WO2022166459A1 - 一种莫匹罗星的发酵方法 - Google Patents

一种莫匹罗星的发酵方法 Download PDF

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WO2022166459A1
WO2022166459A1 PCT/CN2021/141025 CN2021141025W WO2022166459A1 WO 2022166459 A1 WO2022166459 A1 WO 2022166459A1 CN 2021141025 W CN2021141025 W CN 2021141025W WO 2022166459 A1 WO2022166459 A1 WO 2022166459A1
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glucose
culture
fermentation
dihydrogen phosphate
glycerol
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French (fr)
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程赵兵
寇广会
许一鸣
胡炜杰
张宇辰
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Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
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Hangzhou Zhongmei Huadong Pharmaceutical Co Ltd
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Priority to CN202180092966.0A priority Critical patent/CN116867905A/zh
Priority to US18/264,400 priority patent/US20240043888A1/en
Priority to IL305014A priority patent/IL305014A/en
Priority to EP21924452.2A priority patent/EP4273256A4/en
Publication of WO2022166459A1 publication Critical patent/WO2022166459A1/zh
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Definitions

  • the invention belongs to the technical field of medicine, and further belongs to the technical field of fermentation, in particular to a fermentation method for mupirocin, including seed culture, fermentation culture, feeding culture and the like.
  • Mupirocin Pseudomonas acid A, is an aminoacyl transaminase inhibitor antibiotic that can specifically bind to the isoleucine transfer RNA synthetase of bacteria, thereby inhibiting isoleucine-containing bacteria in bacteria protein synthesis. It can fight against various gram-positive bacteria, including Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, etc.; it can also fight against some Gram-negative bacteria, including Haemophilus influenzae Bacillus etc. Mupirocin has no cross-resistance with other antibiotics, and has high medical value.
  • Patent WO2000046389 discloses a method for preparing pseudomonas acid A, including culturing a pseudomonas strain capable of biosynthesizing pseudomonas acid A on a medium of organic nitrogen source or carbon source and completing the cultivation, the method involves The composition of the medium is complex and the titer is unstable.
  • Patent CN201410083050.0 discloses a preparation method of Pseudomonas acid A and a series of culture and fermentation conditions. The steps of this method are complicated and the titer is unstable, and it is uncertain whether it can be put into practical application.
  • Patent CN201810896368.9 discloses a mupirocin fermentation medium. The medium additionally uses a production promoter, but its potency is not good and is not suitable for large-scale production.
  • the fermentation method of mupirocin has unsatisfactory aspects due to harsh process control conditions, environmental requirements and commercial or ecological perspectives. Therefore, there is still a need for a new method for the fermentation of mupirocin that is suitable for industrial mass production and that is environmentally friendly and cost controllable.
  • Mupirocin is a Pseudomonas A (Pseudomonas A) produced by fermentation of Pseudomonas fluorescens.
  • the existing mupirocin fermentation method is unstable and the fermentation titer is low, which cannot meet the status quo of industrialized high-volume production.
  • the present invention provides a mupirocin fermentation method suitable for industrialized large-scale production, comprising shaking bottle seeds Culture, seed tank culture, fermentation culture and fed culture.
  • the fermentation method provided by the invention is suitable for large-scale mass production and is environmentally friendly.
  • the titer of mupirocin fermented by the method provided by the invention can reach more than 8000ug/ml stably.
  • a mupirocin fermentation medium comprising the following components:
  • Mass to volume ratio (W/V) carbon source 5 ⁇ 12% nitrogen source 2 ⁇ 10% inorganic salt 0.1 ⁇ 1.5% defoamer 0.1 ⁇ 0.5%
  • the pH value of the fermentation medium is 6.0-7.5.
  • the fermentation medium is batched according to the above-mentioned process formula, and water is added to the fermentation tank, and the raw materials are put in while stirring;
  • the volume of the fermentation after feeding can be 0-20000L;
  • the culturing time of the fermenter is 48-144 hours, preferably 80-132 hours;
  • the temperature of the fermentation tank is 10-37°C, preferably 20-30°C.
  • the supplement recipe is as follows:
  • Mass to volume ratio (W/V) carbon source 25 ⁇ 60% nitrogen source 1 ⁇ 10% inorganic salt 0.10 ⁇ 0.5% defoamer 0.1 ⁇ 0.5%
  • the pH after feeding is 5.0-6.5.
  • the carbon sources include but are not limited to: glucose, sucrose, fructose, glycerol, dextrin, peptone, wheat bran, soy flour, corn starch, soybean flour, potato flour, tapioca starch, corn pulp, yeast powder, mannitol, etc.
  • the nitrogen source includes but is not limited to: ammonia water, urea, ammonium salt, nitrate, peptone, wheat bran, corn steep liquor, soybean meal powder, peanut meal meal, cottonseed meal meal, yeast powder, etc. .
  • the inorganic salts include but are not limited to: sulfates, phosphates, chlorides and the like.
  • the present invention also provides a kind of fermentation method of mupirocin, comprising the following steps:
  • Shake flask seed culture including the configuration, sub-packaging, sterilization, inoculation and cultivation of shake flask seeds to obtain a shake flask seed bacterial liquid;
  • Seed tank culture including the ingredients, sterilization, inoculation and culture of seed culture to obtain seed bacteria liquid;
  • Fermentation culture including ingredients, sterilization, inoculation and culture of fermentation medium to obtain mupirocin fermentation broth;
  • Feed culture including ingredients, sterilization and culture of feed culture to obtain mupirocin fermentation broth;
  • shake flask seed culture its culture formula is as follows:
  • the pH of the shake flask seed culture is 6.0-7.5;
  • the temperature of the shake flask seed culture is 20-30°C;
  • the culture time of the shake flask seeds is 18-30 hours, preferably 20-24 hours.
  • the inoculation amount of the shake flask seeds is 0.1-10% into the seed tank.
  • cultivation formula of seed tank cultivation is as follows:
  • Mass to volume ratio (W/V) carbon source 1 ⁇ 5% nitrogen source 0.5 ⁇ 1.5% inorganic salt 0.1 ⁇ 0.5% defoamer 0.01 ⁇ 0.1%
  • the pH of the seed tank culture is 6.0-7.5;
  • the temperature of the seed tank cultivation is 20-30°C;
  • the cultivation time of the seed tank cultivation is 12-36 hours, preferably 18-24 hours.
  • the carbon sources described in the culture formula include but are not limited to: glucose, sucrose, fructose, glycerol, dextrin, peptone, wheat bran, soy flour, corn starch, soybean flour, potato flour, tapioca starch, corn steep liquor, yeast powder, mannitol, etc.
  • the nitrogen sources described in the culture formula include but are not limited to: ammonia water, urea, ammonium salt, nitrate, peptone, wheat bran, corn steep liquor, soybean meal powder, peanut meal meal, cottonseed meal meal, yeast powder, etc.
  • the inorganic salts described in the culture formula include but are not limited to: sulfate, phosphate, chloride and the like.
  • the formula of the fermentation medium of the present invention is as follows:
  • bean cake powder 1.5 ⁇ 3.0%; Wheat bran 0.5 ⁇ 2.5%; Urea 0.1 ⁇ 0.2%; glycerin 1.0 ⁇ 2.0%; Ammonium sulfate 0.01 ⁇ 1.0%; Potassium dihydrogen phosphate 0.01 ⁇ 0.25%; Magnesium sulfate 0.01 ⁇ 0.25%; Potassium chloride 0.01 ⁇ 0.25%; Zinc sulfate 0.01 ⁇ 0.25%; Bubble enemies 0.1 ⁇ 0.5%.
  • the fermentation culture method is as follows: take each component according to the proportion for ingredients, add water to the fermentation tank, add the raw materials while stirring, and adjust the pH to 6.8-7.5 with sodium hydroxide; After sterilization and pipe sterilization, the seeds are hydraulically fed into the fermenter with sterile air.
  • the feeding culture formula of the present invention is as follows:
  • the method of fed-feed cultivation is to take each component according to the proportion for batching, and then carry out seeding after sterilization in real tanks and pipelines in sequence; and monitor pH and glucose content in a timely manner after seeding.
