CN102154426B - Industrial fermentation method of riboflavin - Google Patents
Industrial fermentation method of riboflavin Download PDFInfo
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- CN102154426B CN102154426B CN 201010608768 CN201010608768A CN102154426B CN 102154426 B CN102154426 B CN 102154426B CN 201010608768 CN201010608768 CN 201010608768 CN 201010608768 A CN201010608768 A CN 201010608768A CN 102154426 B CN102154426 B CN 102154426B
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Abstract
The invention discloses an industrial fermentation method of riboflavin, which comprises the following steps of: filling a seed culture medium into a 10m<3> seed tank, and performing oxic cultivation; and filling a fermentation medium into a 200m<3> fermentation tank for fermentation, mechanically stirring, adding sterilization starch glucose liquid, stopping fermenting till that the glucose is completely consumed or the dissolved oxygen is raised to be 60%, and extracting the riboflavin from the fermentation liquid. After the industrial fermentation method is used, the conventional riboflavin material-supplementing fermentation technology is changed, the low-concentration raw material cultivating bacteria is adopted, the material is quantificationally supplemented according to the change of the dissolved oxygen of the fermentation solution, the excessive multiplication of the thallus can be effectively controlled, the deviation of the optimum control temperature caused by the excessive supersession can be avoided, the defects of the insufficient ventilation and oxygen supply can be remitted, and the thallus vigor can be kept, so that the indusial fermentation unit of the riboflavin is improved by more than 18g/L, and the yield is increased by 20% compared with the common fermentation.
Description
Technical field
The present invention relates to a kind of method of riboflavin industrial fermentation.
Background technology
The three grade fermemtation method is adopted in the microbial fermentation production of riboflavin.The slant pore of cultivating ripe Strains Producing Riboflavin is made spore suspension with sterilized water, be inoculated in the seed culture medium and cultivate, at last second order fermentation liquid culture transferring to three grade fermemtation tank fermentation is obtained Lactochrome fermentation liquor.The raw material that different IPs flavine manufacturer uses is not quite similar.At present, the main carbon source of microbe fermentation method employing is vegetables oil, glucose, molasses or rice candy etc.; Organic nitrogen source is take peptone, gelatine, fish meal, corn steep liquor as main, and inorganic salt have NaCl, K
2HPO
4, MgSO
4Deng.Used raw material causes cost high in industrial application, and supply is difficult to ensure.
The biosynthesizing of riboflavin is the metabolic process of a complexity with accumulation, is subject to carbon source, nitrogenous source etc. and is permitted multifactorial impact.Therefore, the same with seed selection riboflavin high-yield strains, the optimization for fermentation technology condition also is one of main factor that improves output and economic benefit.
Existing riboflavin fed-batch fermentation technique is a continuous feed supplement process, is how much content according to reducing sugar in the fermented liquid carries out flow feeding.This feed supplement mode is subjected to impact that ambient systems changes greatly, causes the reducing sugar content in the fermented liquid fluctuated, unstable, is unfavorable for the production of riboflavin.
Summary of the invention
The method that the purpose of this invention is to provide a kind of riboflavin industrial fermentation.
Technical scheme of the present invention is: the method for riboflavin industrial fermentation, and its step is as follows:
(1) seed culture medium is packed into 10 m
3Seeding tank, after sterilization is finished, the bacillus subtilis spore suspension that access makes, aerobic cultivation 16 ~ 18 hours, the condition of seed culture medium is: biological nitrogen 15g/L, corn steep liquor 0.2g/L, glucose 5g/L, pH6.8 ~ 7.0;
(2) fermention medium is packed into 200m
3Fermentor tank adds the defoamer that 40kg has sterilized, and the condition of fermention medium is: biological nitrogen 3g/L, corn steep liquor 1.2g/L, trimethyl-glycine 0.01g/L, pH6.8 ~ 7.2;
(3) passing under the aseptic uncontaminated air condition, the starch Glucose Liquid that disposable adding disinfects, make glucose concn reach 2g/L, after the sterilization, move into cultured seed liquor, begin fermentation, keeping leavening temperature is 38 ~ 40 ℃, regulate pH as 6.7 ~ 6.9 take liquefied ammonia, the control ventilation ratio is 0.8, makes dissolved oxygen amount reach 20 ~ 30%.
