits the activity of pancreatic lipase in the gastrointestinal tract, reducing the breakdown and absorption of fat. Orlistat (orlistat), a tetrahydro derivative of lipstatin, has been successfully developed by Roche lnc as an antiobesity drug, cenicron, which has been approved by FDA and is currently the only drug for obesity marketed as a non-central nervous system drug, occupying 2/3 of the antiobesity drug market. Two processes for preparing orlistat are known at present, one is a chemical synthesis method, and the other is a biological fermentation method to obtain lipstatin, and then orlistat is obtained by hydrogenation. Therefore, the large-scale production of lipstatin is of great significance to the development of orlistat medicaments.
The production of lipstatin by fermentation is the mainstream process in recent years. For example, chinese patent CN103555610A discloses an application of lipstatin fermentation medium, wherein through optimized screening of fermentation strains, the fermentation level of a target product can reach 10.8g/L, and the fermentation period is 140h; also, for example, chinese patent CN103468603A discloses a fermentation process and production of lipstatin producing bacteria, the fermentation period is 130h-240h, and the fermentation level is 9606-10860 μ g/ml; for example, chinese patent CN108753861A discloses a fermentation medium for producing lipstatin with high yield, wherein the fermentation level can reach 8970-10910 mu g/ml, and the fermentation period is 144h-168h; in addition, chinese patent CN103320481B discloses that by controlling dissolved oxygen to regulate strain metabolism, the yield of lipstatin is increased; chinese patent No. 103820510B discloses that the fermentation titer of lipstatin is also effectively improved by optimizing the addition combination of fermentation precursors and metal ions; european patents EP0803576 and WO2007134836A1 disclose processes for increasing lipstatin by adding linoleic acid, caprylic acid and N-formyl-L-leucine or leucine, precursors, respectively, during fermentation, but the overall fermentation level is low; US patent US20110014664A1 describes the combined use of linoleic acid and gamma-9 fatty acids, which significantly improves the yield of lipstatin; international patent WO2004003212 describes a medium with different starch substrates, the fermentation level reached 1.486g/L and the fermentation time was still 168 hours.
In summary, in the prior art, although the fermentation level of lipstatin is effectively improved by aiming at fermentation strain improvement, fermentation medium composition optimization and fermentation process control in the fermentation production of lipstatin, the problems of low strain fermentation unit, long fermentation period, unstable fermentation level and the like still exist. Therefore, the strain which meets the industrial production and can produce lipstatin with high yield is positively significant for the large-scale production of lipstatin.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a streptomycin toxytricin strain for producing lipstatin by fermentation, so as to solve the problem that the performance of the fermentation strain of lipstatin is not ideal in the prior art;
the second technical problem to be solved by the invention is to provide the application of the streptomyces toxytricini strain in fermentation production of lipstatin.
In order to solve the technical problems, the Streptomyces toxytricini strain is classified and named as Streptomyces toxytricini FIM-28-93, is preserved in Guangdong province microbial strain preservation center, is located in Experimental building 5 building of Merry 100, jieli, middleway, california, guangdong province, has a preservation number of GDMCC No.61322, and has a preservation date of 2020, 12 and 2 days.
The invention also discloses application of the streptomycin toxotrigine strain in fermentation production of lipstatin.
The invention also discloses a method for producing lipstatin by fermentation, which comprises the step of inoculating the toxotrigine streptomyces strain into a suitable fermentation medium for fermentation culture.
Specifically, the fermentation medium comprises the following components in percentage by mass: 1-3% of glycerol, 3-5% of sunflower seed oil, 0.5-1.5% of corn flour, 2.5-3% of soybean flour, 0.5-1.5% of peptone, 0.5-1.5% of lecithin, 0.05-0.1% of calcium carbonate and pH6.5-7.0.
Preferably, the fermentation medium comprises the following components in percentage by mass: 2% of glycerol, 4% of sunflower seed oil, 1% of corn flour, 2.8% of soybean flour, 1% of peptone, 1% of lecithin, 0.08% of calcium carbonate and pH6.5-7.0.
Specifically, the conditions of the fermentation culture comprise: controlling the rotation speed to be 100-250rpm, and performing fermentation culture at 25-30 ℃ for 120-144h.
