CN108048512B - Fermentation method for preparing pneumocandin B0 - Google Patents
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Abstract
The invention discloses a fermentation method for preparing pneumocandin B0, which comprises the following steps: inoculating a strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid; inoculating the bacteria source liquid into a fermentation tank filled with a second culture medium; pumping sterile air into the second culture medium through the aeration pipe; after fermenting for 24 hours, pumping sterile air containing ammonia gas into the second culture medium through an aeration pipe; after fermentation for 60h, mannitol was added to the second medium by flowing through a microbial culture plate inserted in the second medium; after fermentation for 72h, pneumocandin B0 in the fermentor was collected. The technical scheme provided by the invention can reduce the viscosity of the bacterial liquid and obtain high yield of the pneumocandin B0.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a fermentation method for preparing pneumocandin B0.
Background
Pneumocandin B0, also known as pneumocandin B0The neomycin-B0 is mainly used for synthesizing medicine caspofungin, which is a semi-synthetic lipopeptide (echinocandin) compound and can inhibit the synthesis of beta (1,3) -D-glucan which is a basic component of cell walls of a plurality of filamentous fungi and yeasts, so that the cell walls of the fungi are cracked and lose integrity, the osmotic pressure inside and outside the cells is changed, and the antifungal effect is finally exerted.
In addition to pneumocandin B0, A is produced during the fermentation of Glarea lozoyensis0、C0The method is a key for industrialization of preparing the pneumocandin B0 by using equal components and how to rapidly and efficiently culture the Glarea lozoyensis and simultaneously maximize the content proportion of pneumocandin B0 in the generated secondary metabolite.
In the fermentation process of Glarea lozoyensis, the concentration of bacterial liquid is continuously increased along with the continuous accumulation of filamentous fungi in the fermentation process of the filamentous fungi, so that the fungi and nutrient substances cannot be fully contacted or part of harmful metabolites are excessively accumulated, and the yield is influenced.
Therefore, it is necessary to provide a fermentation method with high culture efficiency and high yield of pneumocandin B0.
Content of application
The invention aims to solve the technical problem that the existing fermentation method for preparing pneumocandin B0 is influenced by the high concentration of the gleanea zoyensis zymocyte liquid of the mould to overcome the defects and shortcomings in the background technology, and the technical problem of influencing the yield of pneumocandin B0 is solved.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a fermentation method for preparing pneumocandin B0 comprises the following steps:
step one, inoculating a strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid;
inoculating the bacterial source liquid prepared in the step one into a fermentation tank filled with a second culture medium for fermentation, wherein the volume ratio of the bacterial source liquid to the second culture medium is 3-5: 100, pumping sterile air into the second culture medium, controlling the dissolved oxygen in the second culture medium to be not less than 45%, the pH value to be 5.0-5.4, the ventilation volume to be 0.5-1.0 VVM and the tank pressure to be 0.04-0.06 Mpa;
thirdly, after the bacterial source liquid prepared in the first step is inoculated into a fermentation tank filled with a second culture medium and fermented for 24 hours, pumping ammonia gas into the second culture medium, and controlling the pH value to be 5.0-5.4;
step four, after the bacterial source liquid prepared in the step one is inoculated in a fermentation tank filled with a second culture medium and fermented for 60 hours, feeding mannitol into the second culture medium through a microorganism culture plate inserted into the second culture medium at a feeding speed of 1.5-2.5%/day so that the total sugar concentration of the fermentation liquid is controlled to be 1-3%, wherein the microorganism culture plate is provided with an electrode for preventing thalli from being accumulated on the surface of the microorganism culture plate;
and fifthly, inoculating the bacteria source liquid prepared in the first step into a fermentation tank filled with a second culture medium, fermenting for 13-15 days, and collecting pneumocandin B0 in the fermentation tank.
Preferably, the first step specifically includes:
inoculating the strain Glarea lozoyensis into a 250ml shake flask filled with 40-50 ml of a first culture medium, and culturing for 2-3 days on a shaking table at 180-260 rpm to obtain a strain source liquid.
