WO2023030496A1 - 一种发酵制备纽莫康定b0的方法 - Google Patents

一种发酵制备纽莫康定b0的方法 Download PDF

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WO2023030496A1
WO2023030496A1 PCT/CN2022/116810 CN2022116810W WO2023030496A1 WO 2023030496 A1 WO2023030496 A1 WO 2023030496A1 CN 2022116810 W CN2022116810 W CN 2022116810W WO 2023030496 A1 WO2023030496 A1 WO 2023030496A1
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fermentation
day
added
volume
mannitol
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侯宏波
张宇
江乾森
葛倩倩
张茂华
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杭州中美华东制药有限公司
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    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • the invention belongs to the field of microbial fermentation, and in particular relates to a preparation method of caspofungin intermediate pneumocontin B0 by microbial fermentation.
  • Caspofungin acetate is a lipopeptide antifungal drug developed by Merck Sharp & Dohme (MSD). In 2001, caspofungin was approved by the FDA for listing. Caspofungin is an antifungal drug that destroys the integrity of fungal cell walls by inhibiting (1,3)- ⁇ -D-glucan synthase, and is suitable for the treatment of patients who are ineffective or intolerant to other treatments Invasive aspergillosis. As a representative of a new class of echinocandin antibacterial drugs, caspofungin has obvious advantages in high selectivity, good antibacterial activity, high safety, and less drug resistance. It has become a research hotspot of systemic antifungal drugs.
  • the echinocandin-like compound of Nimocontin was discovered and named by scientists from Merck & Co.
  • the production strain is a filamentous fungus isolated from a pond near the Lozoa River in Madrid, Spain, and belongs to a new genus of asexual reproduction .
  • pneumocidine B0 is currently the most important research object for optimization.
  • improving its unit is a research hotspot.
  • CN108265096A provides a method for efficient and stable production of pneumocantine B0, through the liquid submerged fermentation of the fungus Glarea lozoyensis, the seed medium and fermentation medium contain suitable carbon sources, nitrogen sources, inorganic salts and trace element sources, wherein The fermentation medium uses sorbitol as the main carbon source, and glucose is added to form a composite carbon source. This method can be stably produced on an industrial-grade fermenter, and the yield of pneumocidine B0 reaches 2.5g/L.
  • CN106755224B relates to a fermentation method of a caspofungin fermentation intermediate.
  • the method adopts a dilute formula medium and adds lactose and ammonia water during the fermentation process, which can effectively reduce the viscosity of the bacteria, improve the aeration effect, and strictly control the pH during the fermentation process.
  • vitamin b5 is supplemented in the fermentation broth, and this method can increase the fermentation unit to 2000-2500mg/L.
  • CN108048512A discloses a fermentation method for preparing pneumocantine B0, inoculating the strain Glarea lozoyensis in a shaker flask equipped with a first medium, and culturing to obtain a bacterial source solution; inoculating the bacterial source solution in a shaker flask equipped with a second medium In the fermenter; pump sterile air into the second culture medium through the aeration pipe; after fermentation to 24h, pump the sterile air containing ammonia through the aeration pipe in the second culture medium; ferment to After 60 hours, mannitol was added to the second medium through the microbial culture plate inserted into the second medium; after 72 hours of fermentation, the pneumocidine B0 in the fermenter was collected.
  • the examples show that the amount of nemocontin B0 collected in the fermented broth after 13 days was 2580 mg/L.
  • the present invention provides a method for preparing pneumocidine B0 by fermentation.
  • a method for fermenting and preparing pneumocidine B0 comprising adding one or any combination of lactic acid, mannitol or gamma-aminobutyric acid during the fermentation process.
  • a method for preparing pneumocidine B0 by fermentation includes adding lactic acid during the fermentation process, the amount of lactic acid added is 0%-0.3% (excluding 0%) of the volume of the fermentation broth, preferably adding 0.1% lactic acid in liquid volume; specifically, 0%-0.3% (V/V) lactic acid is added at one time on the 4th to 7th day after the start of fermentation, preferably 0% of the fermented liquid volume is added at one time on the 6th day of fermentation culture %-0.3% (excluding 0%) lactic acid, more preferably, 0.1% lactic acid of the fermentation broth volume is added at one time on the sixth day of the fermentation culture.
  • a method for fermentatively preparing pneumocantine B0 comprising adding 1%-2.5% (V/V) mannitol once a day on day 3-6 after the initiation of fermentation culture until Fermentation ends; preferably on the 5th day, 1%-2.5% mannitol in the volume of the fermentation broth is added until the end of the fermentation; more preferably, 2% mannitol in the volume of the fermentation broth is added once a day from the 5th day until the end of the fermentation.
  • a method for preparing pneumocantine B0 by fermentation comprising adding gamma-aminobutyric acid in a volume of 0%-0.05% (excluding 0%) of the fermentation broth at one time on the 4th to 7th day after the start of fermentation culture ;
  • 0%-0.05% (excluding 0%) ⁇ -aminobutyric acid is added at one time on the 6th day; more preferably, 0.03% (V/V) ⁇ -aminobutyric acid is added at one time on the 6th day.