Specifically, the method for producing lipstatin by fermentation further comprises the step of inoculating the streptomycin toxytricin strain into a seed culture medium for seed liquid culture;
the seed culture medium comprises the following components in percentage by mass: soybean flour 1-3%, yeast powder 0.3-0.8%, glycerin 1-3%, and pH6.8-7.2.
Preferably, the seed culture medium comprises the following components in percentage by mass: soybean powder 2.0%, yeast powder 0.5%, glycerin 2.0%, and pH6.8-7.2.
Specifically, the conditions of seed liquid culture include: controlling the rotation speed to be 100-200rpm, and carrying out seed liquid culture at 25-30 ℃ for 60-72h.
Specifically, the method for producing lipstatin by fermentation further comprises the step of inoculating the streptomycin toxotrigine strain into a slant culture medium for activation;
the slant culture medium comprises the following components in percentage by mass: 0.5 to 1.5 percent of soluble starch, 0.3 to 0.8 percent of oat flour, 0.1 to 0.3 percent of yeast powder, 0.05 to 0.2 percent of casein, 0.01 to 0.1 percent of dipotassium phosphate, 0.03 to 0.08 percent of magnesium sulfate, 2.0 percent of agar and pH 7.2 to 8.0.
Preferably, the slant culture medium comprises the following components by mass: 1.0% of soluble starch, 0.5% of oat flour, 0.2% of yeast powder, 0.1% of casein, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 2.0% of agar and pH 7.2-8.0.
Specifically, the conditions of the slant culture medium activation step include: culturing at 25-30 deg.C for 6-10 days.
The Streptomyces toxytricini FIM-28-93 (Streptomyces toxytricini) capable of producing lipstatin with high yield is obtained by screening through a normal-pressure room-temperature plasma mutagenesis technology, and can ferment lipstatin with high yield, in a fermentation experiment, the titer of the lipstatin produced by fermenting the Streptomyces toxytricini FIM-28-93 is as high as 13386 mu g/mL, the yield of the lipstatin is greatly improved, the fermentation period is shortened to 108h, and the method is more suitable for industrial fermentation production; the FIM-28-93 strain screened by the invention has good stability, the titer of lipstatin produced by the strain for four generations of continuous passage is basically stable, the same higher level is maintained, and the strain can be used as a production strain for further research and development.
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In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 is a graph of ARTP efflux time versus lethality in mutagenesis experiments;
FIG. 2 is a tree of evolutionary trees of the strain FIM-28-93 of the present invention.
Detailed Description
In the following examples of the present invention, the culture medium includes:
the separation plate culture medium, the slant culture medium and the plate culture medium comprise the following components in percentage by mass: 1.0% of soluble starch, 0.5% of oat flour, 0.2% of yeast powder, 0.1% of casein, 0.05% of dipotassium hydrogen phosphate, 0.05% of magnesium sulfate, 2.0% of agar and the balance of distilled water, wherein the pH value is 7.2-8.0, and the mixture is sterilized by high-pressure steam at 121 ℃ for 30min;
the seed culture medium comprises the following components in percentage by mass: 2.0% of soybean meal, 0.5% of yeast powder, 2.0% of glycerol and the balance of distilled water, wherein the pH value is 6.8-7.2, and the soybean meal is sterilized by high-pressure steam at 121 ℃ for 30min;
the fermentation medium comprises the following components in percentage by mass: 2% of glycerol, 4% of sunflower seed oil, 1% of corn flour, 2.8% of soybean flour, 1% of peptone, 1% of lecithin, 0.08% of calcium carbonate and the balance of distilled water, wherein the pH value is 6.5-7.0, and the high-pressure steam sterilization is carried out at 121 ℃ for 30min.
In the following embodiments of the present invention, the lipstatin content is detected by a high performance liquid chromatography method: adding 4mL of absolute ethyl alcohol into 2mL of fermentation liquor, fully oscillating for 5min, standing for 30min, centrifuging for 10min at 4000r/min, filtering supernate, and taking 2 mu L for HPLC detection. Chromatographic conditions are as follows: c18 (4.6 mm. Times.250mm, 5 μm) column, detection wavelength 195nm, in acetonitrile: water =85:15 (V/V) is a mobile phase, the flow rate is 1.0mL/min, the column temperature: at 40 ℃. And (3) taking a lipstatin standard substance as a reference substance, calculating the titer according to the peak area of the sample/the peak area of the standard solution, the concentration of the standard solution and the dilution factor, and taking the average value of the fermentation titer for 3 times.