  • shake flask seed formula of the present invention is as follows:
  • glycerin 0.2 ⁇ 1.0%; Ammonium sulfate 0.1 ⁇ 0.5%; Magnesium sulfate 0.01 ⁇ 0.1%; Potassium dihydrogen phosphate 0.01 ⁇ 0.1%; Bubble enemies 0.01 ⁇ 0.1%.
  • the method for culturing seeds in shake flasks is as follows: take each formula component according to the proportion, add an appropriate amount of water, stir evenly, adjust the pH to 6.8-7.5 with sodium hydroxide solution, take the inclined surface after sterilization, and use Wash with sterile water, and insert the seed shake flask with an inoculation amount of 0.1-10.0%, wherein the culture temperature of the seed shake flask is 20-30° C., and the culture time is 12-48 hours.
  • the seed tank cultivation formula of the present invention is as follows:
  • the cultivation method of the seed tank is as follows: take each formula component according to the proportion, add water into the seed tank, put in the components under stirring, adjust the pH to 6.8-7.5 with sodium hydroxide, and sterilize after sterilization.
  • the shake flask seed liquid is connected to the seed tank, the tank temperature is 20-30 DEG C during inoculation, the culture time is 12-48 hours, and the culture pH is 6.8-7.5.
  • the titer of the tank is greater than or equal to 8000ng/ml, and stop the monitoring and control of pH and glucose before the tank is put , the fermentation broth is transferred to the pretreatment tank for the next step of extraction and/or purification.
  • the pH when the fermentation is finished and put into the tank is greater than 6.0, and the mass-volume ratio of the glucose content is 0.5-6%.
  • the mass volume ratio of the glucose content is 0.1-5%, preferably 0.5-1.5%; the pH of the shake flask seeds is 6.0-7.0 when inserted into the seed tank.
  • the inoculation amount is 1.0-8.0%, preferably 2.5-5%.
  • the pH is 6.0-6.5; the mass-volume ratio of the glucose content in the fermentation medium is 3.0-7.0, preferably 4.0-6.5%.
  • the method for culturing seeds in shake flasks is as follows: weighing each raw material, in a weight-to-volume ratio, glucose 1.0%, wheat bran 0.5%, glycerol 0.5%, magnesium sulfate 0.05%, phosphoric acid 0.05% Potassium dihydrogen 0.05%, ammonium sulfate 0.2%, soaked enemy 0.05%, the remainder is added to drinking water, and then stirred evenly, and the pH is adjusted to 6.8-7.5 with sodium hydroxide solution. ⁇ 10.0% of the inoculum is inserted into the seed shaker flask, the culture temperature of the seed shaker flask is 20 ⁇ 30°C, and the shaking culture is performed for 12 ⁇ 48 hours.
  • the method for seed tank cultivation is to weigh each raw material, and in a weight-to-volume ratio, glucose 1.0%, wheat bran 0.5%, glycerol 0.5%, magnesium sulfate 0.05%, dihydrogen phosphate Potassium 0.05%, ammonium sulfate 0.2%, foamed enemy 0.05%; add water to the seed tank, put in the raw materials while stirring, adjust the pH to 6.8-7.5 with sodium hydroxide, and sterilize at a tank temperature of 90-150 °C; before inoculation Detect that the glucose content in the medium of the seed tank is 0.5-1.5% and the pH of the medium is 6.0-7.0, insert the shake flask seed liquid into the seed tank, the tank temperature is 20-30 °C, cultivate for 12-36 hours, and the pH is controlled to be 6.5- 7.5; Under the microscope, the cell morphology was rod-shaped, with a large amount of bacteria, deep staining, and no miscellaneous bacteria.
  • the fermentation culture method is as follows: weighing each raw material, in a weight-to-volume ratio, glucose 5.0-6.0%, soybean meal 2.0%, wheat bran 1.5%, glycerol 1.5%, urea 0.15%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.06%, potassium chloride 0.12%, zinc sulfate 0.2%, foam enemy 0.3%; add water to the fermenter, put in the raw materials while stirring, use hydrogen Sodium oxide adjusts pH to 6.8-7.5, sterilizes at 90-150°C tank temperature, and maintains pressure for 0.5-1 hour; before inoculation, the glucose content in the culture medium of the fermenter is detected to be 4.0-6.5% and the pH of the medium is 6.0- 7.0, hydraulically press the seeds into the fermentation tank with sterile air, the inoculation amount is 1.0-8.0%, the tank temperature is 20-30 °C, and the pH and glucose content of the fermentation broth during the fermentation
  • the method of feeding culture is as follows: after the fermentation culture starts, sampling is performed regularly to detect the glucose content and pH, and feeding is started when the glucose content is lower than 1.0%; the configuration ratio of glucose in the feeding is as follows: Glucose 30 ⁇ 50%, wheat bran 1 ⁇ 5%, bean cake flour 1 ⁇ 5%, glycerin 0.5%, magnesium sulfate 0.06%, potassium dihydrogen phosphate 0.05%, zinc sulfate 0.05%, bubble enemy 0.3%; Sterilize at ⁇ 150°C; repeatedly detect and control the glucose content and pH in the tank, start feeding when the glucose content is lower than 1.0%, and control pH at 5.0-6.0 and glucose at 1.0-1.5% after feeding.
  • the method of putting the tank is as follows: fermentation is cultured for 80 to 128 hours, the pot titer is greater than or equal to 8000ug/ml, and the detection and control of pH and glucose are stopped before the tank is put; When the fermentation is over 6.0 and the glucose content is less than 0.5%, the fermentation liquid is transferred to the pretreatment tank, ready to enter the extraction and purification steps of the fermentation liquid.
  • the mupirocin fermentation method provided by the invention comprises the following steps:
  • Shake flask seed culture Weigh each raw material, in terms of weight volume ratio, glucose 1.0%, wheat bran 0.5%, glycerol 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, ammonium sulfate 0.2%, foam 0.05% of nitric acid, the remainder is added to drinking water, and then stirred evenly, adjusted to pH 6.8-7.5 with sodium hydroxide solution, sterilized at 90-150 °C for 0.5-1 hour, then take the slope, wash with sterile water, and rinse with 0.1 ⁇ 10.0% of the inoculum is inserted into the seed shaker flask, the culture temperature of the seed shaker flask is 20 ⁇ 30°C, and the shaking culture is performed for 12 ⁇ 48 hours;
  • Seed tank culture Weigh each raw material, in weight-to-volume ratio, glucose 1.0%, wheat bran 0.5%, glycerol 0.5%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, ammonium sulfate 0.2%, foam enemy 0.05%; add water to the seed tank, put in the raw materials while stirring, adjust the pH to 6.8-7.5 with sodium hydroxide, sterilize at a tank temperature of 90-150°C, and keep the temperature and pressure for 0.5-1 hour; check the seed tank before inoculation
  • the glucose content in the medium is 0.5-1.5% and the pH of the medium is 6.0-7.0, the shake flask seed liquid is connected to the seed tank, the tank temperature is 20-30 ° C, the culture is 12-36 hours, and the pH is controlled at 6.5-7.5;
  • the morphology of the cells observed under the microscope was rod-shaped, with a large amount of bacteria, deep staining, and no bacteria;
  • Fermentation culture Weigh each raw material, in weight-to-volume ratio, glucose 5.0-6.0%, soybean meal 2.0%, wheat bran 1.5%, glycerol 1.5%, urea 0.15%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.1%, 0.06% of magnesium sulfate, 0.12% of potassium chloride, 0.2% of zinc sulfate, and 0.3% of foamed enemy; add water to the fermenter, put in the raw materials while stirring, adjust the pH to 6.8-7.5 with sodium hydroxide, and keep it at the temperature of the tank.