  • the present invention provides a method for fermenting and preparing pneumocantine B0, the method comprising adding 0%-0.3% (excluding 0%) of the fermented broth volume at one time on the 4th to 7th day after the start of fermentation Lactic acid, 0%-0.05% (excluding 0%) gamma-aminobutyric acid, more preferably, on the sixth day of fermentation culture, add 0%-0.3% lactic acid, 0%-0.05 % gamma-aminobutyric acid, most preferably, add 0.1% lactic acid and 0.03% gamma-aminobutyric acid to the fermented broth at one time on the 6th day of fermentation; From 3-6 days, add 1%-2.5% mannitol of the fermentation broth volume once a day until the end of fermentation, preferably from the 5th day after the start of fermentation culture, add 1%-2.5% mannitol once a day, Until the end of the fermentation, more preferably, 2% mannitol of the volume
  • the method for preparing pneumocidine B0 by fermentation according to the present invention includes adding one or any combination of lactic acid, mannitol or gamma-aminobutyric acid during the fermentation process, wherein adding lactic acid, mannitol or gamma-aminobutyric acid When any two or three kinds of butyric acid are used, the amount and method of adding the added substances are the same as when adding a single substance.
  • the bacterial strain used in the fermentation of the present invention is Glarea lozoyensis or its mutagenic strain, preferably Glarea lozoyensis FIM2006071, which is preserved by the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M2012475 and a preservation date of November 23, 2012 .
  • the fermentation medium for implementing the invention comprises a carbon source selected from one or more of mannitol, sorbitol, sucrose, and glucose;
  • the fermentation medium also includes a nitrogen source, which is selected from one or more of cottonseed powder, corn gluten powder, yeast extract powder, yeast powder, and bean cake powder;
  • the fermentation medium also includes trace elements selected from one or more of dipotassium hydrogen phosphate, ferrous sulfate, and magnesium sulfate;
  • the fermentation medium also contains amino acids, and the amino acids are one or more of proline and threonine.
  • the fermentation medium includes mannitol, glucose, bean cake powder, magnesium sulfate, proline, dipotassium hydrogen phosphate, and ferrous sulfate;
  • the fermentation medium contains mannitol 5%-14%, glucose 1%-4%, bean cake powder 1%-3.5%, magnesium sulfate 0.01%-0.2%, proline 0%-3% , Dipotassium hydrogen phosphate 0.25%-1.5%, ferrous sulfate 0.01%-0.2%.
  • the method for fermenting and preparing pneumocidine B0 comprises inoculating seed liquid into a fermenter, cultivating the fermented liquid at 24-26° C., tank pressure at 0.05Mpa-0.1Mpa, and maintaining dissolved oxygen at 30%-40% during the fermentation process.
  • the present invention also provides a method for preparing caspofungin, the preparation method comprising using the above method for preparing pneumocidine B0 to obtain piumocidine B0, and then further preparing caspofungin from pneumocidine B0.
  • the invention provides a stable and efficient method for producing pneumocantine B0 with feeding in the fermentation process, by adding one or more of lactic acid, mannitol or gamma-aminobutyric acid in the fermentation process, can significantly Improve the fermentation level of pneumocidine B0, wherein the optimal combination can increase the fermentation level of pneumocidine B0 to 4000-4500mg/L, which is much higher than the fermentation level of about 2500mg/L disclosed in the prior art, greatly improving the industrial The potency level of pneumocidine B0 is prepared, and the industrial production cost thereof is reduced.
  • Figure 1 is the effect of different amounts of lactic acid added on the fermentation titer of pneumocidine B0.
  • Figure 2 is the effect of lactic acid added at different times on the fermentation titer of pneumocidine B0.
  • Fig. 3 is the effect of different addition amounts of ⁇ -aminobutyric acid on the fermentation titer of pneumocidine B0.
  • Figure 4 is the effect of adding ⁇ -aminobutyric acid at different times on the fermentation titer of pneumocidine B0.
  • Figure 5 is the effect of adding different amounts of mannitol at different times on the fermentation titer of pneumocidine B0.
  • the reagents and instruments used are commonly used reagents and instruments in the art, which can be obtained commercially; the methods used are conventional methods in the art, and those skilled in the art will The content can know how to specifically implement the method and achieve the corresponding result.
  • the bacterial strain used in the examples is Glarea lozoyensis FIM2006071, which is preserved by the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M2012475 and a preservation date of: November 23, 2012.
  • the preparation and characteristics of the strain are recorded in Chinese patent 201310032896.7.
  • the content of the medium components is calculated based on the volume ratio of the total fermentation broth, for example, 8% mannitol means that 8g of mannitol is contained in every 100mL fermentation broth.
  • Embodiment 1 The investigation of lactic acid addition amount
  • the shake flask seed medium formula is: 4% glucose, 2% bean cake powder, 1% cottonseed powder, 0.1% potassium dihydrogen phosphate, 1% solid corn liquor, 1% trace element solution (FeSO 4 7H 2 O 1.0 g/L MnSO 4 4H 2 O 1.0g/L ZnSO 4 7H 2 O 0.2g/L CaCl 2 2H 2 O 0.1g/L CuCl 2 2H 2 O 0.1g/L H 3 BO 3 0.056g/L (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.02g/L), drinking water is prepared and sterilized with a sterilizer at 121°C for 30 minutes.
