CN102268466B - Method for fermentation production of lipstatin and culture medium components thereof - Google Patents
Method for fermentation production of lipstatin and culture medium components thereof Download PDFInfo
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Abstract
The invention provides a method for fermentation production of lipstatin, belonging to the field of fermentation production of microorganisms. The method takes streptomyces toxytricini as an initial bacterium and mainly comprises the steps of preparation of a slope strain, preparation of a seed solution, control of a fermentation process and the like. In the invention, by taking soybean meal, bean oil and glycerol as main nitrogen sources and carbon sources of a fermentation culture medium and regulating and controlling the fermentation process and the components of the culture medium, the production cost of the lipstatin is greatly lowered, and the fermentation titer is increased, thus the defects of high production cost and low fermentation titer in the prior art are overcome.
Description
Technical field
The present invention relates to microorganism fermentation field, be specially a kind of method and media components thereof of fermentative production Lipstatin.
Background technology
The Lipstatin of the fermentative Production the present invention relates to is the intermediate of novel diet pill orlistat.Orlistat is globally unique fat-reducing similar drug at present; it is long-acting and potent specificity gi tract lipase inhibitor; it by with stomach and small intestinal lumen in the active ser position of gastric lipase enzyme and steapsase form covalent linkage and make enzyme deactivation bring into play therapeutic action; the enzyme of inactivation can not, by the fat in food, be mainly that triglyceride hydrolysis is absorbable free fatty acids and monoacylglycerol.Indigested triglyceride level can not be absorbed by health, thereby reduces energy intake, controls body weight.
The production of orlistat has two kinds of methods, fermentation synthesis method and complete synthesizing process.The complete synthesizing process production cost is higher.Fermentation synthesis method production cost is lower, is more suitable for scale operation.
The Lipstatin zymotechnique that U.S. Pat 4598089, US2005089978A1 relate to does not adopt the method for flow feeding and Feeding medium among process, and fermentation level is lower.
The zymotechnique that European patent EP 0S03576A2 and international monopoly WO2007134836A1, WO 2007078263A2 relate to partly adopts stream to add linolic acid and amino acid whose technique, but fermentation level is lower; European patent EP 0803576 fermentation level is lower than 1g/L.
International monopoly WO2004003212 has adopted different starch substrates, and fermentation level is 1.486g/L; International monopoly WO03048335 fermentation level is 1.8g/L.
Summary of the invention
For solving low, the unsettled problem of fermentation level in prior art, the invention provides a kind of substratum and zymotechnique control method and fermention medium moiety of new fermentative production Lipstatin.
Method of the present invention comprises that the slant strains of Streptomyces toxytricini (Streptomyces toxytricini) or derivatives thereof is cultivated, shake-flask seed liquid is cultivated, seed liquor is cultivated and fermentation culture.Described Streptomyces toxytricini (Streptomyces toxytricini) can be ACCC 41038.Specific embodiment is as follows:
A. the preparation of slant strains: the bacterial classification of freeze-drying is equipped with on the inclined-plane of solid medium with coating after the sterilized water dilution, 28 ℃ of temperature, humidity 40～60%, cultivate 8～10 days.Add sterilized water after the product spore and make spore suspension.
B. shake-flask seed liquid preparation: slant pore is inoculated to the shake-flask seed substratum, 250rpm, 28 ℃ of temperature, cultivate 20～30 hours.
C. the preparation of seed liquor: cultured shake-flask seed in step b is inoculated in the seeding tank that contains seed culture medium, 0.1～0.8% (V/V) that inoculum size is the seed culture medium volume, at 28 ℃ of lower air flow 0.8VVM～1.0VVM, the VVM of unit be volume/volume/minute, under the condition of tank pressure 0.04～0.07MPa, cultivate 20～40 hours.
D. fermentation culture: cultured bacterial classification in step c is inoculated in the fermentor tank that contains fermention medium, the amount of inoculation seed liquor accounts for 5～10% (V/V) of fermention medium and seed liquor volume, in temperature, it is 28 ℃, air flow 0.6VVM～1.1VVM, tank pressure 0.06～0.08MPa, in fermenting process, pH is controlled in 6.5～7.5 scopes, fills into nutritive substance in fermenting process, cultivates 160～190 hours.
In step a, used medium composition and content are: glucose 2～8g/L, and malt extract 2～8g/L, yeast extract 4～10g/L, agar 10～20g/L, pH is 6.9～7.4; In step b, used medium composition and content are: glycerine 10g/L, and malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0～6.5; In step c, liquid culture based component used and content are: glycerine 10g/L, and malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0～6.5.In steps d, liquid culture based component used and content are: glycerine 10～20g/L, and defatted soy flour 20～40g/L, soya-bean oil 10～20g/L, defoamer 2～5g/L, the initial pH that ferments is 6.7～7.0.