Preferably, the first culture medium comprises the following components in percentage by mass: 25-35% of mannitol, 13-18% of cottonseed meal cake, 8-12% of glucose and KH2PO40.15-0.25%, and the balance of water, wherein the pH value is 5.5-6.0.
Preferably, the second culture medium in the second step comprises the following components in percentage by mass: 8-12% of mannitol, 2-4% of glucose, 2-4% of peptone, 0.5-1.5% of proline, 0.1-0.25% of inorganic salt and the balance of water, wherein the pH value is 5.0-5.4.
Preferably, the inorganic salt is K2HPO4、MnSO4·4H2O、NaCl、Fe(NH4)2(SO4)2·6H2O、NaNO3、KNO3、MgSO4·7H2O、FeSO4·7H2O、KH2PO4Any one or more of them.
Preferably, the fermentation tank comprises a tank body, a plurality of microorganism culture plates inserted into the tank body, a stirring sheet spirally arranged along the inner wall of the tank body, an aeration pipe arranged at the bottom of the microorganism culture plates, and an air pump arranged outside the tank body, wherein the aeration pipe is provided with a through hole for communicating the aeration pipe with the external space, the air pump is used for pumping sterile air into the second culture medium in the fermentation tank through the aeration pipe, and the stirring sheet and the tank body can rotate relatively.
Preferably, the microorganism culture plate comprises a framework, a first membrane layer and a second membrane layer, wherein the edges of the first membrane layer and the second membrane layer are respectively sealed with the framework to form an accommodating space for accommodating a culture solution, the electrodes are arranged in the accommodating space in a reciprocating bending and extending mode, the two opposite sides of the framework are provided with an inlet and an outlet which are communicated with the accommodating space, the first membrane layer and the second membrane layer are respectively provided with micropores which are suitable for the culture solution to permeate from the accommodating space to the outer space, and the pore diameter of each micropore is smaller than the size of a microorganism cultured in the outer space so as to prevent the microorganism in the outer space from entering the accommodating space.
Preferably, first rete with the second rete is wavy, first rete with the second rete forms a plurality of recesses respectively, the skeleton is including being equipped with the topside of import, with the relative base that sets up of topside, connection the first side and the second side of topside and base, the recess is followed first side to the second side direction extends.
Preferably, the electrode includes a bending portion and a linear portion connected to each other, the linear portion extends along the groove on the first film layer, and the micro-hole is disposed opposite to the linear portion.
Preferably, the fermentation temperature in the fermentation method is 22-26 ℃.
Compared with the prior art, the invention has the advantages that: through detection, in the fermentation method of the pneumocandin B0, the fermentation unit of the pneumocandin B0 can reach more than 2500 mu g/mL, and meanwhile, the fermentation byproducts are relatively less, and the difficulty of post-extraction is reduced, so that the method is beneficial to obtaining high-quality caspofungin through subsequent processing.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic perspective view of a microorganism culture plate according to the present invention;
FIG. 2 is a schematic cross-sectional view of a microorganism culture plate according to the present invention;
FIG. 3 is a schematic flow chart of a fermentation method of pneumocandin B0 provided by the invention.
Detailed Description
In order to facilitate an understanding of the invention, the invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Unless otherwise specifically stated, the species Glarea lozoyensis of the present invention was purchased from Shanghai enzyme research Biotech Co., Ltd, and pneumocandin B0 and pneumocandin B of the present invention0Are the same substance.
The present invention provides a fermenter (not shown). The fermentation tank comprises a tank body (not shown), a plurality of microorganism culture plates 100 inserted into the tank body, a stirring sheet (not shown) spirally arranged along the inner wall of the tank body, an aeration pipe 1 arranged at the bottom of the microorganism culture plates 100 and an air pump arranged outside the tank body, wherein the aeration pipe 1 is provided with a through hole 11 for communicating the aeration pipe 1 with the external space, the air pump is used for pumping sterile air into a second culture medium in the fermentation tank through the aeration pipe 1, and the stirring sheet can rotate relative to the tank body.