  • the formula of fermentation shake flask medium mannitol 8%, glucose 2%, bean cake powder 2%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.25%, ferrous sulfate 0.1%, proline 1%, foam enemy 0.15% , After the drinking water is prepared, use a sterilizer to sterilize at 121°C for 30 minutes.
  • Figure 1 shows that adding 0.05%-0.3% lactic acid on the 6th day after the start of fermentation culture can significantly increase the potency level of pneumocidine B0, and the titer of pneumocidine B0 has the largest increase when the concentration is around 0.1% It was 3421mg/L, compared with the titer level of 2514mg/L without adding lactic acid, an increase of 36%.
  • Embodiment 2 The investigation of lactic acid addition time
  • the shake flask seed medium formula is: 4% glucose, 2% bean cake powder, 1% cottonseed powder, 0.1% potassium dihydrogen phosphate, 1% solid corn liquor, 1% trace element solution (FeSO 4 7H 2 O 1.0 g/L MnSO 4 4H 2 O 1.0g/L ZnSO 4 7H 2 O 0.2g/L CaCl 2 2H 2 O 0.1g/L CuCl 2 2H 2 O 0.1g/L H 3 BO 3 0.056g/L (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.02g/L), drinking water is prepared and sterilized with a sterilizer at 121°C for 30 minutes.
  • the formula of fermentation shake flask medium mannitol 8%, glucose 2%, bean cake powder 2%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.25%, ferrous sulfate 0.1%, proline 1%, foam enemy 0.15% , After the drinking water is prepared, use a sterilizer to sterilize at 121°C for 30 minutes.
  • Figure 2 shows that one-time addition of a certain amount of lactic acid on the 4th to 7th day after the start of fermentation culture can significantly increase the titer level of pneumocidine B0, and the titer of pneumocidine B0 is the largest when added on the 6th day .
  • Fermentation shake flask culture Inoculate 5% shake flask seed liquid into the fermentation shake flask, control the temperature at 25°C, and the constant temperature culture shaker rotates at 250rpm, cultivate for 12 days, and add 0% of the volume of the fermentation broth at one time on the sixth day of fermentation culture , 0.01, 0.03, 0.05% of ⁇ -aminobutyric acid.
  • Fermentation shake flask culture Inoculate 5% shake flask seed liquid into the fermentation shake flask, control the temperature at 25°C, and the constant temperature culture shaker rotates at 250rpm, and cultivate for 12 days. Add ⁇ -aminobutyric acid with a volume of 0.03 of the fermentation broth to the shake flasks at different times.
  • Fermentation shake flask culture Inoculate 5% shake flask seed liquid into the fermentation shake flask, control the temperature at 25°C, and the constant temperature culture shaker speed is 250rpm, and cultivate for 12 days, starting from the 3rd, 4th, 5th and 6th day of fermentation culture respectively Add 1%, 1.5%, 2%, 2.5% mannitol until the end of fermentation.
  • Embodiment 6 enlarged production
  • the first-class shake flask seed medium formula is: 4% glucose, 2% bean cake powder, 1% cottonseed powder, 0.1% potassium dihydrogen phosphate, 1% solid corn steep liquor, and 1% trace element solution (FeSO 4 7H 2 O 1.0g/L MnSO 4 4H 2 O 1.0g/L ZnSO 4 7H 2 O 0.2g/L CaCl 2 2H 2 O 0.1g/L CuCl 2 2H 2 O 0.1g/L H 3 BO 3 0.056g /L(NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.02g/L), drinking water is prepared and then sterilized with a sterilizer at 121°C for 30 minutes.
  • the secondary shake flask seed medium formula is: 4% glucose, 2% bean cake powder, 1% cottonseed fine powder, 0.1% potassium dihydrogen phosphate, 1% solid corn liquor, 1% trace element solution (FeSO 4 7H 2 O 1.0g/L MnSO 4 4H 2 O 1.0g/L ZnSO 4 7H 2 O 0.2g/L CaCl 2 2H 2 O 0.1g/L CuCl 2 2H 2 O 0.1g/L H 3 BO 3 0.056g /L(NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.02g/L), drinking water is prepared and then sterilized with a sterilizer at 121°C for 30 minutes.
  • 700L seed tank culture medium is: glucose 4%, bean cake powder (special for fermentation) 2%, cottonseed fine powder 1%, potassium dihydrogen phosphate 0.1%, solid corn liquor 1%, TE liquid 1% (FeSO 7H 2 O 1.0g/L MnSO 4 4H 2 O 1.0g/L ZnSO 4 7H 2 O 0.2g/L CaCl 2 2H 2 O 0.1g/L CuCl 2 2H 2 O 0.1g/L H 3 BO 3 0.056 g/L (NH 4 ) 6 Mo 7 O 24 ⁇ 4H 2 O 0.02g/L), soak in 0.1% of the enemy, add drinking water to the seed tank, add raw materials while stirring, and constant volume. Carry out steam sterilization, tank temperature 123-126 °C, keep warm for 30 minutes.
  • Embodiment 7 Scale-up culture comparative example
  • the seed preparation of the primary shake flask, the seed preparation of the secondary shake flask and the cultivation in the 700L seed tank are the same as in Example 6.