(4) after fermentation is carried out 6 hours, measure the residual sugar amount, till glucose has consumed to the tank body, kept again 1 ~ 2 hour, then start mechanical stirring, add sterilization starch Glucose Liquid, until glucose consumption is complete or dissolved oxygen amount when rising to 60%, stuck fermentation, fermented liquid enter abstraction process and extract riboflavin.
The defoamer consumption accounts for 0.02% ~ 0.05% of total quality of material in the described step (2).
Start mechanical stirring in the described step (4), flow velocity stream with 600kg/hr adds 60% sterilization starch Glucose Liquid, flow acceleration at the uniform velocity is incremented to 800kg/hr in 15 hours, stirring velocity is incremented to 126rpm by 95rpm, after stream adds 15 hours, flow acceleration remains on about 850kg/hr, stirring velocity keeps 126rpm, in the fed batch fermentation process, keep 37 ~ 39 ℃ of leavening temperatures, regulate pH 7 ~ 7.2, control ventilation ratio VVM1.0 ~ 1.5, keep dissolved oxygen amount more than 20%, from flow feeding, measured a deuterolactoflavin in per 8 hours to tire, until 38 ~ 42 hours given glucose consumptions are complete or dissolved oxygen amount when rising to 60%, stuck fermentation, fermented liquid enter abstraction process and extract riboflavin.
In described step (4) fermenting process, replenish the water yield of evaporation with sterilized water, make volume be controlled at 110 ~ 130 m
3, and keep tank pressure 0.03 ~ 0.10MPa.
The present invention is fermented in defined medium take bacillus subtilis as fermentation strain, namely adopts first the lower concentration raw material to cultivate thalline: the feed supplement thing of disposable interpolation some amount, then inoculation fermentation in the good fermention medium of sterilization; The rear variation according to the fermented liquid dissolved oxygen carries out quantitative material supplementing.Fermenting process is divided into disposable feed supplement stage and continuous and quantitative feed supplement stage.
Its processing condition are as follows:
(1) seed culture medium:
Biological nitrogen 15g/l, corn steep liquor 0.2g/l, glucose 5g/l, pH6.8 ~ 7.0.
(2) fermention medium
Biological nitrogen 3g/l, corn steep liquor 1.2g/l, trimethyl-glycine 0.01g/l, pH6.8 ~ 7.2.
(3) the disposable feed supplement stage: 39 ± 1.0 ℃ of temperature, pH6.8 ± 0.1, pO
2＞=20%, tank pressure 0.03 ~ 0.10MPa, 5 ~ 8 hours time.Pneumatic blending, ventilation ratio V V M is 0.8.
(4) the continuous and quantitative feed supplement stage: 38 ± 1.0 ℃ of temperature, pH7.1 ± 0.1, pO
2＞=20%, tank pressure 0.03 ~ 0.10MPa, 40 ~ 42 hours time.Pneumatic blending and 95 ~ 126rpm mechanical stirring, ventilation ratio VVM1.0 ~ 1.5.
(5) pH regulator: use liquefied ammonia to regulate pH, regulate with phosphoric acid in case of necessity.
(6) defoamer consumption: control by 0.02% ~ 0.05% of total inventory.
(7) feed supplement liquid: 50% ~ 60% W-Gum liquid glucose.
(8) the residual sugar amount requires regulation and control 0.1% ~ 2.0%.
The beneficial effect of the invention is: the present invention is optimized process control condition by starting with from the factors such as temperature, pH value, residual sugar amount, dissolved oxygen amount, logical ammonia amount and feed supplement mode that affect fermentation stability.Changed traditional riboflavin fed-batch fermentation technique, adopt first the lower concentration raw material to cultivate thalline, the rear variation according to the fermented liquid dissolved oxygen carries out quantitative material supplementing, not only can effectively control the thalline hyper-proliferative, avoid the excessive vigorous off-target control temperature of metabolism, alleviate and ventilate for the hypoxgia problem, but also can keep the thalline vigor, the industrial fermentation unit of riboflavin is brought up to more than the 18g/L, approximately than common fermentation volume increase 20%.In addition, riboflavin fed-batch fermentation technique after the optimization also can better allow microcomputer automatically preferentially control, avoid the variation because of external environmental condition and human factor, and affected the process optimization direction, and simplified manual operation program and operative employee's labour intensity, reach stably manufactured, increase production and improve productivity, improve the fed-batch fermentation processing condition of riboflavin, improve resultant velocity, the shortening fermentation period of riboflavin, increase fermentation unit, reduce production costs.
