WO2024027251A1 - 一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法 - Google Patents

一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法 Download PDF

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WO2024027251A1
WO2024027251A1 PCT/CN2023/092689 CN2023092689W WO2024027251A1 WO 2024027251 A1 WO2024027251 A1 WO 2024027251A1 CN 2023092689 W CN2023092689 W CN 2023092689W WO 2024027251 A1 WO2024027251 A1 WO 2024027251A1
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fermentation
penicillium
tank
volume
seed
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PCT/CN2023/092689
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English (en)
French (fr)
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王耀耀
宋盼
王昂
任风芝
张雪霞
李红珍
郝惠云
王亚莉
李业英
路新华
崔晓兰
张雪莲
李国超
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华北制药集团新药研究开发有限责任公司
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Publication of WO2024027251A1 publication Critical patent/WO2024027251A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium

Definitions

  • the present invention relates to a method for synthesizing phthalide structural analogues using fungal metabolites, specifically a method for preparing 7-hydroxybutyphthalide through fermentation of Penicillium vulpinum.
  • cardiovascular and cerebrovascular diseases have gradually become one of the major diseases that threaten modern human health, and the high incidence and mortality of stroke are among the highest.
  • 3-butylphthalide shows multi-target therapeutic approaches, but its overall efficacy is not high and needs to be combined with conventional drugs such as aspirin to enhance its efficacy.
  • conventional drugs such as aspirin to enhance its efficacy.
  • 3-butylphthalide as the lead compound for structural modification, closed-ring derivatives, open-ring derivatives and analogs have been obtained.
  • CN 106214674 A discloses the isolation of three butylphthalide structural analogues from a strain of Penicillium foxguanorum fungus.
  • 7-hydroxy-butylphthalide 4-hydroxy-butylphthalide
  • Both 7-methoxy-butylphthalide and 7-methoxy-butylphthalide have antioxidant, anti-thrombotic and anti-cerebral ischemia effects.
  • 7-hydroxy-butylphthalide has a lower dosage than butylphthalide. It has more significant and excellent pharmacodynamic effects such as anti-platelet aggregation, anti-oxidative stress and anti-cerebral ischemia activity, and has lower toxicity, that is, it has higher drug safety.
  • 7-Hydroxy-butylphthalide is a cultured metabolite of Penicillium vulpinum. Its molecular formula is C 12 H 14 0 3 and its molecular weight is 206.0943. Its structural formula is as follows:
  • Natural Product Research.2021Feb;35(3):421-427 discloses two new phthalide compounds (namely 3-carboxypropyl- 7-hydroxyphthalide and 3-carboxypropyl-7-hydroxyphthalide methyl ester), the antibacterial activity experimental results show that the compound has a moderate inhibitory effect on Bacillus subtilis, Shigella dysenteriae and Enterobacter areogenes.
  • CN104726347A discloses a strain of Penicillium vulpinum NCC3421 (CGMCC NO.9094) and a method for preparing left-handed 7-hydroxybutylphthalide.
  • the levorotatory 7-hydroxybutyphthalide prepared by fermentation by the inventive method reaches 1600 ⁇ g/mL, which shows its industrial development prospects.
  • its technical output level is 1600 ⁇ g/mL in a 50L tank, which is still a certain distance from the industrialization level.
  • the object of the present invention is to provide a method for producing high-concentration 7-hydroxybutylphthalide through fermentation of Penicillium vulpinum, in order to further improve the industrial production efficiency of 7-hydroxybutylphthalide.
  • the method for producing high-concentration 7-hydroxybutylphthalide by fermentation of Penicillium vulpinum includes the following steps:
  • the Penicillium vulpinum NCC3421 (CGMCC NO.9094) disclosed in CN104726347A is used as the fermentation strain (hereinafter referred to as the NCC3421 strain).
  • the slant culture of the strain is inoculated into a shake flask seed medium and cultured at 25°C.
  • the shaker speed is 220 rpm, the culture period is 48 to 52 hours, and the seed culture liquid in the shake flask is obtained;
  • the component ratio and preparation of the culture medium are: dextrin 10.0 ⁇ 20.0g/L, sucrose 20.0 ⁇ 30.0g/L, low-temperature soybean cake powder 10.0 ⁇ 30.0g/L, peptone 5.0 ⁇ 15g/L, and high-phosphorus organic nitrogen Source 2 ⁇ 25.0g/L, ammonium sulfate 2.5 ⁇ 10.0g/L, sodium chloride 2.5 ⁇ 10.0g/L, calcium carbonate 2.0 ⁇ 4.0g/L, add water to adjust the volume to 1000mL, adjust the pH to 6.4 ⁇ 6.8 with sodium hydroxide solution, and sterilize at 121°C for 30 minutes;
  • the high-phosphorus organic nitrogen source described therein can be one or a combination of two or more of fish meal, corn steep liquor, gluten powder, and peanut cake powder. Fish meal, corn steep liquor or a combination of both is preferred.
  • the high-phosphorus organic nitrogen source in the method of the present invention is preferably a composition of fish meal and corn steep liquor, wherein the fish meal dosage is 5.0-10.0 g/L and the corn steep liquor dosage is 8.0-20.0 g/L; the total amount of the composition is controlled at 10.0 g ⁇ 23.0g/L.
  • the more preferred dosage is 8.0g/L of fish meal and 15.0g/L of corn steep liquor.
  • the first-level seed tank culture medium is prepared according to the following method: dextrin 10.0-20g/L, sucrose 20.0-30g/L, low-temperature soybean cake powder 15.0-30g/L, corn steep liquor 2.5-5.0g/L, and peptone 10.0 ⁇ 20g/L, potassium dihydrogen phosphate 3.0 ⁇ 6.0g/L, sodium chloride 2.5 ⁇ 5.0g/L, calcium carbonate 1g ⁇ 2/L, antifoaming agent 1 ⁇ 2mL/L, add water to make the volume to 1000mL, use Adjust the pH of the sodium hydroxide solution to 6.4 ⁇ 6.8 and sterilize at 121°C for 30 minutes.
  • the first-stage tank seed liquid prepared in step (b) into the fermentation medium at an inoculation volume of 3 to 5% by volume.
  • the fermentation temperature is 22 to 25°C
  • the fermentation culture speed is 220 to 250 rpm
  • the tank stirring power is 35 to 40 Hz.
  • the ventilation ratio is 1:0.8 ⁇ 1.5vvm
  • the tank pressure is 0.05 ⁇ 0.01Mpa
  • the dissolved oxygen concentration (hereinafter referred to as DO) is maintained above 20 ⁇ 30%
  • the fermentation cycle is 120-180h;
  • the fermentation culture medium described therein is prepared according to the following method: dextrin 0-40g/L, sucrose 20.0-30.0g/L, low-temperature soybean cake powder 30.0-45g/L, fish meal 10.0-20g/L, corn syrup 16.0- 32.0g/L, potassium dihydrogen phosphate 3.0 ⁇ 6.0g/L, ammonium sulfate 8.0 ⁇ 12.0g/L, sodium chloride 2.5 ⁇ 5.0g/L, magnesium sulfate heptahydrate 2.0 ⁇ 4.0g/L, precursor substance A 0 ⁇ 8.0g/L, precursor B 0 ⁇ 8.0g/L, corn oil 15.0 ⁇ 30mL/L, calcium carbonate 2.0 ⁇ 4.0g/L, antifoaming agent 0 ⁇ 1.0mL/L, add water to make the volume to 1000mL , adjust the pH to 6.4-6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the above-mentioned precursor substance A is methionine (precursor substance A1) or ethionine (precursor substance A1). A2); the precursor substance B is a short-chain fatty alcohol or short-chain fatty acid; the short-chain fatty alcohol can be ethanol, n-propanol, or n-butanol; n-propanol and n-butanol are preferred.