  • 12m3 fermenter culture medium the formula is: mannitol 8%, glucose 2%, bean cake powder 2%, magnesium sulfate 0.1%, dipotassium hydrogen phosphate 0.25%, ferrous sulfate 0.1%, proline 1%, foam Enemy 0.15%.
  • the fermentation tank is sterilized with steam, the tank temperature is 123-126°C, and the temperature is kept for 30 minutes. After steam sterilization, the volume of fermentation liquid is about 5000L.
  • the stirring frequency is the highest at 50HZ, cultivated for 12 days, and the titer of the fermented liquid obtained in the tank is 2564mg/L.
  • Example 6 began to add mannitol when it was fermented to the fifth day, and added 2% (v/v) mannitol every day until the end of fermentation, and on the 6th day after the start of fermentation culture Add 0.03% (v/v) gamma-aminobutyric acid and 0.1% (v/v) lactic acid at one time in the fermenter, when the fermentation finishes and put the tank, the titer of the obtained fermented liquid is 4377mg/L, comparative example The 2564mg/L of 7 has greatly improved the fermentation level of Neomocontin B0.

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Abstract

一种在发酵过程中补料的稳定、高效的生产纽莫康定B0的方法，通过在发酵过程中添加乳酸、甘露醇或γ-氨基丁酸中一种或几种，可显著提高纽莫康定B0的发酵水平，其中最优组合可将纽莫康定B0的发酵水平提高至4000-4500mg/L，远高于现有技术公开的2500mg/L左右的发酵水平，大大提高了工业制备纽莫康定B0的效价水平，降低了其工业化成本。

Description

一种发酵制备纽莫康定B0的方法 技术领域
本发明属于微生物发酵领域，具体涉及卡泊芬净中间体纽莫康定B0的微生物发酵制备方法。
背景技术
醋酸卡泊芬净是由默沙东(Merck Sharp&Dohme，MSD)开发的一种脂肽类抗真菌药物，2001年卡泊芬净获FDA批准上市，商品名为Cancidas(科赛斯)，剂型为注射剂。卡泊芬净是一种抗真菌药，它通过抑制(1，3)-β-D-葡聚糖合成酶，从而破坏真菌细胞壁的完整性，适用于治疗对其它治疗无效或不耐受的侵袭性曲霉菌病。作为新一类棘白菌素类抗菌药物的代表，卡泊芬净具有选择性高、抗菌活性好、安全性高、耐药性少等方面的明显优势，加之产品专利保护接连届满失效，使其成为了全身抗真菌用药的研究热点。
纽莫康定类棘白菌素化合物是默沙东公司科学家发现并命名的，生产菌株是从西班牙马德里的洛索亚流域附近的水塘中分离出来的一株丝状真菌，属于无性繁殖的一个新属。作为卡泊芬净的前体，纽莫康定B0是目前最主要的优化研究对象，关于工业化生产纽莫康定B0，提升其单位则是研究热点。
CN108265096A提供了一种高效、稳定生产纽莫康定B0的方法，通过对霉菌Glarea lozoyensis的液态深层发酵，种子培养基和发酵培养基含有合适的碳源、氮源、无机盐和微量元素源，其中发酵培养基使用山梨醇作为主要碳源，并加入葡萄糖形成复合碳源，该方法能够在工业级发酵罐上稳定地生产，纽莫康定B0的产量达到2.5g/L。
CN106755224B涉及卡泊芬净发酵中间体的发酵方法，该方法采用稀配方培养基，发酵过程中流加乳糖和氨水，能够有效降低菌体粘度，提高通气效果，同时在发酵过程中严格控制pH。与此同时，在发酵液中补加维生素b5，该方法可将发酵单位提高至2000～2500mg/L。
CN108048512A公开了一种制备纽莫康定B0的发酵方法，将菌种Glarea lozoyensis接种于装有第一培养基的摇瓶中，培养得到菌源液；将菌源液接种于装有第二培养基的发酵罐中；通过曝气管向第二培养基中泵送无菌空气；发酵至24h后，通过曝气管向所述第二培养基中泵送含有氨气的无菌空气；发酵至60h后，通过插入所述第二培养基中的微生物 培养板向所述第二培养基中流加甘露醇；发酵至72h后，收集发酵罐中的纽莫康定B0。实施例显示13d后收集所述发酵液中的纽莫康定B0的量为2580mg/L。