Embodiment
Embodiment
The method of riboflavin industrial fermentation, its step is as follows:
(1) seed culture medium is packed into 10 m
3Seeding tank, after sterilization is finished, the bacillus subtilis spore suspension that access makes, aerobic cultivation 16 ~ 18 hours, the condition of seed culture medium is: biological nitrogen 15g/L, corn steep liquor 0.2g/L, glucose 5g/L, pH 6.8 ~ 7.0;
(2) fermention medium is packed into 200m
3Fermentor tank adds the defoamer that 40kg has sterilized, and the defoamer consumption accounts for 0.02% ~ 0.05% of total quality of material, and the condition of fermention medium is: biological nitrogen 3g/L, corn steep liquor 1.2g/L, trimethyl-glycine 0.01g/L, pH6.8 ~ 7.2;
(3) passing under the aseptic uncontaminated air condition, the starch Glucose Liquid that disposable adding disinfects, make glucose concn reach 2g/L, after the sterilization, move into cultured seed liquor, begin fermentation, keeping leavening temperature is 38 ~ 40 ℃, regulate pH as 6.7 ~ 6.9 take liquefied ammonia, the control ventilation ratio is 0.8, makes dissolved oxygen amount reach 20 ~ 30%;
(4) after fermentation is carried out 6 hours, measure the residual sugar amount, till glucose has consumed to the tank body, kept again 1 ~ 2 hour, then start mechanical stirring, flow velocity stream with 600 kg/hr adds 60% sterilization starch Glucose Liquid, and flow acceleration at the uniform velocity is incremented to 800 kg/hr in 15 hours, and stirring velocity is incremented to 126 rpm by 95 rpm, after stream adds 15 hours, flow acceleration remains on about 850 kg/hr, and stirring velocity keeps 126 rpm, in the fed batch fermentation process, keep 37 ~ 39 ℃ of leavening temperatures, regulate pH 7 ~ 7.2, control ventilation ratio VVM 1.0 ~ 1.5 keeps dissolved oxygen amount more than 20%, from flow feeding, measured a deuterolactoflavin in per 8 hours and tire, until 38 ~ 42 hours given glucose consumptions are complete or dissolved oxygen amount when rising to 60%, stuck fermentation, fermented liquid enters abstraction process and extracts riboflavin, in the fermenting process, replenish the water yield of evaporation with sterilized water, make volume be controlled at 110 ~ 130 m
3, and keep tank pressure 0.03 ~ 0.10MPa.