The microorganism culture plate 100 comprises a framework 3, a first film layer 4 and a second film layer 5, wherein the edges of the first film layer 4 and the second film layer 5 are respectively sealed with the framework 3 to form an accommodating space 2 for accommodating mannitol, the electrodes 6 are arranged in the accommodating space 2 in a reciprocating bending and extending manner, the two opposite sides of the framework 3 are provided with an inlet 21 and an outlet 22 which are communicated with the accommodating space 2, the first film layer 4 and the second film layer 5 are respectively provided with micropores suitable for the mannitol to permeate from the accommodating space 2 to the second culture solution, and the pore diameter of the micropores is smaller than the size of the thallus of the Glarea lozoyensis so as to prevent the thallus of the Glarea lozoyensis from entering the accommodating space 2.
By arranging the framework 3, the first film layer 4 and the second film layer 5, the mannitol can permeate out through the first film layer 4 and the second film layer 5, so that the utilization rate of the mannitol is increased, the energy consumption of a system is reduced, and the culture cost is saved; by arranging the electrode 6, under the action of a high-frequency alternating electric field, negative dielectrophoresis force can be utilized to act on a culture solution, so that the thallus of the Glarea lozoyensis close to the first film layer 4 and the second film layer 5 is repelled from the surface of the film, thereby preventing the thallus of the Glarea lozoyensis from gathering on the first film layer 4 and the second film layer 5 and reducing the possibility of micropore blockage.
The electrode 6 comprises a bending part 61 and a linear part 62 which are connected, the linear part 62 extends along the groove 41 on the first film layer 4, and the micropores are arranged opposite to the linear part 62. Further preventing the aggregation of the cells of Glarea lozoyensis before the microwells.
Example one
The invention provides a fermentation method for preparing pneumocandin B0 based on the fermentation tank introduction, which comprises the following steps:
step S1, inoculating the strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid;
specifically, the strain Glarea lozoyensis is inoculated into a 250ml shake flask filled with 40-50 ml of a first culture medium, and cultured on a shaking table at 180-260 rpm for 2-3 days to obtain a strain source liquid.
The first culture medium comprises the following components in percentage by mass: 25-35% of mannitol and cotton13-18% of seed powder cake, 8-12% of glucose and KH2PO40.1-0.25%, and the balance of water, wherein the pH value is 5.5-6.0. In this example, 20% mannitol, 15% cottonseed meal cake, 8% glucose and KH are used2PO41.5～2.5％。
S2, inoculating the bacterial source liquid prepared in the step S1 into a fermentation tank filled with a second culture medium for fermentation, wherein the volume ratio of the bacterial source liquid to the second culture medium is 3-5: 100, pumping sterile air into the second culture medium, controlling the dissolved oxygen content in the second culture medium to be not less than 45%, the pH value to be 5.0-5.40, the ventilation volume to be 0.5-1.0 VVM and the tank pressure to be 0.04-0.06 Mpa;
specifically, the fermentation temperature in the fermentation method is 22-26 ℃. The second culture medium in the second step comprises the following components in percentage by mass: 8-12% of mannitol, 2-4% of glucose, 2-4% of peptone, 0.5-1.5% of proline, 0.1-0.25% of inorganic salt and the balance of water, wherein the pH value is 5.0-5.4. By adding proline as a precursor, the amount of pneumocandin B0 in the secondary metabolite was increased. By adopting mannitol and glucose as carbon sources, the content of pneumocandin B0 in secondary metabolites is increased, meanwhile, the higher metabolic rate of thalli is maintained, and the fermentation period is reduced.
The inorganic salt K2HPO4、MnSO4·4H2O、NaCl、Fe(NH4)2(SO4)2·6H2O、NaNO3、KNO3、MgSO4·7H2O、FeSO4·7H2O、KH2PO4Any one or more of them.
Air is pumped into the second medium through an aeration tube to control aeration and tank pressure.
In this example, mannitol 10%, glucose 3%, peptone 3%, proline 1%, and K were used2HPO4And MnSO4·4H2O is 0.7 percent respectively.
S3, after the bacteria source liquid prepared in the S1 is inoculated into a fermentation tank filled with a second culture medium and fermented for 24 hours, pumping sterile air containing ammonia gas into the second culture medium, and controlling the pH value to be 5.0-5.4;
preferably, the pH is 5.3.