  • the short-chain fatty acids can be acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, norleucine and their sodium salts, of which norleucine and n-butyric acid are preferred.
  • the present invention adds fed feed during the fermentation process of the above step (c), wherein the feed is glucose 300-450g/L or glucose 300-450g/L. It has two components: glycerin 1 ⁇ 150g/L, and the total quality of the two components is controlled at 300 ⁇ 600g/L.
  • the preferred solution for fed feeding during the fermentation process is: during the fermentation process, when the pH of the fermentation broth rises to 7.2-7.3, fed feeding begins, and the feeding flow rate is controlled at 2-4L/h; when the fermentation broth reduces sugar When it is below 1.0%, the feed flow rate is controlled to 0.8-2L/h; after the feed is completed, fermentation continues for 8-12 hours.
  • the preferred preparation method of the shake flask seed culture medium according to the method of the present invention is:
  • Dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, peptone 10.0g/L, high-phosphorus organic nitrogen source 13 ⁇ 25.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0 g/L, calcium carbonate 2.0g/L, add water to make the volume 1000mL, adjust the pH to 6.4 ⁇ 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the amount of precursor substance A added in the method of the present invention is preferably 0.5 to 8.0 g/L.
  • the amount of precursor substance B added in the method of the present invention is preferably 0.5 to 8.0 g/L.
  • the fish meal of the present invention can be commercially available ordinary fish meal, such as commercially available feed grade fish meal.
  • the beneficial effects of the present invention are mainly reflected in: the present invention uses the NCC3421 strain to specially design the shake flask seed culture liquid, the first-level tank seed liquid, and the fermentation medium formula and process parameters, especially adding more to the shake flask seed culture medium.
  • High-phosphorus organic nitrogen sources such as fish meal and corn steep liquor shorten the spore germination time, effectively improve the mycelium reproductive activity in the seed bottle, and enable the bacterial fermentation level to be efficiently expressed.
  • the method of the invention greatly improves the yield of 7-hydroxybutylphthalide fermented by the NCC3421 strain, and its production level can reach 5000--10000 ⁇ g/mL in a 50L tank.
  • FIG. 1 Mycelium morphology ( ⁇ 10) of 48h shake flask seed culture solution in Example 1 of the present invention (medium IV).
  • Figure 2 shows the morphology of mycelium in the seed culture medium of common nitrogen source shake flask after 48 hours ( ⁇ 10).
  • Figure 3 shows the mycelium morphology ( ⁇ 40) of the 2m3 fermentation tank for 48 hours in Example 2 of the present invention (culture medium III).
  • Figure 4 is an HPLC chromatogram of 7-hydroxybutylphthalide produced in Example 6 of the present invention.
  • the present invention will be further described below in conjunction with specific examples.
  • the experimental materials used in the following examples are all industrial grade raw materials unless otherwise specified; the analytical chromatography column Inertsil ODS-SP 5 ⁇ m C18 4.6 ⁇ 250mm was purchased from Shimadzu, Japan, and the chromatography solvent was purchased from Honeywell Trading (Shanghai) Co., Ltd. .
  • the slant culture of strain NCC3421 was inoculated into a 1000 mL Erlenmeyer flask containing the mother bottle culture medium, and cultured at 25°C with a shaker speed of 220 rpm and a culture period of 48 to 52 hours to obtain the seed liquid.
  • the seed culture medium is prepared according to the following method: 10.0g/L dextrin, 20.0g/L sucrose, 15.0g/L low-temperature soybean cake powder, 10.0g/L peptone, and 0-23.0g/L high-phosphorus organic nitrogen source. , ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to adjust the volume to 1000mL, adjust the pH to 6.4 ⁇ 6.8 with 10% sodium hydroxide, and pack into Erlenmeyer flasks according to the filling volume of 200mL , sterilized at 121°C for 30 minutes.
  • shake flask seed culture medium In order to investigate the effect of shake flask seed culture medium on mycelial germination and growth rate and the effect of shake flask seed quality on mycelium development and fermentation level in the first-level seed tank, six different shake flask seed culture media are given in this example, which are respectively :
  • Medium I ordinary organic nitrogen source: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/ L. Calcium carbonate 2.0g/L, add water to make the volume, adjust the pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes;
  • Culture medium II organic nitrogen source is low-amount corn steep liquor: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn liquor 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to make the volume, adjust the pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes;
  • Medium III organic nitrogen source high-amount corn steep liquor: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn liquor 20.0g/L, peptone 10.0g/L, ammonium sulfate 5.0 g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to make the volume, adjust the pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes;
  • Medium IV organic nitrogen source is low-amount fish meal/high-amount corn steep liquor combination: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn liquor 15.0g/L, fish meal 3.0g /L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to dilute to volume, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C 30min;
  • Medium V organic nitrogen source high-amount fish meal/low-amount corn steep liquor combination: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn liquor 8.0g/L, fish meal 10.0g/L L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to dilute to volume, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes ;
  • Medium VI organic nitrogen source medium amount of fish meal/high amount of corn syrup combination: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to adjust to volume. Adjust the pH to 6.8 with 10% sodium hydroxide and sterilize at 121°C for 30 minutes.
  • the culture temperature is 25°C
  • the tank stirring power is 40Hz
  • the ventilation ratio is 1:0.8 ⁇ 1.5vvm
  • the tank pressure is 0.05 ⁇ 0.01 Mpa
  • the culture period is 48h ⁇ 52h
  • the first-level tank seed liquid is obtained;
  • the seed tank culture medium is: dextrin 10.0g/L, sucrose 10.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 5.0g/L, peptone 10.0g/L, potassium dihydrogen phosphate 3.0g/L, Sodium chloride 5.0g/L, calcium carbonate 2.0g/L, defoaming agent 1.0mL/L, add water to make the volume to 1000mL, adjust the pH to 6.4 with 40% sodium hydroxide solution, and sterilize at 121°C for 30 minutes.
  • the above seed liquid was inoculated into the fermentation medium in the fermentation tank at an inoculation amount of 3% by volume.
  • the fermentation temperature was 25°C and the fermentation cycle was 120h to 180h.
  • the stirring power of the fermentation tank was 40Hz and the fermentation culture speed was 220 to 250rpm.
  • the aeration ratio was 1:0.8 ⁇ 1.5vvm, tank pressure 0.05 ⁇ 0.01Mpa, DO maintained above 20%.
  • the fermentation medium was prepared according to the following method: dextrin 40g/L, sucrose 20.0g/L, low-temperature soybean cake powder 30.0g/L, fish meal 10.0g/L, corn steep liquor 16.0g/L, potassium dihydrogen phosphate 6.0 g/L, ammonium sulfate 8.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make up to volume, and use 10% hydrogen Adjust the pH to 6.4-6.8 with sodium oxide, incubate for 120 hours at 25°C and put in bottles.
  • the retention time of L-7-hydroxybutyphthalide is approximately 5.9 minutes.