Claims (1)
1. the method for riboflavin industrial fermentation is characterized in that, its step is as follows:
(1) seed culture medium is packed into 10 m
3Seeding tank, after sterilization is finished, the subtilis spore suspension that access makes, aerobic cultivation 16 ~ 18 hours, seed culture medium is: biological nitrogen 15g/L, corn steep liquor 0.2g/L, glucose 5g/L, pH6.8 ~ 7.0;
(2) fermention medium is packed into 200m
3Fermentor tank adds the defoamer that 40kg has sterilized, and fermention medium is: biological nitrogen 3g/L, and corn steep liquor 1.2g/L, trimethyl-glycine 0.01g/L, pH6.8 ~ 7.2, described defoamer consumption accounts for 0.02% ~ 0.05% of total quality of material;
(3) passing under the aseptic uncontaminated air condition, the starch Glucose Liquid that disposable adding disinfects, make glucose concn reach 2g/L, after the sterilization, move into cultured seed liquor, begin fermentation, keeping leavening temperature is 38 ~ 40 ℃, regulate pH as 6.7 ~ 6.9 take liquefied ammonia, the control ventilation ratio is 0.8 VVM, makes dissolved oxygen amount reach 20 ~ 30%;
(4) after fermentation is carried out 6 hours, measure the residual sugar amount, till glucose has consumed to the tank body, kept again 1 ~ 2 hour, then start mechanical stirring, flow velocity stream with 600kg/hr adds 60% sterilization starch Glucose Liquid, and flow acceleration at the uniform velocity is incremented to 800kg/hr in 15 hours, and stirring velocity is incremented to 126rpm by 95rpm, after stream adds 15 hours, flow acceleration remains on about 850kg/hr, and stirring velocity keeps 126rpm, in the fed batch fermentation process, keep 37 ~ 39 ℃ of leavening temperatures, regulate pH 7 ~ 7.2, dissolved oxygen amount is kept more than 20% in control ventilation ratio VVM1.0 ~ 1.5, from flow feeding, measured a deuterolactoflavin in per 8 hours and tire, until 38 ~ 42 hours given glucose consumptions are complete or dissolved oxygen amount when rising to 60%, stuck fermentation, fermented liquid enters abstraction process and extracts riboflavin, in the fermenting process, replenish the water yield of evaporation with sterilized water, make volume be controlled at 110 ~ 130 m
3, and keep tank pressure 0.03 ~ 0.10MPa.
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| CN 201010608768 CN102154426B (en) | 2010-12-28 | 2010-12-28 | Industrial fermentation method of riboflavin |
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Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102816823B (en) * | 2012-09-18 | 2014-10-22 | 江南大学 | Method for raising output of Bacillus subtilis fermentation produced riboflavin by utilization of multi-stage stirring speed regulation strategy |
| CN103695519B (en) * | 2013-12-23 | 2016-08-17 | 广济药业（孟州）有限公司 | Produce nitrogen source, culture medium and the cultural method of riboflavin |
| CN103952419B (en) * | 2014-04-15 | 2016-06-29 | 天津大学 | Bacillus subtilis adenosine succinic acid synthase mutant gene purA and application |
| CN106566862A (en) * | 2016-06-26 | 2017-04-19 | 宁夏海诚电化信息科技有限公司 | Riboflavin production process |
| CN106434818A (en) * | 2016-12-22 | 2017-02-22 | 广济药业（孟州）有限公司 | Fermentation medium for synthesizing riboflavin by bacillus subtilis |
| CN106434819A (en) * | 2016-12-22 | 2017-02-22 | 广济药业（孟州）有限公司 | Method for increasing yield of riboflavin produced by fermentation of bacillus subtilis |
| CN108795811A (en) * | 2018-06-21 | 2018-11-13 | 赤峰制药股份有限公司 | A kind of riboflavin production bacterium culture medium |
| CN108913747A (en) * | 2018-07-28 | 2018-11-30 | 广济药业（孟州）有限公司 | A kind of high density fermentation vitamin B2Method |
| CN109402210A (en) * | 2018-12-12 | 2019-03-01 | 宁夏启元药业有限公司 | Culture medium and method used in a kind of riboflavin clean manufacturing |
| CN110777184B (en) * | 2019-11-27 | 2023-03-03 | 河北圣雪大成制药有限责任公司 | Fermentation medium for fermenting riboflavin and application method thereof |
| CN112280679A (en) * | 2020-11-04 | 2021-01-29 | 赤峰制药股份有限公司 | Method for producing vitamin B2 by fermentation method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2923666A (en) * | 1954-02-05 | 1960-02-02 | Pabst Brewing Co | Production of vitamin b12 |
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2010
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2923666A (en) * | 1954-02-05 | 1960-02-02 | Pabst Brewing Co | Production of vitamin b12 |
Non-Patent Citations (2)
| Title |
|---|
| 不同流加发酵方式对重组枯草芽胞杆菌生产维生素B2的影响;陈涛 等;《天津大学学报》;20060630;第39卷;7-12 * |
| 陈涛 等.不同流加发酵方式对重组枯草芽胞杆菌生产维生素B2的影响.《天津大学学报》.2006,第39卷7-12. |
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