Example 1 isolation of the hairline FIM-28 Strain
Collecting soil from Jinggang mountain vegetable garden in Jiangxi province, specifically, removing soil floating about 10cm on the surface layer with a sampling shovel, collecting soil with 10cmProcessing the soil sample by 10g-25g; weighing 2g of soil sample, adding 10mL of sterile normal saline, oscillating, standing for 30min, collecting supernatant as stock solution, and performing gradient dilution with sterile normal saline with dilution degree of 10 -2 、10 -3 、10 -4 And 10 -5 A suspension of (2).
0.1mL of the stock solution and the selected suspension are respectively taken and coated on a separation plate culture medium prepared by adding 50mg/L potassium dichromate into water, 3 parallel plates are repeated for each sample, then the coated plate is cultured at 28 ℃, and morphological characteristics such as colony appearance, size, color, edge shape, surface dry and wet state and the like are observed.
Under the aseptic operation condition, selecting single colony with representative colony shape, central protrusion, pink surface and the like according to colony morphology, integrity and abundance after culturing for 6-10 days, streaking and inoculating on a slant of a separation plate culture medium, culturing for 6-10 days at 28 ℃, separating and purifying to obtain a strain, naming the obtained strain as a strain FIM-28, and storing the strain FIM-28 obtained by separation with 20% glycerol at-80 ℃.
EXAMPLE 2 acquisition of the mutagenized Strain FIM-28-93
Taking the screened strain FIM-28, transferring the strain FIM-28 to a slant culture medium, and culturing in a constant temperature incubator for 6-10 days at the temperature of 30 ℃; washing off spores on the slant culture medium with normal saline, scattering glass beads, filtering with warp cloth, and making into 10 6 spores/mL of spore suspension.
10 μ L of the spore suspension prepared above was pipetted by a pipette onto a circular iron plate with a diameter of 1cm, placed in a normal pressure room temperature plasma mutagenesis system with helium as working gas, power supply of 110W, and working gas flow of 10L/min, and a treatment distance of 2mm, treated for 5s, 10s, 15s, 20s, 30s, 45s, and 60s, respectively, and the treated spore suspension was subjected to gradient dilution and plating to prepare a mortality curve (as shown in fig. 1). As can be seen from FIG. 1, there is a clear dose-effect relationship between the mutagenic treatment dose and the lethality of strain FIM-28, which gradually increases with the treatment time.
Selecting two irradiation times with different lethal doses of 15s and 45s according to the lethality curve, and carrying out plasma mutagenesis on spore suspension of the strain FIM-28; and mixing the spore suspensions with different processing time in a test tube filled with normal saline, and uniformly mixing to obtain the mutagenized spore suspension for later use.
Carrying out gradient dilution on the obtained mutagenic spore suspension, and controlling the dilution degree to be 10 respectively -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Choose 10 -4 、10 -5 、10 -6 The spore suspensions with three dilutions are respectively coated on a separation plate culture medium and cultured for 6-10 days at 28 ℃ in the dark.
Inoculating the single colony grown on each separation plate to a slant culture medium for 8 days, inoculating the single colony to a seed culture medium in an inoculation amount of 1%, and culturing at 28 ℃ and 150r/min for 60-72h to obtain a seed solution; inoculating the seed solution to a fermentation culture medium with an inoculation amount of 10%, and culturing at 28 deg.C and 230r/min for 5.5d to obtain a fermentation liquid.
And (3) adding an appropriate amount of anhydrous ethanol into the obtained fermentation liquor, fully oscillating, standing, centrifuging at 4000r/min for 10min, filtering the supernatant with an organic filter membrane, measuring the yield of the lipstatin by using a high performance liquid chromatography, and determining that the product in the fermentation liquor has a correct structure.
By the method, the strain producing the maximum lipstatin is screened, for convenience of description, the screened strain is named as the strain FIM-28-93, and the strain FIM-28-93 is stored in glycerol.
EXAMPLE 3 identification of Strain FIM-28-93
Physiological and biochemical characteristic identification
Streaking the obtained strain FIM-28-93 on a separation culture medium plate, inserting a cover glass, culturing at 28 ℃ for 7-20d, and observing morphological characteristics and hyphae of a single colony by using an optical microscope, a transmission electron microscope and a scanning electron microscope.
