CN108949866A - Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12 - Google Patents

Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12 Download PDF

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CN108949866A
CN108949866A CN201810880607.1A CN201810880607A CN108949866A CN 108949866 A CN108949866 A CN 108949866A CN 201810880607 A CN201810880607 A CN 201810880607A CN 108949866 A CN108949866 A CN 108949866A
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fermentation
agitator
speed
vitamin
fermenting
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胡勇干
王鑫
刘延茂
李会
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GUANGJI PHARMACEUTICAL (MENGZHOU) CO Ltd
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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/42—Cobalamins, i.e. vitamin B12, LLD factor

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Abstract

The invention belongs to technical field of biological fermentation, disclose multistage rotation speed regulating and controlling strategy and improve denitrified pseudomonas fermenting and producing vitamin B12By denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, contain in fermentation medium: 70~85g/L of sucrose, 40~55g/L of corn pulp, 10~20g/L of glycine betaine, 0.05~0.1g/L of 0.02~0.05g/L of auxin, 0.01~0.05g/L of tryptophan and glutamine etc., the 0-25h of fermentation, speed of agitator is 150-280rpm, the 26-90h of fermentation, speed of agitator 300-400rpm, the 91-185h of fermentation, speed of agitator 100-200rpm；Vitamin B is isolated and purified out from fermentation liquid12.By the control to speed of agitator and fermentation medium component, vitamin B is improved12Yield.