In the steps d fermenting process, after inoculation to about 30 hours, after being the glycerine approach exhaustion in fermention medium, start flow feeding linolic acid and leucine solution (leucine solution concentration 70g/L, pH is 11), the feed supplement flow acceleration is 0.2～0.5g/L/h, the g/L/h of unit be grams per liter/hour, after the fermentation inoculation, to 115h, feed supplement speed is adjusted, and the feed supplement flow acceleration is 0.2～0.5g/L/h.Simultaneously, during the fermentation, after inoculation, to about 70 hours, disposable yeast extract and the glycerine that fills into sterilizing, made yeast extract in substratum and the glycerine substrate concn level at 5～10g/L.
Using defatted soy flour in ferment tank substratum of the present invention is major nitrogen source, and glycerine and soya-bean oil are main carbon source, in fermention medium, need not add inorganic salt.On fermentation expression Lipstatin stage continuous feeding linolic acid and amino acid whose basis, disposable a certain amount of glycerine and the yeast extract added of specified phase in fermentation, avoided the situation of high viscosity of the prior art, the bad control of dissolved oxygen, improved the stability of expressing in the fermenting process, improve fermentation titer, reduced fermentation costs.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but the present invention is not limited only to following examples.
Embodiment 1
Slant culture:
Prepare substratum in following ratio: glucose 2g/L, malt extract 8g/L, yeast extract 10g/L, agar 15g/L, pH is 7.1.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 30 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 80mL seed liquor (0.8%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.04MPa, cultivate 20 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 10g/L, defatted soy flour 20g/L, soya-bean oil 10g/L, defoamer 2g/L, pH is 7.0.The rear volume that disappears in fact is 33.25 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 5% (1.75 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MP.After the fermentation inoculation, to 27 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase.Now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.3g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.3g/L/h, after fermentation inoculation to 115h, feed supplement speed is adjusted into linolic acid 0.35g/L/h, leucine solution (concentration 70g/L, pH:11) 0.35g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 350 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 180 hours, fermentation titer is 6.7g/L.
Embodiment 2
Slant culture:
Prepare substratum in following ratio: glucose 8g/L, malt extract 4g/L, yeast extract 4g/L, agar 14g/L, pH is 7.4.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 8 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 20 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 20mL seed liquor (0.2%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 1.0VVM (volume/volume/minute), under the condition of tank pressure 0.07MPa, cultivate 40 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 15g/L, defatted soy flour 30g/L, soya-bean oil 10g/L, defoamer 2g/L, pH is 6.7.The rear volume that disappears in fact is 32.20 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 8% (2.80 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa.After the fermentation inoculation, to 29 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase.Now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.2g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.2g/L/h, after fermentation inoculation to 115h, feed supplement speed is adjusted into linolic acid 0.5g/L/h, leucine solution (concentration 70g/L, pH:11) 0.5g/L/h.To 70 hours, fill into yeast extract 230 grams, glycerine 200 grams after sterilizing after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 190 hours, fermentation titer is 6.6g/L.
Embodiment 3
Slant culture:
Prepare substratum in following ratio: glucose 8g/L, malt extract 2g/L, yeast extract 4g/L, agar 12g/L, pH is 7.2.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.05MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 15g/L, defatted soy flour 20g/L, soya-bean oil 10g/L, defoamer 2g/L, pH is 7.0.The rear volume that disappears in fact is 31.50 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 10% (3.50 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa.After the fermentation inoculation, to 29 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase.Now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.32g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.30g/L/h, after fermentation inoculation to 115h, feed supplement speed is adjusted into linolic acid 0.38g/L/h, leucine solution (concentration 70g/L, pH:11) 0.36g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 210 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 160 hours, fermentation titer is 6.9g/L.
Embodiment 4
Slant culture:
Prepare substratum in following ratio: glucose 6g/L, malt extract 6g/L, yeast extract 6g/L, agar 20g/L, pH is 7.0.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.05MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 15g/L, defatted soy flour 30g/L, soya-bean oil 15g/L, defoamer 5g/L, pH is 6.8.The rear volume that disappears in fact is 31.50 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 10% (3.50 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa.After the fermentation inoculation, to 27 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase.Now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.3g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.3g/L/h, after fermentation inoculation to 115h, feed supplement speed is adjusted into linolic acid 0.35g/L/h, leucine solution (concentration 70g/L, pH:11) 0.35g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 200 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 175 hours, fermentation titer is 6.9g/L.