The main morphological and physiological and biochemical characteristics of the obtained strain FIM-28-93 are as follows: the colony on the flat plate is circular, the center is ancient pink, the outer part is light pink, the hypha in the substrate is colorless, the brown soluble pigment and the aerial hypha are pink, and a large amount of spores are generated; the gelatin is slowly liquefied, and the milk is firstly solidified and then slowly peptonized; can hydrolyze starch without growing on cellulose; nitrate reduction is weak and D-glucose can be utilized. The strain FIM-28-93 is a high oxygen consumption bacterium, the optimal growth temperature is 26-30 ℃, and the optimal growth pH is 5.5-7.0; when the rotating speed of the shaking table is 210-280r/min and the cultivation lasts for 4.5-7d, the yield of lipstatin is highest, and the dissolved oxygen has an obvious effect on the production of lipstatin in the fermentation process.
Molecular biological identification
Sequencing the 16SrDNA sequence of the strain FIM-28-93, comparing the 16SrDNA sequence of the tested strain with the existing sequence in a GenBank database, and performing homology analysis; selecting a corresponding model strain 16SrDNA gene sequence on an LPSN (http:// www. Bacterial. Cic. Fr.) website, comparing the systematic evolution analysis by CLUSTAL-X software, carrying out the systematic evolution analysis on the generated comparison file by a MEGA software adjacency method, wherein the topological analysis is the result of 1000 repeated sampling, and the evolutionary tree of the strain FIM-28-93 is shown in figure 2. Sequence analysis of 16SrDNA showed that the strain FIM-28-93 has 99.86% sequence homology with Streptomyces toxytricini.
By combining the above morphological, physiological and biochemical characteristics and molecular biological identification, the strain FIM-28-93 is finally determined to be the genus Streptomyces toxytricini (Streptomyces toxytricini), which is classified and named as Streptomyces toxytricini FIM-28-93, is preserved in the Guangdong province microbial strain preservation center, is the experimental building 5 th building of Michelia 100, the China area of Guangzhou, and has the preservation number of GDMCC No.61322, and the preservation date is 12 months and 2 days 2020.
Example 4 mutagenesis of Strain FIM-28-93 to ferment lipstatin
Activation of Strain FIM-28-93: transferring the strain FIM-28-93 preserved by adopting glycerol to a slant culture medium, and culturing for 6-10 days in a constant temperature incubator at the culture temperature of 28 ℃.
Preparing a seed solution: inoculating the activated single colony of the strain FIM-28-93 into seed culture medium (150 mL seed culture medium in 500mL triangular flask), and culturing at 28 deg.C and 150r/min for 60-72 hr to obtain seed solution.
Fermentation culture: the prepared seed solution is inoculated into a fermentation medium (50 mL of the fermentation medium is filled in a 500mL triangular flask) in an inoculation amount of 10% (v/v), fermentation culture is carried out for 136 hours under the conditions of 28 ℃ and 230r/min, and then the obtained fermentation liquid is detected.
The detection result shows that the yields of the lipstatin obtained by three batches of shake flask fermentation are 13036 mu g/mL, 13386 mu g/mL and 13213 mu g/mL respectively; further determining that the purity of the target product in the fermentation broth reaches 85.0%, 86.3% and 87.2% respectively, and the effective components contained in the fermentation broth for synthesizing orlistat are not lower than 91%. Therefore, the screened strain can efficiently ferment lipstatin, and the purity of the target product and the effective substances in the fermentation liquid is better.
Example 5 fermentative production of lipstatin by the mutagenized Strain FIM-28-93
And (3) seed culture in a shaking flask: inoculating the strain FIM-28-93 lawn into seed culture medium (250 mL seed culture medium in 1000mL triangular flask), and culturing at 28 deg.C and 150r/min for 63 hr to obtain shake flask seed solution.
Seed tank seed culture: the seed solution in the shake flask is inoculated into a seed culture medium (60L of the seed culture medium is filled in a 100L tank) according to the amount of 1 percent, and the seed solution is cultured at the culture temperature of 28 ℃, the tank pressure of 0.05MPa and the air flow rate of 1: culturing for 48h under the conditions of 1vvm and the stirring speed of 100-120r/min to obtain seed liquid in a seed tank.