Description

Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12
Technical field
The invention belongs to technical field of biological fermentation, and in particular to a kind of multistage rotation speed regulating and controlling strategy raising denitrogenation is false single Born of the same parents' bacillus fermentation produces vitamin B12。
Background technique
Vitamin B12Also known as cobalamin, it is a kind of important animals and humans trophic factors, is widely used in feed, food And pharmaceutical sanitary field, there is remarkable effect at many aspects, such as promotes the development and maturation of red blood cell, prevent pernicious poor Blood；Safeguard nervous system health；Exist in the form of coenzyme, the utilization rate of folic acid can be increased, promotes carbohydrate, fat With the metabolism of protein；Also the synthesis that can promote protein plays an important role to the growth and development of infant；Agitation can also be eliminated Uneasiness focuses on, enhances memory and the sense of equilibrium, and vitamin B12A kind of formation of lipoprotein in nerve fiber is participated in, It is that nervous function perfects indispensable vitamin.
Vitamin B is produced using chemical synthesis12Method it is at high cost, by-product is more, it is difficult to purify.Vitamin B12Work Industry manufacture mainly by microbe fermentation method be made, applied to industrial bacterial strain mainly have anaerobism propionibacterium freudenreichii, De- single Pseudomonas of Xie Shi Propionibacterium and oxygen consumption.Vitamin B is produced by anaerobic fermentation12Yield it is lower, it is practical raw Production is substantially carried out aerobic fermentation.The mainly de- single Pseudomonas of the bacterial strain of aerobic fermentation, culture medium is with beet molasses, malt Sugar, sucrose are carbon source, using corn pulp and yeast extract as nitrogen source, add inorganic salts and Co, and add 5,6- dimethylbenzimidazole And glycine betaine is as precursor.By controlling zymotechnique, vitamin B is produced12, how to improve vitamin B12Yield and production Efficiency is to meet the continuous pursuit that the market demand is the field.
Summary of the invention
For above situation, the object of the present invention is to provide a kind of multistage rotation speed regulating and controlling strategies to improve denitrogenation vacation unit cell bar Bacterium fermenting and producing vitamin B12, by regulating and controlling to the speed of agitator in fermentation process, and the adjustment to fermentation medium, Realize vitamin B12The raising of yield.
The present invention provides a kind of multistage rotation speed regulating and controlling strategy raising denitrified pseudomonas fermenting and producing vitamin B12, packet Include following steps:
1) by denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, vitamin B is produced12, the fermented and cultured of use Contain in base: 70~85g/L of sucrose, 40~55g/L of corn pulp, 10~20g/L of glycine betaine, 0.5~1.5g/L of ammonium sulfate, phosphoric acid 0.5~1g/L of potassium dihydrogen, 0.05~0.1g/L of cobalt chloride, 0.4~0.6g/L of magnesia, 5,6~dimethylbenzimidazole 0.04 ~0.06g/L, 0.05~0.1g/L of zinc sulfate, 0.8~1.2g/L of calcium carbonate, 0.02~0.05g/L of auxin, tryptophan 0.01 0.05~0.1g/L of~0.05g/L and glutamine, the pH of fermentation medium are 7.2-7.4；In fermentation process, to speed of agitator Carry out control by stages, the 0-25h of fermentation, speed of agitator 150-280rpm, the 26-90h of fermentation, speed of agitator 300- 400rpm, the 91-185h of fermentation, speed of agitator 100-200rpm；
2) vitamin B is isolated and purified out from the fermentation liquid that step 1) obtains12。
Under preferable case, contain in the fermentation medium: 75~85g/L of sucrose, 40~50g/L of corn pulp, glycine betaine 12~18g/L, 0.8~1.2g/L of ammonium sulfate, 0.5~0.8g/L of potassium dihydrogen phosphate, 0.05~0.08g/L of cobalt chloride, magnesia 0.4~0.6g/L, 5,6~dimethylbenzimidazole, 0.04~0.06g/L, 0.05~0.08g/L of zinc sulfate, calcium carbonate 0.8~ 1.0g/L, 0.06~0.1g/L of 0.02~0.05g/L of auxin, 0.02~0.05g/L of tryptophan and glutamine, fermented and cultured The pH of base is 7.2-7.4.
The present invention is conducive to the growth hair of denitrified pseudomonas bacterial strain by the fermentation medium of selection specific composition Ferment.Wherein the addition of auxin, tryptophan and glutamine can improve the permeability of denitrified pseudomonas, enhance simultaneously The transmission speed of oxygen improves effective use of the thallus to oxygen.
Under preferable case, the 0-25h of fermentation, speed of agitator 150-220rpm, the 26-90h of fermentation, speed of agitator is 300-400rpm, the 91-185h of fermentation, speed of agitator 100-150rpm.
It is further preferred that the 0-25h, speed of agitator 150-180rpm, the 26-90h of fermentation of fermentation, speed of agitator are 350-380rpm, the 91-185h of fermentation, speed of agitator 100-120rpm.
In the present invention, the condition of fermentation further includes the general parameters condition of denitrified pseudomonas aerobic fermentation process, example If fermentation temperature can be 28-35 DEG C, pH value 6.8-7.2.
Preferably, it is 30-33 DEG C, Ventilation Rate 0.8-1.2LPM, pH value 7- that fermentation condition, which includes: fermentation temperature, 7.2。
The conventional means of this field can be used in the fermentation parameter not being limited in the present invention.
In the present invention, vitamin B is isolated and purified out from fermentation liquid12It is pure that conventional use of separation in the prior art can be used Change method.