  • the inoculum amount is 1.0-8.0%; the fermentation tank is cultured at a tank temperature of 20-30 °C, and the pH and glucose content of the fermentation broth during the fermentation and metabolism process are the main control parameters;
  • Feed culture After the start of fermentation culture, sample regularly to detect glucose content and pH, and start feeding when the glucose content is lower than 1.5%; the proportion of glucose in the feed is 30-50% glucose and 1-5% wheat bran , Soybean cake flour 1 ⁇ 5%, glycerin 0.5%, magnesium sulfate 0.06%, potassium dihydrogen phosphate 0.05%, zinc sulfate 0.05%, bubble enemy 0.3%; Sterilize at a temperature of 90 ⁇ 150°C in a tank, keep the heat preservation pressure 0.5 ⁇ After sterilization, the temperature was lowered to 25-40°C for 1 hour, and the glucose content and pH in the tank were repeatedly detected and controlled. When the glucose content was lower than 1.0%, the feeding was started. After feeding, the pH was controlled at 5.0-6.0 and the glucose was controlled at 1.0 ⁇ 1.5%;
  • the titer of the tank is greater than or equal to 8000ug/ml, and the detection and control of pH and glucose are stopped before the tank is placed; when the pH is above 6.0 and the glucose content is less than 0.5%, the fermentation ends , transfer the fermentation broth to the pretreatment tank, ready to enter the extraction and purification steps of the fermentation broth.
  • the fermentation medium is supplemented, which is based on a fast-acting carbon source glucose, a comprehensive carbon source glycerol, a comprehensive carbon source nitrogen source bean cake powder and wheat bran.
  • the fermentation medium is supplemented, which is based on a fast-acting carbon source glucose, a comprehensive carbon source glycerol, a comprehensive carbon source nitrogen source bean cake powder and wheat bran.
  • Mixed feed of inorganic salt potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate and defoamer foam enemy that is, in fed-batch fermentation, a single carbon source feed is converted into a mixed feed based on the proportion of the medium, and the fermentation time is appropriately extended to achieve an increase in the fermentation titer.
  • the mupirocin fermentation method provided by the present invention is not only suitable for small-scale experiments, but also for industrialized production of thousands of liters.
  • the average level of fermentation titer of this process can reach more than 8000ug/ml, and the quality meets USP41 and EP9.0 standard.
  • the present invention by improving the feeding formula in the fermentation culture process, replaces the basic sugar supplementing method with the mixed feed adjusted based on the fermentation medium culture, and cooperates with the corresponding adjustment of the fermentation cycle to improve
  • the ability of the bacteria to pass and metabolize in the fermentation process is improved, and the time for producing titers is prolonged, and the final output of the fermentation is improved.
  • the present invention will be further described in detail below in conjunction with specific embodiments.
  • the following examples are used to understand the method and core idea of the present invention.
  • any possible changes or substitutions without departing from the concept of the present invention belong to the protection scope of the present invention.
  • the experimental methods without specific conditions are usually conventional conditions, or according to the conditions suggested by the raw material or commodity manufacturers; the raw materials and reagents with unspecified sources are usually conventional ones that can be purchased through commercial channels. reagents.
  • strains are preserved in two forms, glycerol tubes and slants.
  • Preparation, packaging and sterilization Weigh each raw material according to its proportion, add 400ml of drinking water, stir evenly, adjust the pH to 7.0 with sodium hydroxide solution, set the volume to 500ml, and then put it in a 3000ml conical flask, Sterilize at 121°C for 30min.
  • Inoculation and culture take the inclined surface, wash it with sterile water, and insert it into the seed shaker flask at an inoculation amount of 0.1-10.0%. Seed shake flask culture temperature 20 ⁇ 30 °C, shaking culture for 24h.
  • the ingredients are carried out according to the above-mentioned technical formula, water is added to the seed tank, the raw materials are put in while stirring, and the pH is adjusted to 7.0 with 6N sodium hydroxide solution.
  • the tank temperature is 120 °C
  • the tank pressure is 0.09 ⁇ 0.12MPa
  • the heat preservation and pressure are maintained for 30 minutes.
  • the requirements of the seed tank medium after testing and sterilization are as follows: glucose: 0.5-1.5%, pH 6.0-7.0.
  • the seed solution is controlled at a tank pressure of 0.04-0.05MPa, a tank temperature of 20-30°C, and a pH of 6.5-7.5.
  • the cell morphology was observed under the microscope: rod-shaped, abundant bacteria, deep staining, and no bacteria.
  • the ingredients are carried out according to the above-mentioned technical formula, water is added to the fermentation tank, the raw materials are put in while stirring, and the pH is adjusted to 6.5-7.0 with solid sodium hydroxide.
  • the tank temperature is 120 °C
  • the tank pressure is 0.09-0.12MPa
  • the heat preservation and pressure are maintained for 30 minutes; the pipeline sterilization is performed, and the sterilization time is 60 minutes.
  • the fermenter medium after sterilization was detected before inoculation, and the requirements were: glucose 4.0-6.5%, pH 6.0-6.5.
  • the temperature of the fermentation broth is 20-30°C
  • the pressure of the tank is 0.04-0.05MPa
  • the pH and glucose content of the fermentation broth during the fermentation and metabolism are the main control parameters.
  • the proportion of glucose in the feed is glucose 30-50%; wheat bran 2.0%; bean cake flour 2.0%; glycerin 0.5%; magnesium sulfate 0.06%; potassium dihydrogen phosphate 0.05%; zinc sulfate 0.05%;
  • the tank temperature is 120 °C
  • the tank pressure is 0.09-0.12 MPa
  • the temperature is maintained for 30 minutes, and the temperature is lowered to 30-40 °C after sterilization. Then sterilize the sugar supplement pipeline, and the sterilization time is 60min.
  • the purpose of this experiment was to screen the selection of carbon and nitrogen sources in the material formulations of fermentation culture and fed culture.
  • the main carbon sources, nitrogen sources and composite carbon and nitrogen sources screened in this experiment are: dextrin, peptone, wheat bran, bean cake flour, raw bean flour, corn steep liquor, yeast powder, urea, etc.
  • the introduction of peptone into the formula leads to poor fermentation titer, and the excess of urea in the formula leads to poor fermentation titer; it is basically confirmed that the carbon source, nitrogen source and composite carbon and nitrogen source of the fermentor and feed of the present invention can be selected as glucose , glycerin, dextrin, wheat bran, soy flour, raw soy flour, corn steep liquor, yeast powder.
  • the materials in the formula of the fermentation basic material culture medium are respectively glucose, soybean meal powder, wheat bran, urea, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate, potassium chloride, zinc sulfate, glycerin, and foamed enemy.
  • the feed formula is based on the fermentation base medium, which is screened to determine the feed tank formula.
  • magnesium sulfate in the basic material Since the proportion of magnesium sulfate in the basic material is low, only 0.06%, magnesium is an activator of many enzymes, which can promote the metabolism of carbohydrates, the synthesis of nucleic acids, and the conversion of phosphate. It is a basic element and only provides nutrients for basic growth. , so the proportion of magnesium sulfate 0.06% in the feed formula remained unchanged.
  • Foaming is only used to eliminate foam and does not affect the process, so the proportion of the supplementary formula and the fermentation base material remain the same.
  • glycerol As a polar protic solvent, glycerol is completely soluble in water and short-chain alcohols but not in hydrocarbons. It can promote the dissolution of various non-organic acids, bases, salts, and hydrophilic organic compounds in the aqueous phase. In addition, glycerol has good biocompatibility with biological enzymes, which can promote the correct folding of proteins, stabilize the protein structure, and maintain the activity of enzymes, and has a certain protective effect on biological cells and enzymes in the environment of heat and organic solvents. It is therefore necessary to determine the optimum ratio of glycerol in the feed formulation.
  • soybean meal powder, wheat bran, potassium dihydrogen phosphate, zinc sulfate, and glycerol will now be confirmed through the proportions of different proportions, and the formula with high titer will be selected as the mixture in the feeding tank during the fermentation process.
  • the most suitable formula for supplementation The seed tank was cultured at 20-30° C. for 24 hours and then transferred to the existing fermenter for cultivation. After culturing for 6 days, the fermented titer was tested by putting it in a bottle.