Fermentation culture in a fermentation tank: the prepared seed solution was inoculated into a fermentation medium (1 ton of a tank-packed 500L fermentation medium) at an inoculation amount of 10% (v/v), and the mixture was cultured at a culture temperature of 28 ℃, a tank pressure of 0.05MPa, and an air flow rate of 1:0.8-1.5vvm, the stirring speed is 150-250r/min, the dissolved oxygen is controlled to be not less than 30 percent in the process, the fermentation culture is carried out for 120h, and the obtained fermentation liquor is put into a tank for detection.
The detection result shows that the yield of lipstatin produced by three batches of fermentation is 13539 mug/mL, 13052 mug/mL and 13218 mug/mL respectively; the purity of the target product in the fermentation broth is further determined to be not less than 86.0%, 86.6% and 86.1%, and the purity of the effective component containing the synthetic orlistat is more than 91%.
Example 6 fermentative production of lipstatin by the mutagenized Strain FIM-28-93
And (3) seed culture in a shaking flask: inoculating the strain FIM-28-93 lawn into seed culture medium (250 mL seed culture medium in 1000mL triangular flask), and culturing at 28 deg.C and 150r/min for 63 hr to obtain shake flask seed solution.
Seed tank seed culture: inoculating the seed solution in the shake flask into a seed culture medium (60L of the seed culture medium is filled in a 100L tank) according to the amount of 1%, and culturing for 48h under the conditions that the culture temperature is 28 ℃, the tank pressure is 0.05MPa, the air flow is 1vvm, and the stirring speed is 100-120r/min to obtain the seed solution in the seed tank.
Seed culture in a propagation tank: inoculating seed tank seed solution at 3% amount into seed culture medium (200L seed culture medium in 300L tank), and culturing at 28 deg.C, 0.05MPa, air flow rate of 1 1vvm, and stirring speed of 100-120r/min for 24 hr to obtain seed solution in propagation tank.
Fermentation culture in a fermentation tank: the prepared seed solution was inoculated into a fermentation medium (3 tons of a tank-packed 1500L fermentation medium) at an inoculation amount of 10% (v/v), and the mixture was cultured at a culture temperature of 28 ℃, a tank pressure of 0.05MPa, and an air flow rate of 1:0.8-1.5vvm, the stirring speed is 150-250r/min, the dissolved oxygen is controlled to be not less than 30 percent in the process, the fermentation culture is carried out for 108h, and the obtained fermentation liquor is put into a tank for detection.
The detection result shows that the yield of lipstatin produced by three batches of fermentation is 13201 mug/mL, 13163 mug/mL and 13426 mug/mL respectively; the purity of the target product in the fermentation broth is further determined to be not less than 86.5%, 85.7% and 87.1%, and the purity of the target product and the purity of the effective substances in the fermentation broth are respectively determined to be not less than 91% and the effective components containing the synthetic orlistat are all higher than 91%.
Example 7 genetic stability verification of Strain FIM-28-93
The screened high-yield lipstatin strain FIM-28-93 is subjected to 500mL shake flask continuous culture for 5 generations to detect the genetic stability, and the strain passage fermentation experiment result is as follows:
the high-yield resistant strain FIM-28-93 is continuously passaged for 5 times: f1, F2, F3, F4 and F5, measuring the fermentation titer after shaking flask fermentation, taking a primary strain (F0) with good growth as a control, and obtaining the results shown in Table 1.