Compared with prior art, the present invention has following beneficial to point:
The present invention adjusts the composition of fermentation medium by regulating and controlling to the speed of agitator in fermentation process, realizes dimension Raw element B12The raising of yield.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the embodiment of the present invention, it is clear that retouched The embodiment stated is only a part of the embodiment of the present invention, instead of all the embodiments.
Embodiment 1-3 is used to illustrate that multistage rotation speed regulating and controlling strategy of the invention to improve denitrified pseudomonas fermenting and producing Vitamin B12。
Embodiment 1
Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12, comprising the following steps:
1) by denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, vitamin B is produced12, the fermented and cultured of use Contain in base: sucrose 80g/L, corn pulp 45g/L, glycine betaine 14g/L, ammonium sulfate 1g/L, potassium dihydrogen phosphate 0.6g/L, cobalt chloride 0.06g/L, magnesia 0.5g/L, 5,6~dimethylbenzimidazole 0.05g/L, zinc sulfate 0.06g/L, calcium carbonate 0.8g/L, life Long element 0.03g/L, tryptophan 0.04g/L and glutamine 0.08g/L, the pH of fermentation medium are 7.2-7.4；Fermentation process In, control by stages, the 0-25h of fermentation, speed of agitator 160rpm, the 26-90h of fermentation, stirring turn are carried out to speed of agitator Speed is 370rpm, the 91-185h of fermentation, speed of agitator 100rpm；Fermentation condition further include: fermentation temperature is 30 DEG C, ventilation Rate is 1LPM, pH value 7；
2) vitamin B is isolated and purified out from the fermentation liquid that step 1) obtains12。
Using vitamin B obtained by the above method12Fermentation titer be 246 μ g/mL.
Embodiment 2
Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12, comprising the following steps:
1) by denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, vitamin B is produced12, the fermented and cultured of use Contain in base: sucrose 75g/L, corn pulp 40g/L, glycine betaine 12g/L, ammonium sulfate 0.8g/L, potassium dihydrogen phosphate 0.5g/L, chlorination Cobalt 0.05g/L, magnesia 0.4g/L, 5,6~dimethylbenzimidazole 0.04g/L, zinc sulfate 0.05g/L, calcium carbonate 0.8g/L, Auxin 0.02g/L, tryptophan 0.02g/L and glutamine 0.06g/L, the pH of fermentation medium are 7.2-7.4；Fermentation process In, control by stages, the 0-25h of fermentation, speed of agitator 150rpm, the 26-90h of fermentation, stirring turn are carried out to speed of agitator Speed is 300rpm, the 91-185h of fermentation, speed of agitator 100rpm；Fermentation condition further include: fermentation temperature is 30 DEG C, ventilation Rate is 0.8LPM, pH value 7.2；
2) vitamin B is isolated and purified out from the fermentation liquid that step 1) obtains12。
Using vitamin B obtained by the above method12Fermentation titer be 238 μ g/mL.
Embodiment 3
Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12, comprising the following steps:
1) by denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, vitamin B is produced12, the fermented and cultured of use Contain in base: sucrose 85g/L, corn pulp 50g/L, glycine betaine 18g/L, ammonium sulfate 1.2g/L, potassium dihydrogen phosphate 0.8g/L, chlorination Cobalt 0.08g/L, magnesia 0.6g/L, 5,6~dimethylbenzimidazole 0.06g/L, zinc sulfate 0.08g/L, calcium carbonate 1.0g/L, Auxin 0.05g/L, tryptophan 0.05g/L and glutamine 0.1g/L, the pH of fermentation medium are 7.2-7.4；Fermentation process In, control by stages, the 0-25h of fermentation, speed of agitator 200rpm, the 26-90h of fermentation, stirring turn are carried out to speed of agitator Speed is 380rpm, the 91-185h of fermentation, speed of agitator 120rpm；Fermentation condition further include: fermentation temperature is 32 DEG C, ventilation Rate is 1.2LPM, pH value 7；
2) vitamin B is isolated and purified out from the fermentation liquid that step 1) obtains12。
Using vitamin B obtained by the above method12Fermentation titer be 243 μ g/mL.
Comparative example 1
Difference from example 1 is that stirring rate is controlled in 160rpm, remaining step phase during the entire process of fermentation Together.
Using vitamin B obtained by the above method12Fermentation titer be 210 μ g/mL.
Comparative example 2
Difference from example 1 is that stirring rate is controlled in 370rpm, remaining step phase during the entire process of fermentation Together.
Using vitamin B obtained by the above method12Fermentation titer be 223 μ g/mL.
Comparative example 3
Difference from example 1 is that not containing auxin in fermentation medium, remaining step is identical.
Using vitamin B obtained by the above method12Fermentation titer be 234 μ g/mL.
Comparative example 4
Difference from example 1 is that not containing tryptophan and glutamine in fermentation medium, remaining step is identical.
Using vitamin B obtained by the above method12Fermentation titer be 230 μ g/mL.
It compares it is found that the present invention to the speed of agitator progress control by stages in fermentation process, and in fermentation by training It supports and adds auxin, tryptophan and glutamine in base, it can be achieved that vitamin B12The raising of yield.
The embodiment of the present invention is described above, above description is exemplary, and non-exclusive, and also not It is limited to disclosed embodiment.Without departing from the scope and spirit of illustrated embodiment, for the art Many modifications and changes are obvious for those of ordinary skill.