  • the present invention uses fish meal + corn steep liquor as a compound high-phosphorus organic nitrogen source when carrying out shake flask seed culture, which significantly enhances the mycelial development of the seed bottle and improves the cultivation of low-inoculation first-level seed tanks. Quality, it has an outstanding and significant effect on improving the fermentation level of seed bottles.
  • the combination of corn steep liquor 15.0g/L/fish meal 8.0g/L has a more significant effect.
  • the fermentation level of the shake flask seed bottle reaches 2871 ⁇ g/mL
  • the fermentation level of the first-level seed tank reaches 3007 ⁇ g/mL.
  • the slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and cultured at 25°C and a shaker speed of 220 rpm for 52 h to obtain the shake flask seed liquid.
  • Shake flask seeds were inoculated into fermentation shake flasks with an inoculation amount of 5%, and cultured at 25°C and shaker speed of 220 rpm for 120 h and then placed in bottles.
  • the shake flask seed culture medium is: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8g/L, peptone 10.0g/L, ammonium sulfate 5.0 g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to adjust the volume to 1000mL, adjust the pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Example 2 Prepare the seed culture liquid according to the method described in Example 1, inoculate it into the fermentation medium with an inoculation amount of 3% by volume; ferment and synthesize the 7-hydroxybutylphthalide fermentation liquid.
  • the fermentation temperature is 25°C
  • the fermentation cycle is 120h ⁇ 180h
  • the fermentation tank stirring power is 40Hz
  • the fermentation culture speed is 220 ⁇ 250rpm
  • the ventilation ratio is 1:0.8 ⁇ 1.5vvm
  • the tank pressure is 0.05 ⁇ 0.01Mpa
  • DO is maintained above 20%.
  • Medium I (excluding precursor A): dextrin 40.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 30.0g/L, fish meal 10.0g/L, corn steep liquor 16.0g/L, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to dilute to volume, use 10 % sodium hydroxide to adjust the pH to 6.4, and sterilize at 121°C for 30 minutes.
  • Medium II (containing low amount of precursor substance A1): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, methionine 2.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium III (containing medium amount of precursor A1): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium IV (containing high amounts of precursor substance A1): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, methionine 8.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.3 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium V (contains low amount of precursor substance A2) dextrin 40g/L, sucrose 20.0g/L, low-temperature soybean cake powder 30.0g/L, fish meal 10.0g/L, corn steep liquor 16.0g/L, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, ethionine 2.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.5 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • precursor substance A2 dextrin 40g/L
  • sucrose 20.0g/L sucrose 20.0g/L
  • low-temperature soybean cake powder 30.0g/L fish meal 10.0g/L
  • corn steep liquor 16.0g/L
  • dihydrogen phosphate Potassium 6.0g/L ammonium s
  • Medium VI (containing medium amount of precursor A2): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, ethionine 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium VII (containing high amount of precursor substance A2): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, dihydrogen phosphate Potassium 6.0g/L, ammonium sulfate 8.0g/L, ethionine 8.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g /L, add water to volume, adjust pH to 6.7 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the retention time of L-7-hydroxybutyphthalide is approximately 5.9 minutes.
  • the slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and cultured at 25°C and a shaker speed of 220 rpm for 52 h to obtain the shake flask seed liquid.
  • Shake flask seeds were inoculated into fermentation shake flasks with an inoculation amount of 3%, cultured at 25°C, and shaker speed 220 rpm for 120 h and placed in bottles.
  • the shake flask seed culture medium is: dextrin 10.0g/L, sucrose 20.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, add water to make the volume, adjust pH to 6.5 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the seed culture liquid with reference to the method described in Example 1, inoculate it into the fermentation medium with an inoculation amount of 5% by volume; ferment and synthesize the 7-hydroxybutylphthalide fermentation liquid.
  • the fermentation temperature is 22°C
  • the fermentation cycle is 120h ⁇ 180h
  • the fermentation tank stirring power is 40Hz
  • the fermentation culture speed is 220 ⁇ 250rpm
  • the ventilation ratio is 1:0.8 ⁇ 1.5vvm
  • the tank pressure is 0.05 ⁇ 0.01Mpa
  • DO is maintained at 30%.
  • the six culture media are:
  • Medium I (containing low amount of precursor B n-butanol): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup, Potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, carbonic acid Calcium 4.0g/L, add water to volume, adjust pH to 6.5 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes. After elimination, add n-butanol to a final concentration of 2.0g/L.
  • Medium II (containing high amount of precursor substance B n-butanol): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup, Potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, carbonic acid Calcium 4.0g/L, add water to make the volume 1000mL, adjust pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes. After elimination, add n-butanol to a final concentration of 5.0g/L.
  • Medium III (containing low amount of precursor substance B norleucine): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup L, potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, norleucine 2.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g /L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make the volume, adjust the pH to 6.7 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium IV (containing high amount of precursor substance B norleucine): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup , potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, norleucine 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/ L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make the volume, adjust the pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium V (containing low amount of precursor substance B sodium n-butyrate): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup , potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, sodium n-butyrate 2.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/ L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to volume, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • Medium VI (containing high amount of precursor substance B sodium n-butyrate): 40g/L dextrin, 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn syrup , potassium dihydrogen phosphate 6.0g/L, ammonium sulfate 8.0g/L, methionine 5.0g/L, sodium n-butyrate 5.0g/L, sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/ L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make the volume, adjust the pH to 6.4 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the retention time of L-7-hydroxybutyphthalide is approximately 5.9 minutes.
  • the method of the present invention further promotes the synthesis of 7-hydroxybutylphthalide fermentation product by adding a high-phosphorus organic nitrogen source and precursor substance A in combination with precursor substance B.
  • the optimal group of methionine is 5.0 g/L, n-butanol 2.0g/L, The fermentation level reached 6332 ⁇ g/mL.
  • the slant culture of strain NCC3421 was inoculated into the shake flask seed culture medium, and cultured at 25°C and the shaker speed was 220 rpm for 48 hours to obtain the shake flask seed liquid.
  • the shake flask seed culture medium was: dextrin 10.0g/L, sucrose 20.0g/L. L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L , add water to volume, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the seed liquid in the shake flask was inoculated into a first-level seed tank (500L) with an inoculation amount of 0.5%.
  • the volume after inoculation was 300L.
  • the culture temperature was 25°C.
  • the stirring power of the seed tank was 40Hz.
  • the ventilation ratio was 1:1.0vvm.
  • the tank pressure was 0.05 ⁇ 0.01Mpa. , cultivate for 48 hours to obtain first-level seeds.
  • the seed tank culture medium is: dextrin 10.0g/L, sucrose 10.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 5.0g/L, peptone 10.0g/L, potassium dihydrogen phosphate 3.0g/L, Sodium chloride 5.0g/L, calcium carbonate 2g/L, defoaming agent 1mL/L, add water to volume, adjust pH to 6.8 with 40% sodium hydroxide solution, and sterilize at 121°C for 30 minutes.
  • the fermentation tank culture medium is: 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, 6.0g/L potassium dihydrogen phosphate, and 8.0g/L ammonium sulfate.
  • methionine 5.0g/L sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make up to volume, and use 10% hydroxide Adjust the pH to 6.4 with sodium and sterilize at 121°C for 30 minutes. After elimination, add n-butanol to a final concentration of 2.0g/L.
  • the fermentation process is carried out with fed feeding (the feeding is glucose, and the total amount added is 300g/L).