尽管现有技术已经公开了多个关于提升纽莫康定B0的发酵方法，但是似乎纽莫康定B0的发酵水平止步于此，很难进行提高，因此进一步提升纽莫康定B0的发酵水平，降低工业成本仍有着广泛的需求。
发明内容
为了解决上述问题，本发明提供了一种发酵制备纽莫康定B0的方法。
一种发酵制备纽莫康定B0的方法，包括在发酵过程中添加乳酸、甘露醇或γ-氨基丁酸中的一种或任意几种的组合。
具体地，一种发酵制备纽莫康定B0的方法，所述方法包括在发酵过程中添加乳酸，所述乳酸添加量为发酵液体积的0％-0.3％(不含0％)，优选添加发酵液体积0.1％的乳酸；具体地，在发酵开始后的第4-7天一次性添加0％-0.3％(V/V)的乳酸，优选发酵培养的第6天一次性添加发酵液体积0％-0.3％(不含0％)的乳酸，更优选地，发酵培养的第6天一次性添加发酵液体积0.1％的乳酸。
具体地，一种发酵制备纽莫康定B0的方法，所述方法包括在发酵培养开始后的第3-6天开始，每天一次性添加1％-2.5％(V/V)的甘露醇，直至发酵结束；优选第5天开始添加发酵液体积1％-2.5％的甘露醇，直至发酵结束；进一步优选，从第5天开始每天一次性添加发酵液体积2％的甘露醇，直至发酵结束。
具体地，一种发酵制备纽莫康定B0的方法，包括在发酵培养开始后的第4-7天，一次性添加发酵液体积0％-0.05％(不含0％)的γ-氨基丁酸；优选第6天一次性添加0％-0.05％(不含0％)的γ-氨基丁酸；更优选地，第6天一次性添加0.03％(V/V)的γ-氨基丁酸。
进一步地，本发明提供了一种发酵制备纽莫康定B0的方法，所述方法包括在发酵开始后的第4-7天一次性添加发酵液体积0％-0.3％(不含0％)的乳酸、0％-0.05％(不含0％)的γ-氨基丁酸，更为优选地，在发酵培养的第6天一次性添加发酵液体积0％-0.3％的乳酸、0％-0.05％γ-氨基丁酸，最优选地，在发酵培养的第6天一次性添加发酵液体积0.1％的乳酸、0.03％的γ-氨基丁酸；所述方法还包括在发酵培养开始后的第3-6天开始，每天一次性添加发酵液体积1％-2.5％的甘露醇，直至发酵结束，优选在发酵培养开始后的第5天开始每天一次性添加1％-2.5％的甘露醇，直至发酵结束，更为优选地，在发酵培养开始后的第5天开始每天一次性添加发酵液体积2％的甘露醇，直至发酵结束。
本发明所述的发酵制备纽莫康定B0的方法包括在发酵过程中添加乳酸、甘露醇或γ- 氨基丁酸中的一种或任意几种的组合，其中添加乳酸、甘露醇或γ-氨基丁酸中的任意两种或三种时，所添加物质的添加量及添加方式与添加单一物质时相同。
本发明发酵使用的菌株为Glarea lozoyensis或其诱变的菌株，优选Glarea lozoyensis FIM2006071，该菌株由中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2012475，保藏日期为：2012年11月23日。
实施发明的发酵培养基包含碳源，所述碳源选自甘露醇、山梨糖醇、蔗糖、葡萄糖中的一种或多种；
所述发酵培养基还包含氮源，所述氮源选自棉籽精粉、玉米蛋白粉、酵母浸出粉、酵母粉、豆饼粉中的一种或多种；
所述发酵培养基还包含微量元素，所述微量元素选自磷酸氢二钾、硫酸亚铁、硫酸镁中的一种或多种；
所述发酵培养基还包含氨基酸，所述氨基酸为脯氨酸、苏氨酸中的一种或多种。
优选地，所述发酵培养基包括甘露醇、葡萄糖、豆饼粉、硫酸镁、脯氨酸、磷酸氢二钾、硫酸亚铁；
更为优选地，所述发酵培养基含甘露醇5％-14％、葡萄糖1％-4％、豆饼粉1％-3.5％、硫酸镁0.01％-0.2％、脯氨酸0％-3％、磷酸氢二钾0.25％-1.5％、硫酸亚铁0.01％-0.2％。
发酵制备纽莫康定B0的方法，包括种子液接种至发酵罐，发酵液在24-26℃培养，罐压0.05Mpa-0.1Mpa，发酵过程中溶氧维持在30％-40％。
本发明还提供了一种制备卡泊芬净的方法，该制备方法包括使用上述制备纽莫康定B0的方法获取纽莫康定B0，再由莫康定B0进一步制备卡泊芬净。
本发明的有益效果：
本发明提供了一种在发酵过程中补料的稳定、高效的生产纽莫康定B0的方法，通过在发酵过程中添加乳酸、甘露醇或γ-氨基丁酸中一种或几种，可显著提高纽莫康定B0的发酵水平，其中最优组合可将纽莫康定B0的发酵水平提高至4000-4500mg/L，远高于现有技术公开的2500mg/L左右的发酵水平，大大提高了工业制备纽莫康定B0的效价水平，降低了其工业生产成本。
附图说明：
图1为不同添加量的乳酸对纽莫康定B0发酵效价的影响。
图2为不同时间添加的乳酸对纽莫康定B0发酵效价的影响。
图3为不同添加量的γ-氨基丁酸对纽莫康定B0发酵效价的影响。
图4为不同时间添加γ-氨基丁酸对纽莫康定B0发酵效价的影响。
图5为不同时间补加不同量的甘露醇对纽莫康定B0发酵效价的影响。
具体实施方式：
以下将结合具体实施方式详细说明本发明内容，但本发明的范围不限于此。
以下具体实施例中，如无具体说明，使用的试剂和仪器都是本领域常用试剂和仪器，可以通过商购的方式获得；所使用的方法为本领域常规方法，本领域技术人员根据实施例内容可以知道如何具体实现所述方法，并实现相应的结果。