Step S4, after the bacteria-derived liquid prepared in the step S1 is inoculated into a fermentation tank filled with a second culture medium and fermented for 60 hours, mannitol is added into the second culture medium through a microorganism culture plate inserted into the second culture medium in a flowing mode, so that the mass concentration of total sugar in the fermentation liquid is controlled to be 1-3%, and the microorganism culture plate is provided with an electrode for preventing bacteria from being accumulated on the surface of the microorganism culture plate;
and S5, inoculating the bacteria source liquid prepared in the step S1 into a fermentation tank filled with a second culture medium, fermenting for 13-15 days, and collecting pneumocandin B0 in the fermentation tank.
In this example, the amount of pneumocandin B0 collected in the fermentation broth after 13d was 2641 mg/L.
Example two
The invention provides a fermentation method for preparing pneumocandin B0 based on the fermentation tank introduction, which comprises the following steps:
step S1, inoculating the strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid;
specifically, the strain Glarea lozoyensis is inoculated into a 250ml shake flask filled with 40-50 ml of a first culture medium, and cultured on a shaking table at 180-260 rpm for 2-3 days to obtain a strain source liquid.
The first culture medium comprises the following components in percentage by mass: 25% of mannitol, 13% of cottonseed meal cake, 8% of glucose and KH2PO40.1 percent, the balance being water, and the pH value being 5.5-6.0.
S2, inoculating the bacterial source liquid prepared in the step S1 into a fermentation tank filled with a second culture medium for fermentation, wherein the volume ratio of the bacterial source liquid to the second culture medium is 3:100, pumping sterile air into the second culture medium, controlling the dissolved oxygen in the second culture medium to be not less than 45%, the pH value to be 5.0-5.40, the ventilation volume to be 0.5-1.0 VVM and the tank pressure to be 0.04-0.06 Mpa;
specifically, the fermentation temperature in the fermentation process is 24 ℃. The second culture medium in the second step comprises the following components in percentage by mass: 8% of mannitol, 2% of glucose, 2% of peptone, 0.5% of proline, 0.1% of inorganic salt and the balance of water, and the pH value is 5.0. By adding proline as a precursor, the amount of pneumocandin B0 in the secondary metabolite was increased. By adopting mannitol and glucose as carbon sources, the content of pneumocandin B0 in secondary metabolites is increased, meanwhile, the higher metabolic rate of thalli is maintained, and the fermentation period is reduced.
The inorganic salt is K2HPO4、MnSO4·4H2O and NaCl.
Step S3, after the bacteria source liquid prepared in the step S1 is inoculated in a fermentation tank filled with a second culture medium and fermented for 24 hours, pumping sterile air containing ammonia gas into the second culture medium, and controlling the pH value to be 5.0;
step S4, after the bacteria-derived liquid prepared in the step S1 is inoculated into a fermentation tank filled with a second culture medium and fermented for 60 hours, mannitol is added into the second culture medium through a microorganism culture plate inserted into the second culture medium in a flowing mode, so that the mass concentration of total sugar in the fermentation liquid is controlled to be 1-3%, and the microorganism culture plate is provided with an electrode for preventing bacteria from being accumulated on the surface of the microorganism culture plate;
step S5, after the strain source liquid prepared in the step S1 is inoculated in a fermentation tank filled with a second culture medium and fermented for 13d, collecting pneumocandin B0 in the fermentation tank.
In this example, the amount of pneumocandin B0 collected in the fermentation broth after 13d was 2618 mg/L.
EXAMPLE III
The invention provides a fermentation method for preparing pneumocandin B0 based on the fermentation tank introduction, which comprises the following steps:
step S1, inoculating the strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid;
specifically, the strain Glarea lozoyensis is inoculated into a 250ml shake flask filled with 40-50 ml of a first culture medium, and cultured on a shaking table at 180-260 rpm for 2-3 days to obtain a strain source liquid.