  • the fed feeding method is: when the pH of the fermentation broth rises to 7.2, start the sugar feeding flow rate to 2L/h, and reduce sugar in the middle and late stages of fermentation. Drop to below 1.0%, lower the sugar supply flow rate to 0.8mL/h, stop feeding 12 hours before placing in the tank, and the fermentation cycle is 168 hours.
  • the tank fermentation unit was measured to be 8235 ⁇ g/mL.
  • the shake flask seed medium is: dextrin 10.0g/L and sucrose 20.0g/L. , low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, Add water to volume, adjust pH to 6.8 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the seed liquid in the shake flask was inoculated into a first-level seed tank (500L) with an inoculation amount of 0.3%.
  • the volume after inoculation was 300L.
  • the culture temperature was 25°C.
  • the stirring power of the seed tank was 40Hz.
  • the ventilation ratio was 1:1.5vvm.
  • the tank pressure was 0.05 ⁇ 0.01Mpa. , cultivate for 52 hours to obtain first-level seeds.
  • the seed tank culture medium is: dextrin 10.0g/L, sucrose 10.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 5.0g/L, peptone 10.0g/L, potassium dihydrogen phosphate 3.0g/L, Sodium chloride 5.0g/L, calcium carbonate 2g/L, defoaming agent 1.0mL/L, add water to make the volume, adjust pH to 6.4 with 40% sodium hydroxide solution, and sterilize at 121°C for 30 minutes.
  • the fermentation tank culture medium is: 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, 6.0g/L potassium dihydrogen phosphate, and 8.0g/L ammonium sulfate.
  • methionine 5.0g/L sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make up to volume, and use 10% hydroxide Adjust the pH to 6.8 with sodium and sterilize at 121°C for 30 minutes. After elimination, add n-butanol to a final concentration of 2.0g/L.
  • the fermentation process is carried out with fed feeding (the concentration of the feeding liquid is 400g/L glucose and 100g/L glycerol).
  • the concentration of the feeding liquid is 400g/L glucose and 100g/L glycerol.
  • the sugar supply flow rate is started to be 3L/h, and the reducing sugar drops to 3L/h in the middle and late stages of fermentation. Below 1.0%, reduce the sugar supply flow rate to 1L/h, stop feeding 8 hours before placing in the tank, and the fermentation cycle is 170 hours.
  • the measured unit of tank fermentation was 10168 ⁇ g/mL.
  • the slant culture of strain NCC3421 was inoculated into the shake flask seed medium, and cultured at 25°C and the shaker speed 220 rpm for 50 hours to obtain the shake flask seed liquid.
  • the shake flask seed medium was: 10.0g/L dextrin and 20.0g/L sucrose. , low-temperature soybean cake powder 15.0g/L, corn steep liquor 15.0g/L, fish meal 8.0g/L, peptone 10.0g/L, ammonium sulfate 5.0g/L, sodium chloride 5.0g/L, calcium carbonate 2.0g/L, Add water to volume, adjust pH to 6.6 with 10% sodium hydroxide, and sterilize at 121°C for 30 minutes.
  • the seed liquid in the shake flask was inoculated into the first-level seed tank (500L) with an inoculation amount of 0.4%, and the volume after inoculation was 300L.
  • the culture temperature was 25°C
  • the stirring power of the seed tank was 40Hz
  • the ventilation ratio was 1:1.2vvm
  • the tank pressure was 0.05 ⁇ 0.01Mpa. Cultivate for 52 hours to obtain first-level seeds.
  • the seed tank culture medium is: dextrin 10.0g/L, sucrose 10.0g/L, low-temperature soybean cake powder 15.0g/L, corn steep liquor 5.0g/L, peptone 10.0g/L, potassium dihydrogen phosphate 3.0g/L, Sodium chloride 5.0g/L, calcium carbonate 2g/L, defoaming agent 1.0mL/L, add water to make the volume, adjust pH to 6.4 with 40% sodium hydroxide solution, and sterilize at 121°C for 30 minutes.
  • the first-level seed tank seed liquid was inoculated into a 2m 3 fermentation tank with an inoculum volume percentage of 5% for fermentation.
  • the volume after inoculation was 1.2m 3
  • the fermentation temperature was 25°C
  • the fermentation tank stirring power was 40Hz
  • the ventilation ratio was 1:0.8 ⁇ 1.5vvm
  • tank pressure 0.05 ⁇ 0.01Mpa DO maintained above 20%.
  • the fermentation tank culture medium is: 20.0g/L sucrose, 30.0g/L low-temperature soybean cake powder, 10.0g/L fish meal, 16.0g/L corn steep liquor, 6.0g/L potassium dihydrogen phosphate, and 8.0g/L ammonium sulfate.
  • methionine 5.0g/L sodium chloride 5.0g/L, magnesium sulfate heptahydrate 2.0g/L, corn oil 15.0mL/L, calcium carbonate 4.0g/L, add water to make up to volume, and use 10% hydroxide Adjust the pH to 6.8 with sodium and sterilize at 121°C for 30 minutes. After elimination, add n-butanol to a final concentration of 2.0g/L.
  • the fermentation process is carried out with fed feeding (the concentration of the feeding liquid is glucose 450g/L, glycerol 150g/L).
  • concentration of the feeding liquid is glucose 450g/L, glycerol 150g/L.
  • the pH of the fermentation broth rises to 7.3, start the sugar supply flow rate to 4L/h, and the reducing sugar drops to 4L/h in the middle and late stages of fermentation. Below 1.0%, reduce the sugar supply flow rate to 2L/h, stop feeding 12 hours before placing in the tank, and the fermentation cycle is 180 hours.
  • the measured unit of tank fermentation was 10205 ⁇ g/mL. Its HPLC chromatogram is detailed in Figure 4.