  • Zinc sulfate The fermented tank titer of average 1 is 8986.75, which is significantly higher than the other three groups, so the ratio of zinc sulfate is selected as level 1, that is, 0.05%;
  • Bean cake flour The fermented pot titer of average 2 was 9050.75, which was significantly higher than the other three groups, so the proportion of bean cake flour was selected as level 2, that is, 2.0%;
  • Potassium dihydrogen phosphate The fermentation pot titers of mean 1, mean 2, mean 3, and mean 4 were 7800.75, 7763.25, 7792.25 and 6601.25, respectively. There was no significant difference between the first three and the mean 4 showed a significant downward trend. With the increase of the hydrogen-potassium ratio, the pot titer shows a downward trend, indicating that the higher the ratio of potassium dihydrogen phosphate, the greater the risk of the pot titer falling, so the level 1 with the smallest risk and the highest pot titer is selected, namely 0.05%;
  • Glycerol The fermentation pot titer of mean 1 is 8513.00, the fermentation pot titer of mean 4 is 8526.25, the fermentation pot titer of mean 1 and mean 4 is higher and there is no significant difference, given that there is already glucose in the feeding formula , glycerol and glucose are both added as carbon sources, and the proportion of glucose is larger, which plays the role of the main carbon source. If the proportion of glycerol is 0.5% and 2%, there is no significant difference in the titer of fermentation and storage, and a smaller proportion is selected. That is, the second is to consider the cost; and the production cost is lower if the smaller proportion is selected, so the proportion of 0.5% is selected, so the proportion of glycerol is determined to be 0.5%;
  • Wheat bran The fermentation pot titers of mean 1, mean 2, mean 3, and mean 4 are 6545.00, 8113.00, 7142.25, and 8157.25, respectively. With the increase of the proportion of wheat bran, the pot titer showed an upward trend, indicating that the lower the wheat bran ratio, the greater the risk of the decrease of the pot titer, so the level with the least risk and the highest pot titer was selected. i.e. 2.0%.
  • the feeding formula of the present invention is: glucose 30-50%; wheat bran 2.0%; bean cake flour 2.0%; glycerin 0.5%; magnesium sulfate 0.06%; potassium dihydrogen phosphate 0.05%; zinc sulfate 0.05%; 0.3%.
  • this example uses a 4-ton fermenter for testing. According to the titer test results, the stability of the fermentation tank level of the fermentation process was confirmed.
  • Seed medium formula glucose 1.0%, magnesium sulfate 0.05%, potassium dihydrogen phosphate 0.05%, ammonium sulfate 0.2%, buodi 0.05%, glycerol 0.5%, wheat bran 0.5%, pH adjusted to 7.0;
  • Fermentation medium formula: glucose 5.5%, bean cake flour 2.0%, ammonium sulfate 0.5%, potassium dihydrogen phosphate 0.10%, magnesium sulfate 0.06%, potassium chloride 0.12%, zinc sulfate 0.2%, glycerol 1.5%, bubble Di 0.3% , 0.15% urea, 1.0% wheat bran, adjust pH7.0;
  • Supplementary formula glucose 30-50%, bean cake flour 2.0%, potassium dihydrogen phosphate 0.05%, magnesium sulfate 0.06%, zinc sulfate 0.05%, glycerin 0.5%, Paodi 0.3%, wheat bran 2.0%;
  • Shake flasks for seed preparation and seed tank preparation ingredients are made according to the above process formula, the volume after feeding is 220L, the pH is adjusted to 7.0 with sodium hydroxide solution, the temperature of the sterilization tank is 120°C, and the temperature of the sterilization tank is kept at 30min;
  • the seed tank is connected to the pre-sample for inspection, the seed tank is inoculated and the fermentation tank is prepared and eliminated: the ingredients are carried out according to the above process formula, and the volume after feeding is 800 L; tank culture;
  • the seed tank is transplanted and the saccharified liquid is prepared and eliminated, the saccharified liquid is sent for inspection, and the fermentation begins to cultivate;
