CN101402979A - High-efficiency method for fermentation production of L-glutamic acid - Google Patents
High-efficiency method for fermentation production of L-glutamic acid Download PDFInfo
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- CN101402979A CN101402979A CNA2008101528832A CN200810152883A CN101402979A CN 101402979 A CN101402979 A CN 101402979A CN A2008101528832 A CNA2008101528832 A CN A2008101528832A CN 200810152883 A CN200810152883 A CN 200810152883A CN 101402979 A CN101402979 A CN 101402979A
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Abstract
The invention relates to a method for high-efficiency fermentation production of L-glutamic acid. The method can solve the problems of lower acid production, low glucose-acid conversion rate, and the like of the prior production process. The method comprises the following steps: a biotin auxotroph strain is taken as a production strain, according to a certain proportion, a plurality of nutritional components are prepared into a culture medium with rich and balanced nutrients as the fermentation culture medium, the dissolved oxygen is controlled on the appropriate level through the regulation of the stirring speed of a fermentation tank and the wind amount, the pH is controlled by feeding ammonia water, and the residual sugar is controlled on the lower level by feeding glucose solution with a certain concentration, and the fermentation is stopped till 32h. The method can shorten the whole fermentation cycle and greatly improve the yield (more than 150g/l) and the conversion rate (more than 60 percent) of the L-glutamic acid under the situation of not increasing any additional equipment and manpower investment, the whole process has simple operation and lower production cost, thereby being very applicable to industrial production.
Description
[technical field]:
The present invention relates to a kind of method of efficient fermentation production of L-glutamic acid, belong to technical field of producing amino acid by fermentation.
[background technology]:
In whole amino acid manufacture, the production of L-glutamic acid occupies very consequence, and its output accounts for more than 2/3 of amino acid ultimate production.Msg powder type is a monosodium glutamate, has the intensive delicate flavour, is the present seasonings of consumption maximum in the world.In addition, L-glutamic acid is at medicine, makeup with industrially also have a wide range of applications.Glutamic acid fermentation is typical metabolic control fermentation, and people such as nineteen fifty-seven Kinoshita success first is with behind the Production by Microorganism Fermentation L-glutamic acid, and microbe fermentation method becomes the main method that L-glutamic acid is produced, and its industrially scalable also enlarges year by year.At present, the production technology of glutamic acid fermentation is quite ripe.In recent years, along with rising steadily and market competition fierce further of the prices of raw and semifnished materials, in order when improving glutamic acid yield and transformation efficiency, to reduce production costs, still have in a large number and report, mainly concentrate on the aspects such as exploitation of genetic engineering means directive breeding yield glutamic acid strains in high, fermenting process control and optimization and new raw material about the research of glutamic acid fermentation aspect.
Glutamate producing bacterium belongs to auxotrophic mutant, and can accumulate L-glutamic acid outside born of the same parents in a large number is because the metabolism of thalline is in error state (ERST), very responsive to envrionment conditions.Fermentation condition such as vitamin H, carbon source, nitrogenous source, inorganic salt, pH, temperature and dissolved oxygen etc. all have material impact to synthesizing of L-glutamic acid.Wherein, substrate (as glucose) concentration is an important parameter of control bacterial metabolism process, the main pathways metabolism of passing through the synthetic or activity influence thalline of control relevant enzyme.Glucose can check, suppress the synthetic of plurality of enzymes by " glucose effect ", and as amino acid synthetase etc., this to check effect mainly be that its catabolite causes.Be in the glutamic acid fermentation of carbon source with glucose, the catabolite repression of glucose can cause the accumulation of multiple by product such as lactic acid, thereby influences glucose acid invert ratio.In addition, dissolved oxygen also is a vital factor, and the generation of heteroacid and the waste of carbon skeleton and dissolved oxygen concentration are closely related.Therefore must appropriately control dissolved oxygen during the fermentation,, increase the L-glutamic acid production rate, reduce the generation of heteroacid to change the metabolism distributions.