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Abstract

本发明公开了一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法。其采用NCC3421菌株，并对摇瓶种子培养液、一级罐种子液以及发酵培养基配方及工艺参数等进行特别设计，尤其是在摇瓶种子培养基中增加鱼粉、玉米浆等高磷有机氮源，缩短了孢子萌发时间，有效改善了种瓶菌丝繁殖活力，使菌种发酵水平得以高效表达，并且在后续的工业放大环节，采用合理的可操作接种量接种技术得以实现。本发明方法大大提高了NCC3421菌株发酵合成7-羟基丁苯酞的产量，其2m3发酵罐规模上发酵水平达到5000--10250μg/mL。

Description

一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法 技术领域
本发明涉及利用真菌代谢产物合成苯酞类结构类似物的方法，具体地说是利用狐粪青霉菌(Penicillium vulpinum)发酵制备7-羟基丁苯酞的方法。
背景技术
近年来随着人们物质生活水平的提高，心脑血管疾病逐渐成为威胁现代人类健康的主要疾病之一，脑卒中的高发病率和高死亡率更是位居前列。
3-丁苯酞作为治疗缺血性脑卒中的国内一类新药，表现出多靶点治疗途径，但是其总体疗效并不高，需要与阿司匹林等常规药物联用增强疗效。针对此类药物及其衍生物、结构类似物的深入研究成为该领域药物研发的热点。以3-丁苯酞为先导化合物进行结构改造，已获得闭环衍生物、开环衍生物以及类似物等。文献European Journal of Medicinal Chemistry,2010,45(5):1941-1946.进行了一系列卤素取代的苯肽环衍生物的合成及生物活性研究，其中化合物4i(6-溴-丁苯酞),4g(6-氟-丁苯酞)的血管舒张活性优于NBP；小鼠急性毒性实验显示，化合物4g(6-氟-丁苯酞)的LD50值为750.89mg/kg，4i(6-溴-丁苯酞)的LD50值为1000mg/kg，明显高于NBP的LD50值(592.93mg/kg)。文献Journal of medicinal chemistry,2013,56(7):3078-3089.报道了以2-(α-羟正戊基)苯甲酸为先导化合物，杂合异山梨醇，合成了一系列异山梨醇-NBP开环衍生物，其中化合物9a的活性最佳。体外抗血小板聚集试验及体内抗血栓试验显示，化合物9a的抑制活性较(S)-NBP及阿司匹林均显著增强。文献Organic&Biomolecular Chemistry,2011,9(16):5670-5681.以具有抗氧化、抗血小板作用的阿魏酸为连接基团，合成了NO供体型NBP开环衍生物，其中新化合物ZJM-289活性最佳。与NBP相比，化合物ZJM-289具有更好或相当的体内外抗血小板聚集、抗血栓和抗脑缺血活性。
CN 106214674 A公开了从一株狐粪青霉真菌菌株中分离得到三个丁苯酞结构类似物，体内外药效和毒性研究证明：7-羟基-丁苯酞、4-羟基-丁苯酞和7-甲氧基-丁苯酞均具有抗氧化、抗血栓和抗脑缺血等药效作用，其中的7-羟基-丁苯酞与丁苯酞相比，具有更低的用药剂量、更显著、优良的抗血小板凝集、抗氧化应激和抗脑缺血活性等药效作用，而且具有更低的毒性，即具有更高的药物安全性。
7-羟基-丁苯酞为狐粪青霉菌(Penicillium vulpinum)的培养代谢产物，其分子式为C12H1403，分子量为206.0943，结构式如下：
(参见Heterocycles,Vol.48,NO.9,1998：1931-1941)。
来源自真菌狐粪青霉菌(Penicillium vulpinum)的培养代谢产物的研究表现出丰富的天然活性。Natural Product Research.2021Feb；35(3):421-427公开了利用越南槐植株内生真菌狐粪青霉菌(Penicillium vulpinum)所产生得两种新的苯酞类化合物(即3-羧丙基-7-羟基苯酞和3-羧丙基-7-羟基苯酞甲酯)，其抑菌活性实验结果显示该化合物对Bacillus subtilis,Shigella dysenteriae and Enterobacter areogenes具有中等抑制作用。
目前，利用真菌代谢生成苯酞类结构类似物的研究比较活跃，尤其以短密青霉菌(Penicillium brevicompactum)发酵合成具有苯酞环结构的霉酚酸较为深入。
CN104726347A公开了一种狐粪青霉菌(Penicillium vulpinum)NCC3421(CGMCC NO.9094)菌株及利用其制备左旋7-羟基丁苯酞的方法。该发明方法发酵制备的左旋7-羟基丁苯酞达到1600μg/mL，显示了其工业化开发前景，但其产量技术水平为50L罐1600μg/mL，距离工业化水平尚有一定距离。
发明内容
本发明的目的在于提供一种利用狐粪青霉菌(Penicillium vulpinum)发酵产生高浓度7-羟基丁苯酞的方法，以期进一步提高7-羟基丁苯酞的工业化生产效能。
本发明目的是通过以下方式实现的：
本发明所提供的利用狐粪青霉菌(Penicillium vulpinum)发酵产生高浓度7-羟基丁苯酞的方法，包括以下步骤：
(a)制备狐粪青霉菌(Penicillium vulpinum)摇瓶种子培养液：
采用CN104726347A所公开得狐粪青霉菌(Penicillium vulpinum)NCC3421(CGMCC NO.9094)为发酵菌株(以下简称NCC3421菌株)，将该菌株的斜面培养物接种于摇瓶种子培养基，于25℃培养，摇床转速220rpm，培养周期48～52h，获得摇瓶种子培养液；
其中的培养基的成分配比及制备为：糊精10.0～20.0g/L、蔗糖20.0～30.0g/L、低温豆饼粉10.0～30.0g/L、蛋白胨5.0～15g/L、高磷有机氮源2～25.0g/L、硫酸铵2.5～10.0g/L、氯化钠 2.5～10.0g/L、碳酸钙2.0～4.0g/L，加水定容至1000mL，用氢氧化钠溶液调pH为6.4～6.8，121℃灭菌30min；
其中所述的所述高磷有机氮源，可选用鱼粉、玉米浆、_麸质粉、花生饼粉中的一种或两种以上组合。优选鱼粉、玉米浆或二者组合。
本发明方法所述高磷有机氮源为优选鱼粉和玉米浆的组合物，其中鱼粉用量为5.0～10.0g/L、玉米浆用量为8.0～20.0g/L；组合物总量控制在10.0g～23.0g/L.更优选的用量为鱼粉8.0g/L、玉米浆用量为15.0g/L。
(b)制备狐粪青霉菌(Penicillium vulpinum)一级罐种子液：
将(a)所制备的摇瓶种子培养液以体积百分比0.3～0.5％的接种量接种于一级种子培养基上，于培养温度22～25℃，罐搅拌功率35～40Hz，通气比1:0.8～1.5vvm，罐压0.05±0.01Mpa，培养周期48～52h，获得一级罐种子液；
其中所述一级种子罐培养基按以下方法制得：糊精10.0～20g/L、蔗糖20.0～30g/L、低温豆饼粉15.0～30g/L、玉米浆2.5～5.0g/L、蛋白胨10.0～20g/L、磷酸二氢钾3.0～6.0g/L、氯化钠2.5～5.0g/L、碳酸钙1g～2/L、消沫剂1～2mL/L，加水定容至1000mL，用氢氧化钠溶液调pH至6.4～6.8，121℃灭菌30min。
(c)制备狐粪青霉菌(Penicillium vulpinum)发酵液：
将(b)步骤所制备的一级罐种子液以体积百分比3～5％的接种量接种于发酵培养基，发酵温度22～25℃，发酵培养转速为220～250rpm；罐搅拌功率35～40Hz，通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，溶氧浓度(以下简称DO)保持20～30％以上，发酵周期120-180h；
其中所述的发酵培养基按以下方法制得：糊精0～40g/L、蔗糖20.0～30.0g/L、低温黄豆饼粉30.0～45g/L、鱼粉10.0～20g/L、玉米浆16.0～32.0g/L、磷酸二氢钾3.0～6.0g/L、硫酸铵8.0～12.0g/L、氯化钠2.5～5.0g/L、七水硫酸镁2.0～4.0g/L、前体物质A 0～8.0g/L、前体物质B 0～8.0g/L、玉米油15.0～30mL/L、碳酸钙2.0～4.0g/L、消沫剂0～1.0mL/L，加水定容至1000mL，用10％氢氧化钠调pH为6.4～6.8，121℃灭菌30min。
(d)将(c)所制备的发酵液，按照常规方法进行分离、收集，获得7-羟基丁苯酞溶液。
如将(c)所制备的发酵液3000rpm离心15min，弃上清液，留菌丝体；菌丝体中加入95％乙醇，搅拌浸泡25-30min，3000rpm离心15min，过滤，滤液中即富含获得7-羟基丁苯酞。
本发明方法中上述前体物质A为甲硫氨酸(前体物质A1)或乙硫氨酸(前体物质 A2)；所述的前体物质B是短链脂肪醇或短链脂肪酸；短链脂肪醇可选用乙醇、正丙醇、正丁醇；优选正丙醇、正丁醇。短链脂肪酸可选用乙酸、丙酸、异丁酸、丁酸、异戊酸、戊酸、正亮氨酸及其钠盐，其中优选正亮氨酸、正丁酸。
为进一步提增强菌种发酵产物的高效表达，本发明在上述(c)步骤的发酵过程中增加流加补料，其中所述的补料为葡萄糖300～450g/L或葡萄糖300～450g/L与甘油1～150g/L两种组分，两种组分的总质量控制在300～600g/L。