实施例中使用的菌株为Glarea lozoyensis FIM2006071，该菌株由中国典型培养物保藏中心保藏，保藏号为CCTCC NO：M2012475，保藏日期为：2012年11月23日，有关该菌株的制备及特征记载在中国专利201310032896.7。
培养基组分的含量基于总发酵液的体积比来计算，如8％甘露醇是指每100mL发酵液中含8g甘露醇。
实施例1.乳酸添加量的考察
(1)摇瓶种子制备
取工作菌种库中一支甘油管，解冻后接入摇瓶种子培养基中，控制温度25℃，恒温培养振荡器转速250rpm，培养3天。
其中摇瓶种子培养基配方为：葡萄糖4％，豆饼粉2％，棉籽精粉1％，磷酸二氢钾0.1％，固体玉米浆1％，微量元素溶液1％(FeSO 4·7H 2O 1.0g/L MnSO 4·4H 2O 1.0g/L ZnSO 4·7H 2O 0.2g/L CaCl 2·2H 2O 0.1g/L CuCl 2·2H 2O 0.1g/L H 3BO 3 0.056g/L(NH 4) 6Mo 7O 24·4H 2O 0.02g/L)，饮用水配制后用灭菌器121℃灭菌30分钟。
(2)发酵摇瓶培养
接种5％的摇瓶种子液至30mL发酵摇瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养12天，发酵培养开始后第6天时分别一次性加入发酵液体积0％、0.05％、0.08％、0.1％、0.3％、0.5％的乳酸，结果如图1所示。
其中发酵摇瓶培养基配方：甘露醇8％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％，饮用水配制后用灭菌器121℃灭菌30分钟。
图1表明，在发酵培养开始后第6天时添加0.05％-0.3％的乳酸可显著提升纽莫康定B0的效价水平，其中添加浓度在0.1％左右时纽莫康定B0的效价提升幅度最大为3421mg/L， 对比不添加乳酸的效价水平2514mg/L，增加了36％。
实施例2.乳酸添加时间的考察
(1)摇瓶种子制备
取工作菌种库中一支甘油管，解冻后接入摇瓶种子培养基中，控制温度25℃，恒温培养振荡器转速250rpm，培养3天。
其中摇瓶种子培养基配方为：葡萄糖4％，豆饼粉2％，棉籽精粉1％，磷酸二氢钾0.1％，固体玉米浆1％，微量元素溶液1％(FeSO 4·7H 2O 1.0g/L MnSO 4·4H 2O 1.0g/L ZnSO 4·7H 2O 0.2g/L CaCl 2·2H 2O 0.1g/L CuCl 2·2H 2O 0.1g/L H 3BO 3 0.056g/L(NH 4) 6Mo 7O 24·4H 2O 0.02g/L)，饮用水配制后用灭菌器121℃灭菌30分钟。
(2)发酵摇瓶培养
接种5％的摇瓶种子液至30mL发酵摇瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养12天，发酵培养开始后第4-7天时分别一次性加入发酵液体积0.1％的乳酸，结果如图2所示。
其中发酵摇瓶培养基配方：甘露醇8％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％，饮用水配制后用灭菌器121℃灭菌30分钟。
图2表明，在发酵培养开始后第4-7天时一次性添加一定量的乳酸可显著提升纽莫康定B0的效价水平，其中在第6天添加时纽莫康定B0的效价提升幅度最大。
实施例3γ-氨基丁酸添加量的考察
(1)摇瓶种子制备
同实施例2。
(2)发酵摇瓶培养
配制30mL发酵摇瓶培养基：甘露醇8％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％，饮用水配制后用灭菌器121℃灭菌30分钟。
发酵摇瓶培养：接种5％的摇瓶种子液至发酵摇瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养12天，发酵培养第6天时分别一次性加入发酵液体积的0％、0.01、0.03、0.05％的γ-氨基丁酸。
结果如图3所示，发酵开始后添加一定量的γ-氨基丁酸的对纽莫康定B0效价的提升具 有显著促进作用，其添加浓度在0.03％时纽莫康定B0的效价为3358mg/L，对比不添加γ-氨基丁酸时的发酵效价2546mg/L，增加了32％左右。
实施例4γ-氨基丁酸添加时间的考察
(1)摇瓶种子制备
同实施例2。
(2)发酵摇瓶培养
配制30mL发酵摇瓶培养基：甘露醇8％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％，饮用水配制后用灭菌器121℃灭菌30分钟。
发酵摇瓶培养：接种5％的摇瓶种子液至发酵摇瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养12天，在发酵培养开始后第4、5、6、7天同一时间分别向摇瓶中加入发酵液体积0.03的γ-氨基丁酸。
结果如图4所示，在发酵培养第4-7天一次性添加一定量的γ-氨基丁酸对纽莫康定B0效价的提升具有一定的促进作用，其中第6天添加0.03％的γ-氨基丁酸提升纽莫康定B0的效价水平到3358mg/L，对比不添加γ-氨基丁酸时的发酵效价2546mg/L，提升效果较明显。