  • Fermentation culture The fermentation broth was cultured at a temperature of 20-30°C in the tank. The next morning after the transplant, samples were taken every 2 hours to detect glucose and pH. When the glucose was lower than 1.0%, sugar supplementation was started. After sugar supplementation, the pH was controlled to 5.6-6.5, and the glucose was controlled below 1.5%. After that, pH, glucose and titer were regularly detected from 8:00 every day.
  • the pH and glucose were measured at 8:00 in the morning and when the tank was put in the tank, and the titer was measured every 2 hours from 8:00. Fermentation culture for 80 to 132 hours.
  • the above-mentioned amplification experimental scheme was repeated three times, and the numbers were F190304FJ, F190401FJ, and F1900402FJ.
  • the growth curve was recorded with the fermentation time as the abscissa and the titer as the coordinate.
  • the recording of growth curve started at 8:00 in the morning on the third day after batch inoculation, the titer was detected every 4 hours, and the pH and glucose were detected at 8:00 and 16:00 every day.
  • the pH and glucose were tested at 8:00 on the day of the canning and the canning, and the titer was detected every two hours from 12:00 until the canning was placed.

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Abstract

一种莫匹罗星的发酵方法，包括摇瓶种子培养、种子罐培养、发酵培养、补料培养。发酵方法既适用于工业化放量生产，同时也提高了莫匹罗星的发酵效价，可稳定保持在8000ug/ml以上。

Description

一种莫匹罗星的发酵方法 技术领域
本发明属于医药技术领域，更进一步的属于发酵技术领域，具体涉及一种莫匹罗星的发酵方法，包括种子培养、发酵培养、补料培养等。
背景技术
莫匹罗星，即假单胞菌酸A，是一种氨基酰转氨酶抑制剂类抗生素，能特异性地结合到细菌的异亮氨酸转移RNA合成酶，从而抑制细菌中含异亮氨酸蛋白质的合成。其能够对抗各种革兰氏阳性菌，包括金黄色葡萄球菌、酿胺链球菌、表皮葡萄球菌、肺炎链球菌、化脓性链球菌等；也能够对抗一些革兰氏阴性菌，包括流感嗜血杆菌等。莫匹罗星与其他抗生素无交叉耐药性，具有较高的医疗价值，早已被临床应用，其商品名为百多邦或莫匹罗星软膏。
专利WO2000046389公开了制备假单胞菌酸A的方法，包括在有机氮源或碳源的培养基上培养能够生物合成假单胞菌酸A的假单胞菌算株并完成培养，此方法涉及的培养基成分复杂且效价不稳定。专利CN201410083050.0公开了假单胞菌酸A的制备方法及一系列培养及发酵条件，此方法的步骤繁复且效价不稳定，不确定能否投入实际应用。专利CN201810896368.9公开了一种莫匹罗星的发酵培养基，该培养基附加使用了生产促进剂，但其效价不佳，不适用于大规模生产的情况。
莫匹罗星的发酵方法因工艺控制条件苛刻、环境需求及商业或生态角度上都存在难以令人满意方面。因此，仍然需要适用于工业化大规模生产且环境友好、成本可控的的莫匹罗星的发酵新方法。
发明内容
莫匹罗星是一种由荧光假单胞菌(Pseudomonas fluorescens)发酵生产的假单胞菌酸A(Pseudomonas A)。现有的莫匹罗星的发酵方法不稳定且发酵效价较低，不能满足工业化放量生产的现状，本发明提供一种适用于工业化大规模生产的莫匹罗星发酵方法，包括摇瓶种子培养、种子罐培养、发酵培养和补料培养。
本发明提供的发酵方法适合大规模放量生产，且对环境友好。采用本发明提供的方法发酵的莫匹罗星的效价稳定可达8000ug/ml以上。
一种莫匹罗星的发酵培养基，包括以下组分：
发酵培养基组分 质量体积比(W/V)
碳源 5～12％
氮源 2～10％
无机盐 0.1～1.5％
消泡剂 0.1～0.5％
所述发酵培养基的pH值为6.0～7.5。
发酵培养基按上述工艺配方进行配料，发酵罐内加水，边搅拌边投入原料；
所述的发酵投料后的体积可为0～20000L；
所述的发酵罐培养的时间为48～144小时，优选为80～132小时；
所述的发酵罐温度为10～37℃，优选为20～30℃。
发酵培养24小时开始补料，补料后若pH上升则适当增加补糖量，若pH平稳则维持或适当减少补糖量；
补料配方如下：
补料组分 质量体积比(W/V)
碳源 25～60％
氮源 1～10％
无机盐 0.10～0.5％
消泡剂 0.1～0.5％
所述的补料后pH为5.0～6.5。
作为一种具体的实施方式，所述的碳源包括但不限于：葡萄糖、蔗糖、果糖、甘油、糊精、蛋白胨、麦麸、豆饼粉、玉米淀粉、黄豆粉、马铃薯粉、木薯淀粉、玉米浆、酵母粉、甘露醇等。
作为一种具体的实施方式，所述的氮源包括但不限于：氨水、尿素、铵盐、硝酸盐、蛋白胨、麦麸、玉米浆、豆饼粉、花生饼粉、棉籽饼粉、酵母粉等。
作为一种具体的实施方式，所述的无机盐包括但不限于：硫酸盐、磷酸盐、氯化物等。
本发明还提供一种莫匹罗星的发酵方法，包括以下步骤：
1.摇瓶种子培养：包括摇瓶种子的配置、分装、灭菌、接种和培养，得到摇瓶种子菌液；
2.种子罐培养：包括种子培养的配料、灭菌、接种和培养，得到种子菌液；
3.发酵培养：包括发酵培养基的配料、灭菌、接种和培养，得到莫匹罗星发酵液；
4.补料培养：包括补料培养的配料、灭菌和培养，得到莫匹罗星发酵液；
5.放罐。
其中，摇瓶种子培养其培养配方如下：
摇瓶种子培养组分 质量体积比(W/V)
碳源 1～5％
氮源 0.5～1.5％
无机盐 0.1～0.5％
消泡剂 0.02～0.1％
所述摇瓶种子培养的pH为6.0～7.5；
所述摇瓶种子培养的温度为20～30℃；
所述摇瓶种子的培养时间为18～30小时，优选为20～24小时。
其中，摇瓶种子的接种量为0.1～10％接入种子罐。
其中，种子罐培养的培养配方如下：
种子罐培养组分 质量体积比(W/V)
碳源 1～5％
氮源 0.5～1.5％
无机盐 0.1～0.5％
消泡剂 0.01～0.1％
所述种子罐培养的pH为6.0～7.5；
所述种子罐培养的温度为20～30℃；
所述种子罐培养的培养时间为12～36小时，优选为18～24小时。
其中，培养配方中所述的碳源包括但不限于：葡萄糖、蔗糖、果糖、甘油、糊精、蛋白胨、麦麸、豆饼粉、玉米淀粉、黄豆粉、马铃薯粉、木薯淀粉、玉米浆、酵母粉、甘露醇等。
其中，培养配方中所述的氮源包括但不限于：氨水、尿素、铵盐、硝酸盐、蛋白胨、麦麸、玉米浆、豆饼粉、花生饼粉、棉籽饼粉、酵母粉等。
其中，培养配方中所述的无机盐包括但不限于：硫酸盐、磷酸盐、氯化物等。
作为一种具体的实施方式，本发明的发酵培养基的配方如下：
发酵培养基组分 质量体积比(W/V)
葡萄糖 4.0～10.0％；
豆饼粉 1.5～3.0％；
麦麸 0.5～2.5％；
尿素 0.1～0.2％；
甘油 1.0～2.0％；
硫酸铵 0.01～1.0％；
磷酸二氢钾 0.01～0.25％；
硫酸镁 0.01～0.25％；
氯化钾 0.01～0.25％；
硫酸锌 0.01～0.25％；
泡敌 0.1～0.5％。
作为一种具体的实施方式，发酵培养的方式为，按配比取各组分进行配料，发酵罐内加入水，边搅拌边加入原料，用氢氧化钠调节pH至6.8～7.5；依次进行实罐灭菌和管道灭菌后，用无菌空气把种子液压入发酵罐。
作为一种具体的实施方式，本发明的补料培养配方如下：
补料组分 质量体积比(W/V)
葡萄糖 25～60％；
豆饼粉 1～5％；
麦麸 1～5％；
甘油 0.1～1.0％；
磷酸二氢钾 0.01～0.1％；
硫酸镁 0.01～0.1％；
硫酸锌 0.01～0.1％；
泡敌 0.1～0.5％。
作为一种具体的实施方式，补料培养的方式为，按配比取各组分进行配料，在依次进行实罐灭菌和管道灭菌后进行移种；移种后适时监测pH和葡萄糖含量。