所述的发酵过程中流加补料的优选方案是：发酵过程中，发酵液pH回升至7.2～7.3时，开始流加补料，补料流速控制在2～4L/h；当发酵液还原糖在1.0％以下时，补料流速控制0.8～2L/h；补料完毕后继续发酵8～12h。
本发明方法所述摇瓶种子培养基的优选制备方法为：
糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、蛋白胨10.0g/L、高磷有机氮源13～25.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容1000mL，用10％氢氧化钠调pH为6.4～6.8，121℃灭菌30min。
本发明所述方法中前体物质A的加入量优选0.5～8.0g/L。
本发明所述方法中前体物质B的加入量优选0.5～8.0g/L。
本发明所述鱼粉可选用市售普通鱼粉，如市售的饲料级鱼粉。
本发明的有益效果主要体现在：本发明采用NCC3421菌株，对摇瓶种子培养液、一级罐种子液以及发酵培养基配方及工艺参数的进行特别设计，尤其是在摇瓶种子培养基中增加鱼粉、玉米浆等高磷有机氮源，缩短了孢子萌发时间，有效改善了种瓶菌丝繁殖活力，使菌种发酵水平得以高效表达，并且在后续的工业放大环节，采用合理的可操作接种量接种技术得以实现；在发酵培养基中添加生物合成前体物质(甲硫氨酸、正丁醇等)，进一步促进了目的产物的合成；在发酵过程中增加了组合碳源流加补料工艺，进一步增强了菌种发酵产物的高效表达。
本发明方法大大提高NCC3421菌株发酵合成7-羟基丁苯酞的产量，其生产水平可达到50L罐5000--10000μg/mL。
附图说明
图1本发明实施例1(培养基IV)摇瓶种子培养液48h菌丝形态(×10)。
图2为普通氮源摇瓶种子培养液48h菌丝形态(×10)。
图3为本发明实施例2(培养基Ⅲ)2m3发酵罐48h菌丝形态(×40)。
图4为本发明实施例6所产生的7-羟基丁苯酞HPLC色谱图。
具体实施方式
以下结合具体实施例，对本发明作进一步说明。下列实施例中所用的实验材料如无特殊说明均为工业级原料；分析色谱柱Inertsil ODS-SP 5μm C18 4.6×250mm购自日本岛津，色谱溶剂购自霍尼韦尔贸易(上海)有限公司。
实施例1
a、制备狐粪青霉菌(Penicillium vulpinum)的摇瓶种子培养液：
将菌株NCC3421的斜面培养物接种于分装有母瓶培养基的1000mL三角瓶中，于25℃培养，摇床转速220rpm，培养周期48～52h获得种子液。
其中所述种子培养基按以下方法制得：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、蛋白胨10.0g/L、高磷有机氮源0～23.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容至1000mL，用10％氢氧化钠调pH为6.4～6.8，按照装量200mL分装三角瓶，121℃灭菌30min。
为了考察摇瓶种子培养基对菌丝萌发生长速度以及摇瓶种子质量对一级种子罐菌丝发育、发酵水平的影响，本实施例中给出六个不同摇瓶种子培养基，其分别为：
培养基I(普通有机氮源)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min；
培养基II(有机氮源为低量玉米浆)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆8.0g/L，蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min；
培养基III(有机氮源高量玉米浆)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆20.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min；
培养基IV(有机氮源为低量鱼粉/高量玉米浆组合)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L、鱼粉3.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min；
培养基V(有机氮源高量鱼粉/低量玉米浆组合)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆8.0g/L，鱼粉10.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min；
培养基VI(有机氮源中量鱼粉/高量玉米浆组合)：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉 15.0g/L、玉米浆15.0g/L、鱼粉8.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。
b、制备狐粪青霉菌(Penicillium vulpinum)一级罐种子液：
将步骤a所制备的摇瓶种子培养液以体积百分比0.3％的接种量接种于种子罐内，于培养温度25℃，罐搅拌功率40Hz，通气比1:0.8～1.5vvm，罐压0.05±0.01Mpa，培养周期48h～52h，获得一级罐种子液；
其中种子罐培养基为：糊精10.0g/L、蔗糖10.0g/L、低温豆饼粉15.0g/L、玉米浆5.0g/L、蛋白胨10.0g/L、磷酸二氢钾3.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，消沫剂1.0mL/L，加水定容至1000mL，用40％氢氧化钠溶液调pH至6.4，121℃灭菌30min。
c、制备狐粪青霉菌(Penicillium vulpinum)合成7-羟基丁苯酞发酵液
将上述种子液以体积百分比3％的接种量接种于发酵罐中的发酵培养基，发酵温度25℃，发酵周期120h～180h；发酵罐搅拌功率40Hz，发酵培养转速为220～250rpm；通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持20％以上。
其中，发酵培养基按以下方法制得：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L，玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.4～6.8，于25℃，培养120h放瓶。
发酵结束后，取发酵液10mL，3000rpm离心15min，弃上清，菌丝体加入95％乙醇10mL，搅拌浸泡30min，3000rpm离心15min，上清液经0.45μm滤膜过滤，滤液用岛津高效液相色谱仪LC2030C进行产物含量检测。
左旋7-羟基丁苯酞的液相色谱检测条件：
色谱柱：Inertsil ODS-SP 5μm C18 4.6×250mm
流动相：乙腈/水＝60/40，含0.05％磷酸；流速1mL/min；检测器：UV，210nm。
上述条件下，左旋7-羟基丁苯酞的保留时间约为5.9min。
检测结果如下表：
由上述结果可见，本发明在进行摇瓶种子培养时所采用鱼粉+玉米浆作为复合高磷有机氮源，显著增强了种瓶菌丝发育，改善了低接种量进罐一级种子罐的培养质量，对于提升种瓶发酵水平具有突出的显著效果。尤其玉米浆15.0g/L/鱼粉8.0g/L组合效果更为显著，其摇瓶种瓶发酵水平达到2871μg/mL，一级种子罐发酵水平达到3007μg/mL。
从图1、图2也可看出，本发明与普通氮源(对照工艺)菌丝相比较，本发明所制备的菌丝镜检形态，菌丝粗壮、美兰染色深，菌体量较大。
实施例2
将菌株NCC3421菌株斜面培养物接种于摇瓶种子培养基，于25℃，摇床转速220rpm培养52h获得摇瓶种子液。摇瓶种子以接种量5％接种于发酵摇瓶，于25℃，摇床转速220rpm培养120h放瓶。
其中摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L、鱼粉8g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容至1000mL，用10％氢氧化钠调pH为6.6，121℃灭菌30min。
按照实施例1所述方法制备种子培养液，以体积百分比3％的接种量接种于发酵培养基中；发酵合成7-羟基丁苯酞发酵液。发酵温度25℃，发酵周期120h～180h；发酵罐搅拌功率40Hz，发酵培养转速为220～250rpm；通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持20％以上。
其中本实施例制备了七种发酵培养基(以验证本发明所提出的前体物质A对产物7-羟基丁苯酞的影响)，发酵瓶装液量为30mL/250mL，培养基组成分别为：
培养基I(不含前体物质A)：糊精40.0g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min。
培养基II(含低量前体物质A1)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸2.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min。
培养基III(含中量前体物质A1)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min。