实施例5甘露醇补加量及补加时间的考察
(1)摇瓶种子制备
同实施例2。
(2)发酵摇瓶培养
配制30mL发酵摇瓶培养基：甘露醇5％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％，饮用水配制后用灭菌器121℃灭菌30分钟。
发酵摇瓶培养：接种5％的摇瓶种子液至发酵摇瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养12天，分别在发酵培养3、4、5、6天开始每天分别补入1％、1.5％、2％、2.5％的甘露醇，直至发酵培养结束。
结果如图5所示，在第5天开始每天补入2％的甘露醇的实验组涨幅最高，纽莫康定B0的效价水平到达3791mg/L，与对照组2509mg/L相比增长了51％左右。
实施例6放大生产
纽莫康定B0的12m 3罐发酵生产
(1)取工作菌种库中一支甘油管，解冻后接入一级种瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养3天。
其中一级摇瓶种子培养基配方为：葡萄糖4％，豆饼粉2％，棉籽精粉1％，磷酸二氢钾0.1％，固体玉米浆1％，微量元素溶液1％(FeSO 4·7H 2O 1.0g/L MnSO 4·4H 2O 1.0g/L ZnSO 4·7H 2O 0.2g/L CaCl 2·2H 2O 0.1g/L CuCl 2·2H 2O 0.1g/L H 3BO 3 0.056g/L(NH 4) 6Mo 7O 24·4H 2O 0.02g/L)，饮用水配制后用灭菌器121℃灭菌30分钟。
(2)取一级种子，以5％的接种量接入二级种瓶中，控制温度25℃，恒温培养振荡器转速250rpm，培养3天。
其中二级摇瓶种子培养基配方为：葡萄糖4％，豆饼粉2％，棉籽精粉1％，磷酸二氢钾0.1％，固体玉米浆1％，微量元素溶液1％(FeSO 4·7H 2O 1.0g/L MnSO 4·4H 2O 1.0g/L ZnSO 4·7H 2O 0.2g/L CaCl 2·2H 2O 0.1g/L CuCl 2·2H 2O 0.1g/L H 3BO 3 0.056g/L(NH 4) 6Mo 7O 24·4H 2O 0.02g/L)，饮用水配制后用灭菌器121℃灭菌30分钟。
(3)将二级摇瓶种子以1.0％的接种量接入700L种子罐。种子液于罐压0.05Mpa，罐温25℃，空气流量1vvm，搅拌频率20Hz，培养3天。
其中700L种子罐培养基配方为：葡萄糖4％，豆饼粉(发酵专用)2％，棉籽精粉1％，磷酸二氢钾0.1％，固体玉米浆1％，TE液l％(FeSO 4·7H 2O 1.0g/L MnSO 4·4H 2O 1.0g/L ZnSO 4·7H 2O 0.2g/L CaCl 2·2H 2O 0.1g/L CuCl 2·2H 2O 0.1g/L H 3BO 3 0.056g/L(NH 4) 6Mo 7O 24·4H 2O 0.02g/L)，泡敌0.1％，种子罐内加入饮用水，边搅拌边加入原料，定容。进行蒸汽灭菌，罐温123-126℃，保温30分钟。
(4)12m 3发酵罐培养：
配制12m 3发酵罐培养基，配方为：甘露醇5％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％。配料罐内加入饮用水，边搅拌边加入可配制5000L的发酵培养基原料，搅拌均匀后，打入发酵罐，定容至4000L左右。发酵罐进汽实罐灭菌，罐温123-126℃，保温30分钟，蒸汽灭菌后发酵液体积5000L左右。灭菌前将PH调至7.0左右。
取种子罐5％种子液接种至12m 3发酵罐，控制罐温在25℃，发酵培养的初始罐压0.05Mpa，空气流量1vvm，搅拌频率5HZ，溶氧与转速、空气流量、罐压联动，发酵过程中在溶氧下降至30％以下时依次逐渐提高转速、空气流量和罐压，使溶氧维持在30％-40％，搅拌频率最高为50HZ，最高空气流量2.4VVM，最高罐压为0.1Mpa。
发酵至第五天时开始补加甘露醇，每天补入2％(v/v)的甘露醇，直至发酵结束，发酵 培养开始后的第6天向发酵罐中一次性加入0.03％(v/v)的γ-氨基丁酸，0.1％(v/v)的乳酸，发酵培养共12天，放罐所得发酵液效价为4377mg/L。
实施例7.放大培养对比实施例
纽莫康定B0的12m 3罐发酵生产
一级摇瓶种子制备、二级摇瓶种子制备和700L种子罐培养同实施例6。
(4)12m 3发酵罐培养：
配制12m 3发酵罐培养基，配方为：甘露醇8％，葡萄糖2％，豆饼粉2％，硫酸镁0.1％，磷酸氢二钾0.25％，硫酸亚铁0.1％，脯氨酸1％，泡敌0.15％。配料罐内加入饮用水，边搅拌边加入原料，搅拌均匀后，打入发酵罐，定容至4000L左右。发酵罐进汽实罐灭菌，罐温123-126℃，保温30分钟，蒸汽灭菌后发酵液体积5000L左右。
取步骤(3)种子罐5％种子液接种至12m 3发酵罐，控制罐温在25℃，发酵培养的初始罐压0.05Mpa，空气流量1vvm，搅拌频率5HZ，溶氧与转速联动，发酵过程中在溶氧下降至30％以下时依次逐渐提高转速，使溶氧维持在30％-40％，搅拌频率最高为50HZ，培养12天，放罐所得发酵液效价为2564mg/L。