作为一种具体的实施方式，本发明的摇瓶种子配方如下：
摇瓶种子组分 质量体积比(W/V)
葡萄糖 0.5～5％；
麦麸 0.2～1.0％；
甘油 0.2～1.0％；
硫酸铵 0.1～0.5％；
硫酸镁 0.01～0.1％；
磷酸二氢钾 0.01～0.1％；
泡敌 0.01～0.1％。
作为一种具体的实施方式，摇瓶种子的培养方法为，按配比取各配方组分，加入适量水，搅拌均匀，用氢氧化钠溶液调节pH至6.8～7.5，灭菌后取斜面，用无菌水洗，以0.1～10.0％的接种量接入种子摇瓶，其中，种子摇瓶的培养温度为20～30℃，培养时间为12～48小时。
作为一种具体的实施方式，本发明的种子罐培养配方如下：
种子罐培养组分 质量体积比(W/V)
葡萄糖 0.5～5％；
麦麸 0.2～1.0％；
甘油 0.2～1.0％；
硫酸铵 0.1～0.5％；
硫酸镁 0.01～0.1％；
磷酸二氢钾 0.01～0.1％；
泡敌 0.1～0.5％。
作为一种具体的实施方式，种子罐的培养方法为，按配比取各配方组分，种子罐内加入水，在搅拌下投入组分，用氢氧化钠调节pH至6.8～7.5，灭菌后将摇瓶种子液接入种子罐，接种时罐温为20～30℃，培养时间为12～48小时，培养的pH为6.8～7.5。
作为一种具体的实施方式，在发酵培养和/或补料培养80～132小时后，开始放罐，放罐效价大于等于8000ng/ml，并在放罐之前停止pH和葡萄糖的监测和控制，将发酵液转移至预处理罐，用于下一步提取和/或纯化。
作为一种具体的实施方式，发酵结束放罐时的pH大于6.0，葡萄糖含量的质量体积比为0.5～6％。
作为一种具体的实施方式，摇瓶种子接入种子罐时，葡萄糖含量的质量体积比为0.1～5％，优选为0.5～1.5％；摇瓶种子接入种子罐时pH为6.0～7.0。
作为一种具体的实施方式，种子液压入发酵罐时，接种量为1.0～8.0％，优选为2.5～5％。
作为一种具体的实施方式，种子液压入发酵罐前，pH为6.0～6.5；发酵培养基的葡萄糖含 量的质量体积比为3.0～7.0，优选为4.0～6.5％。
作为一种具体的实施方式，所述的摇瓶种子培养的方法为，称取各原料，以重量体积比计为，葡萄糖1.0％、麦麸0.5％、甘油0.5％、硫酸镁0.05％、磷酸二氢钾0.05％、硫酸铵0.2％、泡敌0.05％，余量加入饮用水后搅拌均匀，用氢氧化钠溶液调节pH至6.8～7.5，灭菌后取斜面，用无菌水洗，以0.1～10.0％的接种量接入种子摇瓶，种子摇瓶的培养温度20～30℃，振荡培养12～48小时。
作为一种具体的实施方式，所述的种子罐培养的方法为称取各原料，以重量体积比计为，葡萄糖1.0％、麦麸0.5％、甘油0.5％、硫酸镁0.05％、磷酸二氢钾0.05％、硫酸铵0.2％、泡敌0.05％；种子罐内加入水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，于罐温90～150℃下灭菌；接种前检测种子罐培养基中葡萄糖含量为0.5～1.5％且培养基pH为6.0～7.0，将摇瓶种子液接入种子罐，罐温20～30℃，培养12～36小时，pH控制为6.5～7.5；在显微镜下观察细胞形态为杆状、菌量多、染色深、无杂菌。
作为一种具体的实施方式，所述的发酵培养的方法为，称取各原料，以重量体积比计为，葡萄糖5.0～6.0％、豆饼粉2.0％、麦麸1.5％、甘油1.5％、尿素0.15％、硫酸铵0.5％、磷酸二氢钾0.1％、硫酸镁0.06％、氯化钾0.12％、硫酸锌0.2％、泡敌0.3％；发酵罐内加入水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，于罐温90～150℃下灭菌，保温保压0.5～1小时；接种前检测发酵罐培养基中葡萄糖含量为4.0～6.5％且培养基pH为6.0～7.0，用无菌空气把种子液压入发酵罐，接种量为1.0～8.0％，罐温20～30℃，发酵代谢过程中的发酵液pH和葡萄糖含量为主要控制参数。
作为一种具体的实施方式，所述的补料培养的方法为，发酵培养开始后定时取样检测葡萄糖含量和pH，当葡萄糖含量低于1.0％时开始补料；补料中葡萄糖的配置比例为葡萄糖30～50％、麦麸1～5％、豆饼粉1～5％、甘油0.5％、硫酸镁0.06％、磷酸二氢钾0.05％、硫酸锌0.05％、泡敌0.3％；于罐温90～150℃下灭菌；反复检测并控制罐内葡萄糖含量和pH，当葡萄糖含量低于1.0％时开始补料，补料后pH控制在5.0～6.0且葡萄糖控制在1.0～1.5％。
作为一种具体的实施方式，所述的放罐的方法为，发酵培养80～128小时，放罐效价大于等于8000ug/ml，在放罐之前停止pH和葡萄糖的检测和控制；当pH至6.0以上且葡萄糖含量小于0.5％时发酵结束，将发酵液转移至预处理罐，准备进入发酵液的提取和纯化步骤。
本发明提供的莫匹罗星发酵方法，包括以下步骤：
1.摇瓶种子培养：称取各原料，以重量体积比计为，葡萄糖1.0％、麦麸0.5％、甘油0.5％、硫酸镁0.05％、磷酸二氢钾0.05％、硫酸铵0.2％、泡敌0.05％，余量加入饮用水后搅拌均匀， 用氢氧化钠溶液调节pH至6.8～7.5，置于90～150℃下灭菌0.5～1小时，后取斜面，用无菌水洗，以0.1～10.0％的接种量接入种子摇瓶，种子摇瓶的培养温度20～30℃，振荡培养12～48小时；
2.种子罐培养：称取各原料，以重量体积比计为，葡萄糖1.0％、麦麸0.5％、甘油0.5％、硫酸镁0.05％、磷酸二氢钾0.05％、硫酸铵0.2％、泡敌0.05％；种子罐内加入水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，于罐温90～150℃下灭菌，保温保压0.5～1小时；接种前检测种子罐培养基中葡萄糖含量为0.5～1.5％且培养基pH为6.0～7.0，将摇瓶种子液接入种子罐，罐温20～30℃，培养12～36小时，pH控制为6.5～7.5；在显微镜下观察细胞形态为杆状、菌量多、染色深、无杂菌；
3.发酵培养：称取各原料，以重量体积比计为，葡萄糖5.0～6.0％、豆饼粉2.0％、麦麸1.5％、甘油1.5％、尿素0.15％、硫酸铵0.5％、磷酸二氢钾0.1％、硫酸镁0.06％、氯化钾0.12％、硫酸锌0.2％、泡敌0.3％；发酵罐内加入水，边搅拌边投入原料，用氢氧化钠调节pH至6.8～7.5，于罐温90～150℃下灭菌，保温保压0.5～1小时；接种前检测发酵罐培养基中葡萄糖含量为4.0～6.5％且培养基pH为6.0～7.0，用无菌空气把种子液压入发酵罐，接种量为1.0～8.0％；发酵罐于罐温20～30℃培养，发酵代谢过程中的发酵液pH和葡萄糖含量为主要控制参数；
4.补料培养：发酵培养开始后定时取样检测葡萄糖含量和pH，当葡萄糖含量低于1.5％时开始补料；补料中葡萄糖的配置比例为葡萄糖30～50％、麦麸1～5％、豆饼粉1～5％、甘油0.5％、硫酸镁0.06％、磷酸二氢钾0.05％、硫酸锌0.05％、泡敌0.3％；于罐温90～150℃下灭菌，保温保压0.5～1小时，灭菌后降温至25～40℃保温；反复检测并控制罐内葡萄糖含量和pH，当葡萄糖含量低于1.0％时开始补料，补料后pH控制在5.0～6.0且葡萄糖控制在1.0～1.5％；
5.放罐：发酵培养80～132小时后，放罐效价大于等于8000ug/ml，在放罐之前停止pH和葡萄糖的检测和控制；当pH至6.0以上且葡萄糖含量小于0.5％时发酵结束，将发酵液转移至预处理罐，准备进入发酵液的提取和纯化步骤。
莫匹罗星发酵工艺的现有技术中，有补料分批发酵的生产方式，在发酵培养过程中持续向发酵体系中补入葡萄糖以供给菌体分裂传代、生长产抗阶段的碳源支持。本发明提供的莫匹罗星发酵工艺，在微生物培养过程中，补入由发酵培养基为基础的，包含速效碳源葡萄糖、综合性碳源甘油、综合性碳源氮源豆饼粉和麦麸、无机盐磷酸二氢钾、硫酸镁、硫酸锌及消泡剂泡敌的混合补料。即，在补料分批发酵中，将单一碳源补料转变为以培养基比例为基础的混合补料，适当延长发酵时间，达到发酵效价的提升。
本发明的有益效果为：
1.本发明提供的莫匹罗星发酵方法，既适用于小试级别的实验，也适用于千升级别的工业 化生产，本工艺发酵效价平均水平可达8000ug/ml以上，且质量符合USP41和EP9.0标准。
2.本发明相较于现有技术，通过改进发酵培养过程中的补料配方，用以发酵培养基培养为基础调整的混合补料代替基础的补糖方法，配合发酵周期的相应调整，提高了菌体在发酵过程中传代和代谢的能力，并延长产效价的时间，提高了发酵的最终产出。
附图说明
图1-实施例7发酵工艺放大实验的菌种生长趋势图
具体实施方式
下面结合具体实施例对本发明作进一步的详细说明。以下实施例用于理解本发明的方法和核心思想，对于本领域的技术人员来说，在不脱离本发明构思的前提下，进行任何可能的变化或替换，均属于本发明的保护范围。本发明实施例中未注明具体条件的实验方法，通常为常规条件，或按照原料或商品制造厂商所建议的条件；未注明来源的原料和试剂，通常为通过商业途径可购得的常规试剂。
菌种保存方法
菌种以甘油管和斜面两种形式保藏。
实施例1摇瓶种子的制备(菌种培养)
配方(W/V)：葡萄糖1.0％；麦麸0.5％；硫酸镁0.05％；磷酸二氢钾0.05％；硫酸铵0.2％；泡敌0.05％；甘油0.5％。
配制、分装、灭菌：按其配比所需称取各原料，加饮用水400ml，搅拌均匀，用氢氧化钠溶液调pH至7.0，定容至500ml，后装于3000ml三角瓶中，121℃灭菌30min。