培养基IV(含高量前体物质A1)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸8.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.3，121℃灭菌30min。
培养基V：(含低量前体物质A2)糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、乙硫氨酸2.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.5，121℃灭菌30min。
培养基VI(含中量前体物质A2)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、乙硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L，玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。
培养基VII(含高量前体物质A2)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、乙硫氨酸8.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L，玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.7，121℃灭菌30min。
发酵结束后，取发酵液10mL，3000rpm离心15min，弃上清液，菌丝体加入95％乙醇10mL，搅拌浸泡30min，3000rpm离心15min，上清液经0.45μm滤膜过滤，滤液用岛津高效液相色谱仪LC2030C进行产物含量检测。
左旋7-羟基丁苯酞的液相色谱检测条件：
色谱柱：Inertsil ODS-SP 5μm C18 4.6×250mm
流动相：乙腈/水＝60/40，含0.05％磷酸；流速1mL/min；检测器：UV，210nm。
上述条件下，左旋7-羟基丁苯酞的保留时间约为5.9min。
结果如下表：
由上述结果可见，发酵培养中添加前体物质A进一步显著促进了7-羟基丁苯酞发酵产物的合成，尤其是添加中等计量的甲硫氨酸(5.0g/L)，发酵水平达到5274μg/mL。
从图3也可以看出，本发明实施例2(培养基Ⅲ)2m3发酵罐48h菌丝形态(×40)，显示种子罐菌丝在移种至发酵罐后，菌丝发育迅速，代谢旺盛。
实施例3
将菌株NCC3421菌株斜面培养物接种于摇瓶种子培养基，于25℃，摇床转速220rpm培养52h获得摇瓶种子液。摇瓶种子以接种量3％接种于发酵摇瓶，于25℃，摇床转速220rpm培养120h放瓶。其中摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L、鱼粉8.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.5，121℃灭菌30min。
参照实施例1所述方法制备种子培养液，以体积百分比5％的接种量接种于发酵培养基中；发酵合成7-羟基丁苯酞发酵液。发酵温度22℃，发酵周期120h～180h；发酵罐搅拌功率40Hz，发酵培养转速为220～250rpm；通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持30％。
为了考察发酵培养基中前体物质B对产物7-羟基丁苯酞的影响，本实施例制备了六种发酵培养基，发酵瓶装液量为30mL/250mL。
六种培养基分别为：
培养基I(含低量前体物质B正丁醇)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.5，121℃灭菌30min。消后加入正丁醇终浓度2.0g/L。
培养基II(含高量前体物质B正丁醇)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容至 1000mL，用10％氢氧化钠调pH为6.4，121℃灭菌30min。消后加入正丁醇终浓度5.0g/L。
培养基III(含低量量前体物质B正亮氨酸)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、正亮氨酸2.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.7，121℃灭菌30min。
培养基IV(含高量前体物质B正亮氨酸)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、正亮氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。
培养基V(含低量前体物质B正丁酸钠)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、正丁酸钠2.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L，玉米油15.0mL/L,碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min。
培养基VI(含高量前体物质B正丁酸钠)：糊精40g/L、蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、正丁酸钠5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min。
发酵结束后，取发酵液10mL，3000rpm离心15min，弃上清，菌丝体加入95％乙醇10mL，搅拌浸泡30min，3000rpm离心15min，上清液经0.45μm滤膜过滤，滤液用岛津高效液相色谱仪LC2030C进行产物含量检测。
左旋7-羟基丁苯酞的液相色谱检测条件：
色谱柱：Inertsil ODS-SP 5μm C18 4.6×250mm
流动相：乙腈/水＝60/40，含0.05％磷酸；流速1mL/min；检测器：UV，210nm。
上述条件下，左旋7-羟基丁苯酞的保留时间约为5.9min。
结果如下表：
由上述结果可见，本发明方法在添加高磷有机氮源以及前体物质A的基础上组合添加前体物质B进一步促进7-羟基丁苯酞发酵产物的合成，最优组甲硫氨酸5.0g/L，正丁醇2.0g/L， 发酵水平达到6332μg/mL。
实施例4
将菌株NCC3421菌株斜面培养物接种于摇瓶种子培养基，于25℃，摇床转速220rpm培养48h获得摇瓶种子液，其中摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L，鱼粉8.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min。
摇瓶种子液以接种量0.5％接种于一级种子罐(500L)内，接后体积300L，培养温度25℃，种子罐搅拌功率40Hz，通气比为1:1.0vvm，罐压0.05±0.01Mpa，培养48h获取一级种子。其中种子罐培养基为：糊精10.0g/L、蔗糖10.0g/L、低温豆饼粉15.0g/L、玉米浆5.0g/L、蛋白胨10.0g/L、磷酸二氢钾3.0g/L、氯化钠5.0g/L、碳酸钙2g/L，消沫剂1mL/L，加水定容，用40％氢氧化钠溶液调pH至6.8，121℃灭菌30min。
将一级种子罐种子液以体积百分比5％的接种量接种于2m3发酵罐，接后体积1.2m3，发酵温度25℃，发酵罐搅拌功率40Hz，通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持20％以上。其中发酵罐培养基为：蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.4，121℃灭菌30min。消后加入正丁醇终浓度为2.0g/L。
发酵过程进行流加补料(补料为葡萄糖，添加总量为300g/L)，流加方式为：当发酵液pH升至7.2时，启动补糖流速为2L/h，发酵中后期还原糖降至1.0％以下，下调补糖流速为0.8mL/h，放罐前12h停止补料，发酵周期168h。测得放罐发酵单位为8235μg/mL。
实施例5
将菌株NCC3421斜面培养物接种于摇瓶种子培养基，于25℃，摇床转速220rpm培养52h获得摇瓶种子液，其中摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L、鱼粉8.0g/L、蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。
摇瓶种子液以接种量0.3％接种于一级种子罐(500L)内，接后体积300L，培养温度25℃，种子罐搅拌功率40Hz，通气比为1:1.5vvm，罐压0.05±0.01Mpa，培养52h获取一级种子。其中种子罐培养基为：糊精10.0g/L、蔗糖10.0g/L、低温豆饼粉15.0g/L、玉米浆5.0g/L、蛋白胨10.0g/L、磷酸二氢钾3.0g/L、氯化钠5.0g/L、碳酸钙2g/L、消沫剂1.0mL/L，加水定容，用40％氢氧化钠溶液调pH至6.4，121℃灭菌30min。