Embodiment 5
Slant culture:
Prepare substratum in following ratio: glucose 5g/L, malt extract 5g/L, yeast extract 6g/L, agar 15g/L, pH is 6.9.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.5.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.5.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.9VVM (volume/volume/minute), under the condition of tank pressure 0.06MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 20g/L, defatted soy flour 40g/L, soya-bean oil 20g/L, defoamer 2g/L, pH is 6.7.The rear volume that disappears in fact is 31.85 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 9% (3.15 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa.After the fermentation inoculation, to 32 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase.Now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.35g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.35g/L/h, after fermentation inoculation to 115h, feed supplement speed is adjusted into linolic acid 0.35g/L/h, leucine solution (concentration 70g/L, pH:11) 0.35g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 240 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 183 hours, fermentation titer is 7.5g/L.
Embodiment 6
Slant culture:
Prepare substratum in following ratio: glucose 4g/L, malt extract 4g/L, yeast extract 6g/L, agar 10g/L, pH is 6.9.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.2.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.05MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 15g/L, defatted soy flour 40g/L, soya-bean oil 20g/L, defoamer 2g/L, pH is 7.0.The rear volume that disappears in fact is 31.50 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 10% (3.50 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa after fermentation inoculation to 30 hours, glycerine has been utilized, start to enter the Lipstatin synthesis phase, now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.32g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.32g/L/h, after the fermentation inoculation, to 115h, feed supplement speed is adjusted into linolic acid 0.35g/L/h, leucine solution (concentration 70g/L, pH:11) 0.35g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 280 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 180 hours, fermentation titer is 7.9g/L.
Comparative Examples 1
Slant culture:
Prepare substratum in following ratio: glucose 4g/L, malt extract 4g/L, yeast extract 6g/L, agar 10g/L, pH is 6.9.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.2.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.05MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum: glycerine 45g/L, bean powder 11g/L, soya-bean oil 11g/L, defoamer 0.55ml, calcium carbonate 2.2g/L, linolic acid 2.2g/L, ferrous sulfate 0.09g/L, zinc sulfate 0.045g/L, tetrahydrate manganese chloride 0.13g/L, magnesium sulfate heptahydrate 0.13g/L, pH is 6.5.The rear volume that disappears in fact is 31.50 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 10% (3.50 liters), under the condition that is 28 ℃ in temperature, in whole process, by adjusting tank pressure, rotating speed, air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa after fermentation inoculation to 32 hours, glycerine has been utilized, start to enter the Lipstatin synthesis phase, now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.32g/L/h, (concentration is 70g/L to leucine solution, PH:11) 0.32g/L/h, after the fermentation inoculation, to 115h, feed supplement speed is adjusted into linolic acid 0.35g/L/h, leucine solution (concentration 70g/L, pH:11) 0.35g/L/h.To 70 hours, fill into yeast extract after sterilizing, glycerine 280 grams respectively after fermentation inoculation.In culturing process, with 20%NaOH, 20%H
2sO
4control PH in 6.5～7.5 scopes.Cultivate 180 hours, fermentation titer is 6.0g/L.
Comparative Examples 2
Slant culture:
Prepare substratum in following ratio: glucose 4g/L, malt extract 6g/L, yeast extract 6g/L, agar 10g/L, pH is 7.0.300 milliliters of substratum of preparation, stir after heating agar melts and pack in 10 Boiling tubes, sterilizing, and beveling is standby.By 5 milliliters of dissolvings of sterilized water for the Streptomyces toxytricini bacterial classification of freeze-drying, get 4 milliliters and be uniformly coated on 10 inclined-planes, 28 ℃ of temperature, humidity 40～60%, cultivate 10 days, produces a large amount of spores.Add sterilized water and make spore suspension.
The shake-flask seed liquid preparation:
Prepare substratum in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.200 milliliters of preparation substratum, dress 4 bottles of triangular flasks of 500mL (every bottled liquid measure 50mL), sterilizing, standby.Spore suspension 2mL is inoculated in shaking flask.28 ℃ of temperature, cultivate 27 hours.
Seed tank culture:
Adopt 15 liters of tanks to carry out seed culture.Prepare seeding tank liquid nutrient medium used (by the real rear 10 liters of preparations that disappear) in following ratio: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0.The rear volume that disappears in fact is 10 liters, when temperature is down to 28 ℃, 50mL seed liquor (0.5%) is inoculated in seeding tank, under the condition that is 28 ℃ in temperature, 400～450 rev/mins of mixing speed, air flow 0.8VVM (volume/volume/minute), under the condition of tank pressure 0.05MPa, cultivate 28 hours.