The first culture medium comprises the following components in percentage by mass: 35% of mannitol, 18% of cottonseed meal cake, 12% of glucose and KH2PO40.25 percent, the balance being water, and the pH value being 6.0. In this example, 20% mannitol, 15% cottonseed meal cake, 8% glucose and KH are used2PO41.5～2.5％。
S2, inoculating the bacterial source liquid prepared in the step S1 into a fermentation tank filled with a second culture medium for fermentation, wherein the volume ratio of the bacterial source liquid to the second culture medium is 5:100, pumping sterile air into the second culture medium, controlling the dissolved oxygen in the second culture medium to be not less than 45%, the pH value to be 5.0-5.40, the ventilation volume to be 0.5-1.0 VVM and the tank pressure to be 0.04-0.06 Mpa;
specifically, the fermentation temperature in the fermentation process is 26 ℃. The second culture medium in the second step comprises the following components in percentage by mass: 12% of mannitol, 4% of glucose, 4% of peptone, 1.5% of proline, 0.25% of inorganic salt and the balance of water, and the pH value is 5.4. By adding proline as a precursor, the amount of pneumocandin B0 in the secondary metabolite was increased. By adopting mannitol and glucose as carbon sources, the content of pneumocandin B0 in secondary metabolites is increased, meanwhile, the higher metabolic rate of thalli is maintained, and the fermentation period is reduced.
The inorganic salt is Fe (NH)4)2(SO4)2·6H2O、NaNO3And KNO3。
Step S3, after the bacteria source liquid prepared in the step S1 is inoculated in a fermentation tank filled with a second culture medium and fermented for 24 hours, pumping sterile air containing ammonia gas into the second culture medium, and controlling the pH value to be 5.4;
step S4, after the bacteria-derived liquid prepared in the step S1 is inoculated into a fermentation tank filled with a second culture medium and fermented for 60 hours, mannitol is added into the second culture medium through a microorganism culture plate inserted into the second culture medium in a flowing mode, so that the mass concentration of total sugar in the fermentation liquid is controlled to be 1-3%, and the microorganism culture plate is provided with an electrode for preventing bacteria from being accumulated on the surface of the microorganism culture plate;
and S5, inoculating the bacteria source liquid prepared in the step S1 into a fermentation tank filled with a second culture medium, fermenting for 13-15 days, and collecting pneumocandin B0 in the fermentation tank.
In this example, the amount of pneumocandin B0 collected in the fermentation broth after 13d was 2580 mg/L.
Claims (9)
1. A fermentation method for preparing pneumocandin B0 is characterized by comprising the following steps:
step one, inoculating a strain Glarea lozoyensis into a shake flask filled with a first culture medium, and culturing to obtain a strain source liquid;
inoculating the bacterial source liquid prepared in the step one into a fermentation tank filled with a second culture medium for fermentation to obtain fermentation liquid, wherein the volume ratio of the bacterial source liquid to the second culture medium is 3-5: 100, pumping sterile air into the second culture medium, controlling the dissolved oxygen in the second culture medium to be not less than 45%, the pH value to be 5.0-5.4, the ventilation volume to be 0.5-1.0 VVM and the tank pressure to be 0.04-0.06 Mpa;
thirdly, after the bacterial source liquid prepared in the first step is inoculated into a fermentation tank filled with a second culture medium and fermented for 24 hours, pumping ammonia gas into the fermentation liquid, and controlling the pH value to be 5.0-5.4;
step four, after the bacterial source liquid prepared in the step one is inoculated in a fermentation tank filled with a second culture medium and fermented for 60 hours, feeding mannitol into the fermentation liquid through a microbial culture plate inserted into the second culture medium, so that the total sugar mass concentration of the fermentation liquid is controlled to be 1-3%, wherein the microbial culture plate is provided with an electrode for preventing thalli from being accumulated on the surface of the microbial culture plate;
fifthly, after the bacteria source liquid prepared in the first step is inoculated into a fermentation tank filled with a second culture medium and fermented for 13-15 days, collecting pneumocandin B0 in the fermentation liquid;
the fermentation tank comprises a tank body and a plurality of microorganism culture plates inserted into the tank body, wherein each microorganism culture plate comprises a framework, a first membrane layer and a second membrane layer, the edges of the first membrane layer and the second membrane layer are respectively sealed with the framework and enclose an accommodating space for accommodating mannitol in the step four, the electrodes are arranged in the accommodating space in a reciprocating bending and extending mode, micropores suitable for allowing the mannitol to permeate from the accommodating space to fermentation liquor are respectively arranged on the first membrane layer and the second membrane layer, and the pore diameter of each micropore is smaller than the size of thallus of the Glarea lozoyensis; the fermentation tank further comprises a stirring sheet spirally arranged along the inner wall of the tank body, an aerator pipe arranged at the bottom of the microorganism culture plate and an air pump arranged on the outer side of the tank body, wherein a through hole communicated with the aerator pipe and the external space is formed in the aerator pipe, the air pump is used for pumping sterile air into a second culture medium in the fermentation tank through the aerator pipe, and the stirring sheet and the tank body can rotate relatively.