接种、培养：取斜面，用无菌水洗下，以0.1～10.0％的接种量接入种子摇瓶。种子摇瓶培养温度20～30℃，振荡培养24h。
镜检摇瓶种子要求：菌体杆状、菌量多、染色深、无杂菌。
实施例2种子罐的制备(种子培养)
种子罐培养基配方(W/V)：葡萄糖1.0％；麦麸0.5％；硫酸镁0.05％；磷酸二氢钾0.05％；硫酸铵0.2％；泡敌0.05％；甘油0.5％。
按上述工艺配方进行配料,种子罐内加水，边搅拌边投入原料，用6N氢氧化钠溶液调节pH至7.0。
进汽实罐灭菌，罐温120℃，罐压0.09～0.12MPa，保温保压30min。
把经上一步骤制备的摇瓶种子液接入种子罐。接前检测灭菌后种子罐培养基要求为，葡萄糖：0.5～1.5％，pH6.0～7.0。种液于罐压0.04～0.05MPa，罐温20～30℃，pH控制为6.5～7.5。显微镜下观察细胞形态：杆状、菌量多、染色深、无杂菌。
实施例3发酵罐的制备(发酵培养)
发酵培养基配方(W/V)：葡萄糖5.0～6.0％；豆饼粉2.0％；麦麸1.5％；尿素0.15％；硫酸铵0.5％；磷酸二氢钾0.10％；硫酸镁0.06％；氯化钾0.12％；硫酸锌0.2％；甘油1.5％；泡敌0.3％。
按上述工艺配方进行配料，发酵罐内加水，边搅拌边投入原料，用固体氢氧化钠调节pH至6.5～7.0。
进汽实罐灭菌，罐温120℃，罐压0.09～0.12MPa，保温保压30min；进行管道灭菌，灭菌时间60min。用无菌空气把种子液压入发酵罐(接种量为1.0～8.0％)，接种前检查并记录种子情况。接种前检测灭菌后发酵罐培养基，要求：葡萄糖4.0～6.5％，pH6.0～6.5。
发酵液于罐温20～30℃，罐压0.04～0.05MPa，发酵代谢过程中的发酵液pH和葡萄糖含量为主要控制参数。
实施例4补料的制备(补料培养)
补料中葡萄糖配制比例为葡萄糖30～50％；麦麸2.0％；豆饼粉2.0％；甘油0.5％；硫酸镁0.06％；磷酸二氢钾0.05％；硫酸锌0.05％；泡敌0.3％。
进汽实罐灭菌，罐温120℃，罐压0.09～0.12MPa，保温保压30min，灭菌后降温至30～40℃保温。后进行补糖管道灭菌，灭菌时间60min。
发酵培养开始后定时取样检测葡萄糖和pH，当葡萄糖低于1.0％时开始补糖，补糖后pH控制到5.0～6.0，葡萄糖控制在1.5％以下；保持定时取样检测葡萄糖和pH。
实施例5发酵罐与补料配方中碳源与氮源的考察
实验设计：
本次实验意在筛选发酵培养与补料培养物料配方中碳源和氮源的选择。
本实验主要筛选的碳源、氮源及复合碳氮源有：糊精、蛋白胨、麦麸、豆饼粉、生豆粉、玉米浆、酵母粉、尿素，等。
下述每个筛选的实验方案，除发酵罐与补料配方中的碳源氮源的组分与比例外，其余方案均与实施例1～4相同。
实验方案：
实验1
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，酵母粉2.0％，麦麸1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，酵母粉1.5％，麦麸1.5％，泡敌0.3％。
实验2
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，豆饼粉2.0％，麦麸1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，豆饼粉1.5％，麦麸1.5％，泡敌0.3％。
实验3
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，蛋白胨2.0％，麦麸1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，蛋白胨1.5％，麦麸1.5％，泡敌0.3％。
实验4
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，生豆粉2.0％，麦麸1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，生豆粉1.5％，麦麸1.5％，泡敌0.3％。
实验5
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，豆饼粉2.0％，糊精1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，豆饼粉1.5％，糊精1.5％，泡敌0.3％。
实验6
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，豆饼粉2.0％，玉米浆1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，豆饼粉1.5％，玉米浆1.5％，泡敌0.3％。
实验7
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，豆饼粉2.0％，尿素1.0％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，豆饼粉1.5％，尿素0.5％，泡敌0.3％。
实验8
发酵培养基配方(W/V)：葡萄糖5.0～6.0％，生豆粉2.0％，糊精1.5％，尿素0.15％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％；
补料配方(W/V)：葡萄糖30～50％，硫酸锌0.05％，硫酸镁0.06％，磷酸二氢钾0.05％，甘油0.5％，生豆粉1.5％，糊精1.5％，泡敌0.3％。
实验结果：
Figure PCTCN2021141025-appb-000001
实验结论：配方中引入蛋白胨导致发酵效价不佳，配方中尿素过量导致发酵效价不佳；基本确认本发明发酵罐与补料的碳源、氮源、复合碳氮源的选择可以为葡萄糖、甘油、糊精、麦麸、豆饼粉、生豆粉、玉米浆、酵母粉。
实施例6补料配方的考察
发酵基础料培养基配方中物料分别有葡萄糖、豆饼粉、麦麸、尿素、硫酸铵、磷酸二氢钾、硫酸镁、氯化钾、硫酸锌、甘油、泡敌。补料配方是基于发酵基础料培养基，经过筛选进而确定补料罐配方。
由于基础料中硫酸镁比例较低，仅为0.06％，镁元素是许多酶的活化剂,能促进碳水化合物的新陈代谢、核酸的合成、磷酸盐的转化等属于基本元素，只提供基本生长的营养，故补料配方中硫酸镁0.06％比例保持不变。
泡敌只用于消除泡沫，不影响工艺，故补料配方中比例和发酵基础料保持相同。
豆饼粉和麦麸均属于廉价有机氮源，除提供氮源外，还提供大量的无机盐及生长因子。考虑微生物的同化能力，因此需讨论豆饼粉和麦麸在补料配方中的最适比例。
甘油作为一种极性质子溶剂，能完全溶于水和短链醇而不溶于烃类，它可促进水相中多种非有机酸、碱、盐，以及亲水性有机化合物的溶解。此外，甘油与生物酶具有较好的生物相容性，可促进蛋白的正确折叠，稳定蛋白结构，并保持酶的活性，对生物细胞和酶在热和有机溶剂环境下具有一定的保护作用。因此确定甘油在补料配方中的最适比具有必要性。
硫酸锌适量的存在对发酵单位的提高具有一定的促进作用，但加入过量对发酵单位提高没有促进作用，因此需要考虑硫酸锌在补料配方中的最适比例。
实验方案：
现将通过不同比例的配比确认豆饼粉、麦麸、磷酸二氢钾、硫酸锌、甘油对莫匹罗星发酵效价的影响，选取效价高的配方作为发酵过程中补料罐中混合补料最适配方。种子罐在20～30℃条件下培养24h后转接至现有发酵罐中培养，培养6天后放瓶检测发酵效价。
Figure PCTCN2021141025-appb-000002
Figure PCTCN2021141025-appb-000003
按照上述方案开展16次试验，得到16个试验的放罐效价，用正交分析软件进行分析，得出五单因素变量的也影响，
实验结果(发酵效价)：
Figure PCTCN2021141025-appb-000004
Figure PCTCN2021141025-appb-000005
实验分析：
Figure PCTCN2021141025-appb-000006
硫酸锌：均值1的发酵放罐效价8986.75，明显高于其他三组水平，所以硫酸锌的配比选定为水平1，即0.05％；
豆饼粉：均值2的发酵放罐效价9050.75，明显高于其他三组水平，所以豆饼粉的配比选定为水平2，即2.0％；
磷酸二氢钾：均值1、均值2、均值3、均值4的发酵放罐效价分别是7800.75、7763.25、7792.25和6601.25，前三者无明显差异，均值4呈明显下降趋势，随着磷酸二氢钾配比的增加，放罐效价呈现下降趋势，表明磷酸二氢钾配比越高，放罐效价下降的风险增大，所以选择风险最小且放罐效价最高的水平1，即0.05％；
甘油：均值1的发酵放罐效价是8513.00，均值4的发酵放罐效价是8526.25，均值1和均值4的发酵放罐效价较高且无明显差异，鉴于补料配方中已有葡萄糖，甘油和葡萄糖均作为碳源补入，且葡萄糖的比例更大，起到主要碳源的作用，如果甘油使用0.5％和2％比例，发酵放罐效价无明显差异，选择较小的比例即可，二是从成本上考虑；且选择较小的比例生产成本更低，所以选择0.5％比例，因此确定甘油的比例为0.5％；
麦麸：均值1、均值2、均值3、均值4的发酵放罐效价分别是6545.00、8113.00、7142.25、8157.25。随着麦麸配比的增加，放罐效价呈现上升趋势，表明麦麸配比越低，放罐效价下降的风险增大，所以选择选择风险最小且放罐效价最高的水平4，即2.0％。