Claims (5)

1. multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12, which is characterized in that including with Lower step:
1) by denitrified pseudomonas bacterial strain fermented and cultured in the fermentation medium, vitamin B is produced12, the fermented and cultured of use Contain in base: 70~85g/L of sucrose, 40~55g/L of corn pulp, 10~20g/L of glycine betaine, 0.5~1.5g/L of ammonium sulfate, phosphoric acid 0.5~1g/L of potassium dihydrogen, 0.05~0.1g/L of cobalt chloride, 0.4~0.6g/L of magnesia, 5,6~dimethylbenzimidazole 0.04 ~0.06g/L, 0.05~0.1g/L of zinc sulfate, 0.8~1.2g/L of calcium carbonate, 0.02~0.05g/L of auxin, tryptophan 0.01 0.05~0.1g/L of~0.05g/L and glutamine, the pH of fermentation medium are 7.2-7.4；In fermentation process, to speed of agitator Carry out control by stages, the 0-25h of fermentation, speed of agitator 150-280rpm, the 26-90h of fermentation, speed of agitator 300- 400rpm, the 91-185h of fermentation, speed of agitator 100-200rpm；
2) vitamin B is isolated and purified out from the fermentation liquid that step 1) obtains12。
2. multistage rotation speed regulating and controlling strategy according to claim 1 improves denitrified pseudomonas fermenting and producing vitamin B12, it is characterised in that: contain in the fermentation medium: 75~85g/L of sucrose, 40~50g/L of corn pulp, glycine betaine 12~ 18g/L, 0.8~1.2g/L of ammonium sulfate, 0.5~0.8g/L of potassium dihydrogen phosphate, 0.05~0.08g/L of cobalt chloride, magnesia 0.4~ 0.6g/L, 5,6~dimethylbenzimidazole, 0.04~0.06g/L, 0.05~0.08g/L of zinc sulfate, 0.8~1.0g/ of calcium carbonate L, 0.06~0.1g/L of 0.02~0.05g/L of auxin, 0.02~0.05g/L of tryptophan and glutamine, fermentation medium PH is 7.2-7.4.
3. multistage rotation speed regulating and controlling strategy according to claim 1 improves denitrified pseudomonas fermenting and producing vitamin B12, it is characterised in that: the 0-25h of fermentation, speed of agitator 150-220rpm, the 26-90h of fermentation, speed of agitator 300- 400rpm, the 91-185h of fermentation, speed of agitator 100-150rpm.
4. multistage rotation speed regulating and controlling strategy according to claim 3 improves denitrified pseudomonas fermenting and producing vitamin B12, it is characterised in that: the 0-25h of fermentation, speed of agitator 150-180rpm, the 26-90h of fermentation, speed of agitator 350- 380rpm, the 91-185h of fermentation, speed of agitator 100-120rpm.
5. multistage rotation speed regulating and controlling strategy according to claim 1 improves denitrified pseudomonas fermenting and producing vitamin B12, which is characterized in that fermentation condition includes: that fermentation temperature is 30-33 DEG C, Ventilation Rate 0.8-1.2LPM, pH value 7- 7.2。
CN201810880607.1A 2018-08-04 2018-08-04 Multistage rotation speed regulating and controlling strategy improves denitrified pseudomonas fermenting and producing vitamin B12 Pending CN108949866A (en)

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Application publication date: 20181207