TABLE 1 Effect of passages on lipstatin production by FIM-28-93 Strain
| Algebra of strains | F0 | F1 | F2 | F3 | F4 | F5 |
| Relative potency (%) | 100 | 101.1 | 102.8 | 99.6 | 98.5 | 90.8 |
As can be seen from the table 1, the level of fermentation of the screened strain FIM-28-93 in the fourth generation has no obvious influence, the titer of lipstatin produced is basically stable and is maintained at the same higher level, thereby indicating that the strain FIM-28-93 has better genetic stability; the highest lipstatin yield of the target strain, namely the strain FIM-28-93 can reach 13539 mug/mL, the fermentation period of a 3-ton tank is only 108 hours, the lipstatin purity in the fermentation liquid is not lower than 85 percent, the impurity content is effectively controlled, and the influence on the chemical synthesis of orlistat at the later stage is avoided. It is expected that the mutant strain Streptomyces toxytricini FIM-28-93 (Streptomyces toxytricini) screened by the invention tends to have higher fermentation performance and efficiency in a larger fermentation system. As can be seen, the mutant strain Streptomyces toxytricini FIM-28-93 (Streptomyces toxytricini) screened by the invention can be used as a production strain for further research and development.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (6)
1. The Streptomyces toxytricini strain for producing lipstatin through high-efficiency fermentation is characterized in that the Streptomyces toxytricini strain is Streptomyces toxytricini FIM-28-93 which is classified and named as Streptomyces toxytricini and is deposited in Guangdong province microbial culture collection center with the collection number of GDMCC No.61322 and the collection date of 2020, 12 months and 2 days.
2. Use of the streptomycin toxytricin strain of claim 1 for the fermentative production of lipstatin.
3. A method for producing lipstatin by fermentation, which comprises the steps of inoculating the streptomycin toxytricin strain of claim 1 in a slant culture medium for activation, inoculating in a seed culture medium for seed liquid culture, and inoculating in a fermentation culture medium for fermentation culture;
the slant culture medium comprises the following components in percentage by mass: 0.5-1.5% of soluble starch, 0.3-0.8% of oat flour, 0.1-0.3% of yeast powder, 0.05-0.2% of casein, 0.01-0.1% of dipotassium phosphate, 0.03-0.08% of magnesium sulfate, 2.0% of agar and the balance of water, wherein the pH value is 7.2-8.0;
the seed culture medium comprises the following components in percentage by mass: 1-3% of soybean meal, 0.3-0.8% of yeast powder, 1-3% of glycerol and the balance of water, wherein the pH value is 6.8-7.2;
the fermentation medium comprises the following components in percentage by mass: 1-3% of glycerol, 3-5% of sunflower seed oil, 0.5-1.5% of corn flour, 2.5-3% of soybean flour, 0.5-1.5% of peptone, 0.5-1.5% of lecithin, 0.05-0.1% of calcium carbonate and the balance of water, wherein the pH value is 6.5-7.0;
the fermentation time of the fermentation culture step is 120-144h.
4. The process for the fermentative production of a lipstatin according to claim 3, wherein the conditions of the fermentation culture comprise: controlling the rotation speed to be 100-250rpm, and performing fermentation culture at 25-30 ℃.
5. The process for the fermentative production of lipstatin according to claim 3 or 4, wherein the conditions of the seed broth comprise: controlling the rotation speed to be 100-200rpm, and culturing the seed liquid at 25-30 ℃ for 60-72h.
6. The process for the fermentative production of lipstatin according to claim 3 or 4, wherein the conditions of the slant medium activation step comprise: culturing at 25-30 deg.C for 6-10 days.
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|---|---|---|---|---|
| CN103468603A (en) * | 2013-07-17 | 2013-12-25 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces toxytricini and applications of streptomyces toxytricini in preparation of lipstatin |
| CN103555610A (en) * | 2013-09-23 | 2014-02-05 | 南京博优康远生物医药科技有限公司 | Streptomyces toxytricini for high yield of lipstatin and fermentation medium thereof |
| CN108753861A (en) * | 2018-06-08 | 2018-11-06 | 福建省微生物研究所 | A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin |
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2021
- 2021-02-03 CN CN202110150965.9A patent/CN112725238B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468603A (en) * | 2013-07-17 | 2013-12-25 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces toxytricini and applications of streptomyces toxytricini in preparation of lipstatin |
| CN103555610A (en) * | 2013-09-23 | 2014-02-05 | 南京博优康远生物医药科技有限公司 | Streptomyces toxytricini for high yield of lipstatin and fermentation medium thereof |
| CN108753861A (en) * | 2018-06-08 | 2018-11-06 | 福建省微生物研究所 | A kind of culture medium and method of Streptomyces toxytricini fermentation high yield Lipstatin |
Non-Patent Citations (1)
| Title |
|---|
| 利普司他汀产生菌的菌种选育及培养基优化;刘顺娜等;《中国医药工业杂志》;20170724;第48卷(第07期);第1006-1011页，摘要 * |
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