实验结论：确认本发明补料配方为，葡萄糖30～50％；麦麸2.0％；豆饼粉2.0％；甘油0.5％；硫酸镁0.06％；磷酸二氢钾0.05％；硫酸锌0.05％；泡敌0.3％。
实施例7发酵工艺稳定性的考察
对发酵工艺进行放大的验证，本实施例使用了4吨发酵罐进行试验。根据效价检测结果，确认本发酵工艺的发酵放罐水平的稳定性。
实验方案：
种子培养基配方：葡萄糖1.0％，硫酸镁0.05％，磷酸二氢钾0.05％，硫酸铵0.2％，泡敌0.05％，甘油0.5％，麦麸0.5％，调节pH7.0；
发酵培养基配方：葡萄糖5.5％，豆饼粉2.0％，硫酸铵0.5％，磷酸二氢钾0.10％，硫酸镁0.06％，氯化钾0.12％，硫酸锌0.2％，甘油1.5％，泡敌0.3％，尿素0.15％，麦麸1.0％，调节pH7.0；
补料配方：葡萄糖30～50％，豆饼粉2.0％，磷酸二氢钾0.05％，硫酸镁0.06％，硫酸锌0.05％，甘油0.5％，泡敌0.3％，麦麸2.0％；
摇瓶备种及种子罐配消：按上述工艺配方进行配料,投料后体积为220L，用氢氧化钠溶液调节pH至7.0，灭菌罐温120℃，保温保压30min；
种子罐接前样送检，种子罐接种及发酵罐配消：按上述工艺配方进行配料，投料后体积为800L；用固体氢氧化钠调节pH至7.0，发酵罐接前样送检，进行种子罐培养；
种子罐移种及糖化液配消，糖化液送检，发酵开始培养；
发酵培养：发酵液于罐温20～30℃下培养，移种后第二天早上8:00开始每隔2小时取样检测葡萄糖和pH，当葡萄糖低于1.0％时开始补糖。补糖后pH控制到5.6～6.5，葡萄糖控制在1.5％以下。之后每天8:00起定时检测pH，葡萄糖，效价。
放罐当天早上8:00和放罐时测pH，葡萄糖，8:00开始每隔2小时测效价。发酵培养80～132小时。
实验设计：上述放大化实验方案重复三次，编号为F190304FJ、F190401FJ、F1900402FJ，以发酵时间为横坐标，效价为坐标记录生长曲线。生长曲线的记录于批移种后第三天早上8:00开始，每4小时检测效价，每天8:00和16:00检测pH、葡萄糖。放罐当天8:00和放罐检测pH、葡萄糖，12:00开始每两小时检测效价，直至放罐。
实验结论：从附图1可知，三次放大实验的放罐效价稳定达到8000ug/ml，生产过程稳定可控。

Claims (11)

  1. 一种莫匹罗星的发酵方法，其包括发酵培养和补料培养步骤，所述的补料培养步骤添加的培养基按质量体积比包括碳源25～60％，氮源1～10％，无机盐0.10～0.25％，消泡剂0.1～0.5％，所述的碳源选自葡萄糖、蔗糖、果糖、甘油、糊精、蛋白胨、麦麸、豆饼粉、玉米淀粉、黄豆粉、马铃薯粉、木薯淀粉、玉米浆、酵母粉或甘露醇中的一种或多种；所述的氮源选自氨水、尿素、铵盐、硝酸盐、蛋白胨、麦麸、玉米浆、豆饼粉、花生饼粉、棉籽饼粉或酵母粉中的一种或多种；所述的无机盐选自硫酸盐和/或磷酸盐。
  2. 一种如权利要求1所述的莫匹罗星发酵方法，所述的碳源选自葡萄糖、甘油、糊精、麦麸、豆饼粉、玉米浆或酵母粉中的一种或多种；所述的氮源选自麦麸、豆饼粉、玉米浆或酵母粉中的一种或多种。
  3. 一种如权利要求2任意一项所述的莫匹罗星发酵方法，所述的补料培养步骤添加的培养基按质量体积比包括葡萄糖25～60％，豆饼粉1～5％，麦麸1～5％，甘油0.1～1.0％；
    或，葡萄糖25～60％，酵母粉1～5％，麦麸1～5％，甘油0.1～1.0％；
    或，葡萄糖25～60％，生豆粉1～5％，麦麸1～5％，甘油0.1～1.0％；
    或，葡萄糖25～60％，豆饼粉1～5％，糊精1～5％，甘油0.1～1.0％；
    或，葡萄糖25～60％，豆饼粉1～5％，玉米浆1～5％，甘油0.1～1.0％；
    或，葡萄糖25～60％，生豆粉1～5％，糊精1～5％，甘油0.1～1.0％。
  4. 一种如权利要求1任意一项所述的莫匹罗星发酵方法，所述的补料培养步骤添加的培养基中无机盐为磷酸二氢钾、硫酸镁和硫酸锌，所述的补料培养步骤添加的培养基中按质量体积比包括磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％。
  5. 一种如权利要求1任意一项所述的莫匹罗星发酵方法，所述的补料培养步骤添加的培养基中消泡剂为泡敌，所述的补料培养步骤添加的培养基按质量体积比包括泡敌0.1～0.5％。
  6. 一种如权利要求1所述的莫匹罗星发酵方法，所述的补料培养步骤pH为5.0～6.5。
  7. 一种如权利要求1所述的莫匹罗星发酵方法，所述的发酵培养步骤添加的培养基按质量百分比包括碳源5.0～12.0％，氮源2.0～10.0％，无机盐0.10～1.5％，消泡剂0.1～0.5％；
    所述碳源和氮源选自葡萄糖、甘油、糊精、麦麸、豆饼粉、生豆粉、玉米浆、酵母粉中的一种或多种；
    和/或，无机盐选自硫酸盐、磷酸盐和氯化物；
    和/或，所述发酵培养的pH值为6.0～7.5，优选为6.8～7.5；
    和/或，所述发酵投料后的体积可为0～20000L；
    和/或，所述发酵罐培养的时间为48～144小时，优选为80～132小时；
    和/或，所述发酵罐温度为10～37℃，优选为20～30℃。
  8. 一种如权利要求1所述的莫匹罗星发酵方法，具体包括以下步骤，
    a.摇瓶种子培养：摇瓶种子配置于pH6.0～7.5、分装、灭菌、以0.1～10.0％的接种量接入种子摇瓶并于20～30℃培养12～48小时，得到摇瓶种子菌液；
    b.种子罐培养：将步骤a得到的种子培养配置于pH6.0～7.5、灭菌、种液并于罐温20～30℃培养12～36小时，得到种子菌液；
    c.发酵培养：将步骤b得到的种子菌液接种至发酵培养，调节pH至6.8～7.5，于20～30℃下培养，监控pH和葡萄糖含量并补料，收集产物，得莫匹罗星发酵液。
  9. 一种如权利要求8所述的莫匹罗星发酵方法，所述的发酵培养步骤中接种的种子菌液是经过1～2级种子培养得到的种子菌液，接种的种子菌液的接种量按体积比为1.0～8.0％。
  10. 一种如权利要求7-9任意一项所述的莫匹罗星发酵方法，所述的发酵培养步骤的培养基按质量体积比包括：葡萄糖5.0～6.0％，豆饼粉1.5～3.0％，麦麸0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％；
    或，葡萄糖5.0～6.0％，酵母粉1.5～3.0％，麦麸0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％；
    或，葡萄糖5.0～6.0％，生豆粉1.5～3.0％，麦麸0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％；
    或，葡萄糖5.0～6.0％，豆饼粉1.5～3.0％，糊精0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％；
    或，葡萄糖5.0～6.0％，豆饼粉1.5～3.0％，玉米浆0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％；
    或，葡萄糖5.0～6.0％，生豆粉1.5～3.0％，糊精0.5～2.5％，尿素0.1～0.2％，甘油1.0～2.0％，硫酸铵0.01～1.0％，磷酸二氢钾0.01～0.25％，硫酸镁0.01～0.25％，氯化钾0.01～0.25％，硫酸锌0.01～0.25％，泡敌0.1～0.5％。
  11. 培养基在制备莫匹罗星中的应用，所述的培养基按质量体积比包括：葡萄糖25～60％，豆饼 粉1～5％，麦麸1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％；
    或，所述的补料培养的配方为葡萄糖25～60％，酵母粉1～5％，麦麸1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％；
    或，所述的补料培养的配方为葡萄糖25～60％，生豆粉1～5％，麦麸1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％；
    或，所述的补料培养的配方为葡萄糖25～60％，豆饼粉1～5％，糊精1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％；
    或，所述的补料培养的配方为葡萄糖25～60％，豆饼粉1～5％，玉米浆1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％；
    或，所述的补料培养的配方为葡萄糖25～60％，生豆粉1～5％，糊精1～5％，甘油0.1～1.0％，磷酸二氢钾0.01～0.1％，硫酸镁0.01～0.1％，硫酸锌0.01～0.1％，泡敌0.1～0.5％。
PCT/CN2021/141025 2021-02-07 2021-12-24 一种莫匹罗星的发酵方法 Ceased WO2022166459A1 (zh)

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