将一级种子罐种子液以体积百分比3％的接种量接种于2m3发酵罐，接后体积1.2m3，发酵温度25℃，发酵罐搅拌功率40Hz，通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持20％以上。其中发酵罐培养基为：蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。消后加入正丁醇终浓度为2.0g/L。
发酵过程进行流加补料(补料料液浓度为葡萄糖400g/L，甘油100g/L)，当发酵液pH升至7.3时，启动补糖流速为3L/h，发酵中后期还原糖降至1.0％以下，下调补糖流速为1L/h，放罐前8h停止补料，发酵周期170h。测得放罐发酵单位为10168μg/mL。
实施例6
将菌株NCC3421斜面培养物接种于摇瓶种子培养基，于25℃，摇床转速220rpm培养50h获得摇瓶种子液，其中摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、玉米浆15.0g/L，鱼粉8.0g/L，蛋白胨10.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容，用10％氢氧化钠调pH为6.6，121℃灭菌30min。
摇瓶种子液以接种量0.4％接种于一级种子罐(500L)，接后体积300L，培养温度25℃，种子罐搅拌功率40Hz，通气比为1:1.2vvm，罐压0.05±0.01Mpa，培养52h获取一级种子。其中种子罐培养基为：糊精10.0g/L、蔗糖10.0g/L、低温豆饼粉15.0g/L、玉米浆5.0g/L、蛋白胨10.0g/L、磷酸二氢钾3.0g/L、氯化钠5.0g/L、碳酸钙2g/L、消沫剂1.0mL/L，加水定容，用40％氢氧化钠溶液调pH至6.4，121℃灭菌30min。
将一级种子罐种子液以体积百分比5％的接种量接种于2m3发酵罐中进行发酵，接后体积1.2m3，发酵温度25℃，发酵罐搅拌功率40Hz，通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，DO保持20％以上。其中发酵罐培养基为：蔗糖20.0g/L、低温黄豆饼粉30.0g/L、鱼粉10.0g/L、玉米浆16.0g/L、磷酸二氢钾6.0g/L、硫酸铵8.0g/L、甲硫氨酸5.0g/L、氯化钠5.0g/L、七水硫酸镁2.0g/L、玉米油15.0mL/L、碳酸钙4.0g/L，加水定容，用10％氢氧化钠调pH为6.8，121℃灭菌30min。消后加入正丁醇终浓度为2.0g/L。
发酵过程进行流加补料(补料料液浓度为葡萄糖450g/L，甘油150g/L)，当发酵液pH升至7.3时，启动补糖流速为4L/h，发酵中后期还原糖降至1.0％以下，下调补糖流速为2L/h，放罐前12h停止补料，发酵周期180h。测得放罐发酵单位为10205μg/mL。其HPLC色谱图详见图4。

Claims (10)

  1. 一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，包括以下步骤：
    (a)制备狐粪青霉菌(Penicillium vulpinum)摇瓶种子培养液
    采用狐粪青霉菌(Penicillium vulpinum)NCC3421(CGMCC NO.9094)为发酵菌株，将该菌株的斜面培养物接种于摇瓶种子培养基，于25℃培养，摇床转速220rpm，培养周期48～52h，获得摇瓶种子培养液；其中培养基成分配比及制备为：糊精10.0～20.0g/L、蔗糖20.0～30.0g/L、低温豆饼粉10.0～30.0g/L、蛋白胨5.0～15g/L、高磷有机氮源2～25.0g/L、硫酸铵2.5～10.0g/L、氯化钠2.5～10.0g/L、碳酸钙2.0～4.0g/L，加水定容至1000mL，用氢氧化钠溶液调pH为6.4～6.8，121℃灭菌30min；
    (b)制备狐粪青霉菌(Penicillium vulpinum)一级罐种子液
    将(a)所制备的摇瓶种子培养液以体积百分比0.3～0.5％的接种量接种于一级种子培养基上，于培养温度22～25℃，罐搅拌功率35～40Hz，通气比1:0.8～1.5vvm，罐压0.05±0.01Mpa，培养周期48～52h，获得一级罐种子液；
    其中所述一级种子罐培养基按以下方法制得：糊精10.0～20g/L、蔗糖10.0～30g/L、低温豆饼粉15.0～30g/L、玉米浆2.5～5.0g/L、蛋白胨10.0～20g/L、磷酸二氢钾3.0～6.0g/L、氯化钠2.5～5.0g/L、碳酸钙1～2g/L、消沫剂1～2mL/L，加水定容至1000mL，用氢氧化钠溶液调pH至6.4～6.8，121℃灭菌30min；
    (c)制备狐粪青霉菌(Penicillium vulpinum)发酵液
    将(b)所制备的一级罐种子液以体积百分比3～5％的接种量接种于发酵培养基，发酵温度22～25℃，发酵周期120h～180h，发酵培养转速为220～250rpm；罐搅拌功率35～40Hz，通气比为1:0.8～1.5vvm，罐压0.05±0.01Mpa，溶氧浓度保持20～30％以上；
    其中所述的发酵培养基按以下方法制得：糊精0～40g/L、蔗糖20.0～30.0g/L、低温黄豆饼粉30.0～45g/L、鱼粉10.0～20g/L、玉米浆16.0～32.0g/L、磷酸二氢钾3.0～6.0g/L、硫酸铵8.0～12.0g/L、氯化钠2.5～5.0g/L、七水硫酸镁2.0～4.0g/L、前体物质A 0～8.0g/L、前体物质B 0～8.0g/L、玉米油15.0～30mL/L、碳酸钙2.0～4.0g/L、消沫剂0～1.0mL/L，加水定容至1000mL，用10％氢氧化钠调pH为6.4～6.8，121℃灭菌30min；
    其中所述的前体物质A为甲硫氨酸或乙硫氨酸；所述的前体物质B是短链脂肪醇或短链脂肪酸；
    (d)将(c)制备的发酵液，按照常规分离、收集方法，获得7-羟基丁苯酞溶液。
  2. 根据权利要求1所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，(a)步所述高磷有机氮源为鱼粉或玉米浆。
  3. 根据权利要求1或2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，(c)步骤发酵过程中流加补料，所述的补料为葡萄糖300～450g/L或葡萄糖300～450g/L与甘油1～150g/L的两种组分。
  4. 根据权利要求1或2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，所述摇瓶种子培养基为：糊精10.0g/L、蔗糖20.0g/L、低温豆饼粉15.0g/L、蛋白胨10.0g/L、高磷有机氮源13～23.0g/L、硫酸铵5.0g/L、氯化钠5.0g/L、碳酸钙2.0g/L，加水定容1000mL，用10％氢氧化钠调pH为6.4～6.8，121℃灭菌30min。
  5. 根据权利要求1或2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，所述前体物质A为正亮氨酸、正丁酸、异丁酸、异戊酸中的任意一种或其钠盐。
  6. 根据权利要求1或2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，所述前体物质B为正丙醇或正丁醇。
  7. 根据权利要求1或2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，所述前体物质A加入量为0.5～8.0g/L；所述前体物质B加入量为0～8.0g/L。
  8. 根据权利要求1所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，(a)步所述高磷有机氮源为鱼粉和玉米浆两种组分，其中鱼粉用量为5.0～10.0g/L、玉米浆用量为8.0～20.0g/L；两种组分的总质量控制在13.0g～23.0g/L。
  9. 根据权利要求2所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，(c)步骤所述的发酵过程中流加补料为：发酵过程中，发酵液pH回升至7.2～7.3时，开始流加补料，补料流速控制在2～4L/h；当发酵液还原糖在1.0％以下时，补料流速控制0.8～2L/h；补料完毕后继续发酵8～12h。
  10. 根据权利要求8所述的利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法，其特征在于，(a)步所述高磷有机氮源为鱼粉8.0g/L、玉米浆15.0g/L。
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CN115141861A (zh) * 2022-08-03 2022-10-04 华北制药集团新药研究开发有限责任公司 一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法

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CN115141861A (zh) * 2022-08-03 2022-10-04 华北制药集团新药研究开发有限责任公司 一种利用狐粪青霉菌发酵生产7-羟基丁苯酞的方法

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