Fermentor cultivation:
Adopt 50 liters of fermentor tanks to be cultivated.In following ratio obtaining liq substratum (by real disappear and culture transferring after 35 liters of preparations): glycerine 15g/L, defatted soy flour 40g/L, soya-bean oil 15g/L, defoamer 2g/L, pH is 6.9.The rear volume that disappears in fact is 31.5 liters, when temperature is down to 28 ℃, cultured seeding tank bacterial classification is inoculated in fermentor tank, and inoculum size is 10% (3.5 liters), under the condition that is 28 ℃ in temperature, in whole process, by regulating tank pressure rotating speed air flow, control dissolved oxygen in 30～40%.Mixing speed is 300～540 rev/mins, air flow 0.6VVM～1.1VVM (volume/volume/minute), tank pressure 0.06～0.08MPa, use 20%NaOH, 20%H in fermenting process
2sO
4control PH in 6.5～7.5 scopes.After the fermentation inoculation, to 30 hours, glycerine was utilized, and starts to enter the Lipstatin synthesis phase, now start feed supplement linolic acid and leucine solution, the feed supplement flow velocity is linolic acid 0.6g/L/h, leucine solution (concentration is 70g/L, PH:11) 0.6g/L/h.Cultivate 190 hours, fermentation titer is 5.3g/L.
Claims (3)
1.
a kind of method of fermentative production Lipstatin, comprise the preparation of the slant strains of Streptomyces toxytricini ACCC41038, the preparation of shake-flask seed liquid, preparation and the fermentation culture of seed liquor, it is characterized in that comprising the following steps:
a. the preparation of slant strains: the bacterial classification of freeze-drying is equipped with on the inclined-plane of solid medium with coating after the sterilized water dilution, 28 ℃ of temperature, humidity 40～60%, cultivate 8～10 days, adds sterilized water after the product spore and make spore suspension;
b. shake-flask seed liquid preparation: slant pore is inoculated to the shake-flask seed substratum, 250rpm, 28 ℃ of temperature, cultivate 20～30 hours;
c. the preparation of seed liquor: cultured shake-flask seed in step b is inoculated in the seeding tank that contains seed culture medium, inoculum size is 0.1～0.8% of seed culture medium volume, at 28 ℃ of lower air flow 0.8VVM～1.0VVM, the VVM of unit be volume/volume/minute, under the condition of tank pressure 0.04～0.07MPa, cultivate 20～40 hours; Wherein seed culture based component and content are: glycerine 10g/L, and malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0～6.5;
d. fermentation culture: cultured bacterial classification in step c is inoculated in the fermentor tank that contains fermention medium, the amount of inoculation seed liquor accounts for 5～10% of fermention medium and seed liquor volume, in temperature, it is 28 ℃, air flow 0.6VVM～1.1VVM, tank pressure 0.06～0.08MPa, in fermenting process, pH is controlled in 6.5～7.5 scopes, cultivate 160～190 hours, after fermentation inoculation to 30 hours about, after glycerine approach exhaustion in fermention medium, starting stream adds and fills into linolic acid and leucine solution, wherein the concentration of leucine solution is 70g/L, pH is 11, the feed supplement flow velocity of the two is 0.2～0.5g/L/h, the g/L/h of unit be grams per liter/hour, after the fermentation inoculation, to 115 hours, feed supplement speed was adjusted, and feed supplement speed is at 0.2～0.5g/L/h, after inoculation, to 70 hours, disposable yeast extract and the glycerine that fills into sterilizing, made yeast extract in substratum and the glycerine substrate concn level at 5～10g/L, wherein fermentation culture based component and content are: glycerine 10～20g/L, and defatted soy flour 20～40g/L, soya-bean oil 10～20g/L, defoamer 2～5g/L, pH is 6.7～7.0.
2.
the method of fermentative production Lipstatin according to claim 1, the composition and the content that it is characterized in that solid medium in described step a are: glucose 2～8g/L, malt extract 2～8g/L, yeast extract 4～10g/L, agar 10～20g/L, pH is 6.9～7.4.
3.
the method of fermentative production Lipstatin according to claim 1, it is characterized in that in described step b that shake-flask seed medium component and content are: glycerine 10g/L, malt extract 10g/L, yeast extract 5g/L, defatted soy flour 20g/L, pH is 6.0～6.5.
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| CN103468603A (en) * | 2013-07-17 | 2013-12-25 | 杭州华东医药集团生物工程研究所有限公司 | Streptomyces toxytricini and applications of streptomyces toxytricini in preparation of lipstatin |
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| CN102965407A (en) * | 2012-11-28 | 2013-03-13 | 广州明新医药科技有限公司 | Production process of Lipstatin |
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| CN108753861B (en) * | 2018-06-08 | 2022-02-01 | 福建省微生物研究所 | Culture medium and method for producing lipstatin by fermenting streptomyces toxytricini |
| CN111979277A (en) * | 2020-07-27 | 2020-11-24 | 河北圣雪大成制药有限责任公司 | Culture medium for producing lipstatin and using method thereof |
| CN112852898B (en) * | 2021-03-19 | 2022-07-22 | 大邦(湖南)生物制药有限公司 | Lipstatin fermentation method and fermentation medium |
| CN114958625B (en) * | 2022-07-07 | 2023-12-19 | 福安药业集团烟台只楚药业有限公司 | Culture and preservation method of aspergillus terreus for producing lovastatin by fermentation |
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