2. The fermentation method for preparing pneumocandin B0 according to claim 1, wherein the first step specifically comprises:
inoculating the strain Glaarloftyensis into a 250ml shake flask filled with 40-50 ml of a first culture medium, and culturing for 2-3 days on a shaking table at 180-260 rpm to obtain a strain source liquid.
3. The fermentation process for preparing pneumocandin B0 according to claim 1, wherein the first culture medium comprises the following components in percentage by mass: 25-35% of mannitol, 13-18% of cottonseed meal cake, 8-12% of glucose and KH2PO40.1-0.25%, and the balance of water, wherein the pH value is 5.5-6.0.
4. The fermentation process for preparing pneumocandin B0 according to claim 1, wherein the second culture medium in the second step comprises the following components in percentage by mass: 8-12% of mannitol, 2-4% of glucose, 2-4% of peptone, 0.5-1.5% of proline, 0.1-0.25% of inorganic salt and the balance of water, wherein the pH value is 5.0-5.4.
5. The fermentation process for the preparation of pneumocandin B0 of claim 4, wherein the inorganic salt is K2HPO4、MnSO4·4H2O、NaCl、Fe(NH4)2(SO4)2·6H2O、NaNO3、KNO3、MgSO4·7H2O、FeSO4·7H2O、KH2PO4Any one or more of them.
6. The fermentation method for preparing pneumocandin B0, according to claim 1, wherein the frame has an inlet and an outlet on opposite sides for communicating the receiving space.
7. The fermentation process of claim 6, wherein the first membrane layer and the second membrane layer are corrugated, the first membrane layer and the second membrane layer each define a plurality of grooves, the frame includes a top edge defining the inlet, a bottom edge disposed opposite the top edge, and first and second side edges connecting the top and bottom edges, the grooves extending along the first side edge in a direction toward the second side edge.
8. The fermentation process of claim 7 for preparing nemocardin B0, wherein the electrode comprises a bent portion and a straight portion connected, the straight portion extending along a groove on the first membrane layer, the micro-hole being disposed opposite the straight portion.
9. The fermentation method for preparing pneumocandin B0 according to any one of claims 1 to 8, wherein the fermentation temperature in the fermentation method is 22-26 ℃.
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| CN103087928B (en) * | 2013-01-25 | 2014-09-17 | 杭州华东医药集团新药研究院有限公司 | Fungus Glarea lozoyensis and application thereof for controlling microbial metabolite Pneumocandin Kangding B0 |
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| CN104531535B (en) * | 2014-10-17 | 2017-08-04 | 南京工业大学 | One kind produces knob not Kangding B0Genetic recombination bacterial strain and selection and application |
| CN105061699A (en) * | 2015-08-06 | 2015-11-18 | 石河子大学 | Preparation method of surface molecularly imprinted polymer of PN (pneumocandins)B0 |
| CN205473735U (en) * | 2015-12-29 | 2016-08-17 | 恒盛高科（天津）生物技术有限公司 | Aeration type medicine fermentation cylinder |
| CN106755223A (en) * | 2017-01-20 | 2017-05-31 | 信泰制药（苏州）有限公司 | A kind of fermentation process of Pneumocandin B0 |
| CN106755224B (en) * | 2017-01-20 | 2017-12-22 | 信泰制药（苏州）有限公司 | The fermentation process of Caspofungin fermentation intermediate |
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