与实施例7相比，实施例6在发酵至第五天时开始补加甘露醇，每天补入2％(v/v)的甘露醇，直至发酵结束，并且在发酵培养开始后的第6天向发酵罐中一次性加入0.03％(v/v)的γ-氨基丁酸和0.1％(v/v)的乳酸，发酵结束放罐时，所得发酵液效价为4377mg/L，对比实施例7的2564mg/L，大大提升了纽莫康定B0的发酵水平。
尽管本发明已经对上述各实施案例进行了描述，但并非因此限制本发明的专利保护范围。因此，基于本发明的创新理念，对本文所述实施案例进行的变更和修改，或利用本发明说明书内容所作的等效结构或等效流程变换，直接或间接地将以上技术方案运用在其他相关技术领域，均包括在本发明的专利保护范围之内。

Claims (11)

  1. 一种发酵制备纽莫康定B0的方法，其特征在于在发酵过程中添加乳酸、甘露醇或γ-氨基丁酸中的一种或任意几种的组合。
  2. 如权利要求1所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵过程中添加乳酸，优选乳酸添加量为发酵液体积的0％-0.3％，进一步优选为0.1％。
  3. 如权利要求2所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵开始后的第4-7天一次性添加0％-0.3％(V/V)的乳酸，优选发酵培养的第6天一次性添加0％-0.3％(V/V)的乳酸，更优选地，发酵培养的第6天一次性添加发酵液体积0.1％的乳酸。
  4. 如权利要求1所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵过程中添加甘露醇，优选在发酵培养开始后的第3-6天开始，每天一次性添加发酵液体积1％-2.5％的甘露醇，直至发酵结束；
    更优选地，在发酵培养第5天开始，每天一次性添加发酵液体积1％-2.5％的甘露醇，直至发酵结束；
    最优选地，在发酵培养第5天开始，每天一次性添加发酵液体积2％的甘露醇，直至发酵结束。
  5. 如权利要求1所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵过程中添加γ-氨基丁酸；
    优选地，γ-氨基丁酸添加量为发酵液体积的0％-0.05％(V/V)，优选0.03％；
    更为优选地，在发酵开始后的第4-7天一次性添加发酵液体积0％-0.05％的γ-氨基丁酸；优选第6天一次性添加发酵液体积0％-0.05％的γ-氨基丁酸；最优选地，发酵开始后的第6天一次性添加发酵液体积0.03％的γ-氨基丁酸。
  6. 如权利要求1所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵开始后的第4-7天一次性添加发酵液体积0％-0.05％的γ-氨基丁和0％-0.3％的乳酸；优选第6天一次性添加发酵液体积0％-0.05％的γ-氨基丁酸和0％-0.3％(V/V)的乳酸；更优选地，发酵开始后的第6天一次性添加发酵液体积0.03％的γ-氨基丁酸和0.1％的乳酸。
  7. 如权利要求6所述的发酵制备纽莫康定B0的方法，其特征在于，在发酵培养后的第3-6天开始，每天一次性添加发酵液体积1％-2.5％的甘露醇，直至发酵结束；
    更优选地，在发酵培养第5天开始，每天一次性添加发酵液体积1％-2.5％的甘露醇，直至发酵结束；
    最优选地，在发酵培养第5天开始，每天一次性添加发酵液体积2％的甘露醇，直至发 酵结束。
  8. 如权利要求1-7任意一项所述的发酵制备纽莫康定B0的方法，其特征在于，发酵使用的菌株为Glarea lozoyensis或其诱变的菌株；优选Glarea lozoyensis FIM2006071；
    发酵培养基包含碳源，所述碳源选自甘露醇、山梨糖醇、蔗糖、葡萄糖中的一种或多种，发酵培养基包含氮源，所述氮源选自棉籽精粉、玉米蛋白粉、酵母浸出粉、酵母粉、豆饼粉中的一种或多种，
    发酵培养基包含微量元素，所述微量元素选自磷酸氢二钾、硫酸亚铁、硫酸镁中的一种或多种，
    发酵培养基还包含氨基酸，所述氨基酸为脯氨酸、苏氨酸中的一种或多种；
    或者
    发酵培养基包括甘露醇、葡萄糖、棉籽精粉和玉米蛋白粉、脯氨酸、磷酸氢二钾、硫酸亚铁、硫酸镁；
    或者
    发酵培养基含甘露醇5％-14％、葡萄糖1％-4％、棉籽精粉1％-3.5％、玉米蛋白粉1％-3.5％、脯氨酸0％-3％、磷酸氢二钾0.25％-1.5％、硫酸亚铁0.01％-0.2％、硫酸镁0.01％-0.2％；
    或者
    发酵培养基包括甘露醇5％，葡萄糖2％，棉籽精粉2.8％，玉米蛋白粉1％，脯氨酸2.5％，磷酸氢二钾1％，硫酸镁0.1％，硫酸亚铁0.05％。
  9. 如权利要求8所述的发酵制备纽莫康定B0的方法，其特征在于，所述制备方法包括发酵液在24-26℃培养，罐压0.05Mpa-0.1Mpa，发酵过程中溶氧维持在30％-40％。
  10. 如权利要求1-9任意一项所述制备纽莫康定B0方法获得的发酵液或发酵产物。
  11. 一种制备卡泊芬净的方法，其特征在于，制备方法包括权利要求1-9任意一项所述制备纽莫康定B0的方法。
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