At present, China's glutamic acid fermentation industry mainly adopts the batch fermentation pattern.In this production model, it is extremely important for the fermentation of L-glutamic acid to keep certain substrate (as glucose) concentration.For reaching the requirement of economic technology, improve glutamic acid yield in the single batch of fermenting process and will improve substrate (as glucose) concentration in the initial medium, but substrate (as glucose) excessive concentration causes that osmotic pressure is excessive, influences the output of the eubolism and the L-glutamic acid of thalline.Otherwise, the low growth that helps thalline of initial glucose concentration, but the residual sugar in the later stage fermentation liquid exhausts rapidly, and its throughput can not be brought into play to greatest extent.Meanwhile, in present production model, oxyty often can not be well controlled, and is not that the oxygen supply deficiency is exactly that oxygen supply is excessive, and dissolved oxygen level float very big, can not be for a long time stable maintain on the appropriate level.Under the low excessively condition of dissolved oxygen, TCA cyclic metabolism flow reduces, and is not enough to balance glucose glycolysis speed, thereby has stimulated the enzyme of serum lactic dehydrogenase to live, and the metabolism circulation is generated to lactic acid, causes the lactic acid accumulation; And dissolved oxygen is when too high, and the glutamate dehydrogenase enzyme is lived and obviously reduced, and the TCA circular flow strengthens, and generates a large amount of CO
2, causing the carbon source loss, two kinds of situations are unfavorable for that all L-glutamic acid generates.In addition, existing a lot of L-glutamic acid fermentation culture medium prescription all can not well remove to satisfy the growth metabolism of its corresponding production bacterial strain to greatest extent and produce sour requirement.
[summary of the invention]:
The objective of the invention is to overcome defectives such as existing L-glutamic acid production method fermentation and acid amount is lower, glucose acid invert ratio is low, a kind of method of efficient fermentation production of L-glutamic acid is provided.Under the situation that does not increase extras and human input, realized the shortening of whole fermentation period and the significantly raising of L-glutamic acid yield and transformation efficiency, be suitable for suitability for industrialized production.
The objective of the invention is to be achieved through the following technical solutions:
The method of a kind of efficient fermentation production of L-glutamic acid provided by the invention, comprise seed culture and carried out the hair care ferment, it is characterized in that: in the fermenting process, by regulating fermentor tank mixing speed and air quantity dissolved oxygen is controlled on the proper level, make whole fermentation process neither produce more lactic acid, also do not cause generating a large amount of CO because the TCA circular flow strengthens
2And cause carbon source to run off in a large number, by stream ammonification water management pH, add an amount of bubble enemy froth breaking by stream, and add certain density glucose solution by stream residual sugar is controlled at certain level, make neither to produce glucose in the whole fermentation process and suppress, also do not constitute the substrate restriction, fermenting to 32h stops.
The concrete steps of this method are as follows:
(1) cultivates seed: preparation seed culture medium [dense 2%, the K of glucose 2.5%, corn steep liquor 3%, beans
2HPO
43H
2O 0.2%, MgSO
47H
2O 0.1%, urea 0.025%], 115 ℃ of sterilization 15min; Bacterial classification is inserted in the seed culture medium, and inoculum size is generally 5%; Under suitable temperature, pH and dissolved oxygen condition, control automatically and be cultured to logarithmic phase in the fermentor tank in 5L.
(2) aerobic fermentation: the kind liquid that step (1) is made is with 10%～20% inoculum size inoculation fermentation, control fermented liquid temperature employing order temperature raising pattern: 0～5h is 34 ℃, and 5～10h is 35 ℃, and 10～18h is 36 ℃, 18～26h is 37 ℃, and 26～32h is 38 ℃; Feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is that 20%, 10～32h is 5%; Control pH 7.0～7.2 by auto-feeding ammoniacal liquor; Add an amount of bubble enemy froth breaking by stream; And to add concentration by stream be that the glucose solution of 500～800g/L is controlled at 0.5～2.0% with residual sugar, ferments to 32h to stop.
Advantage of the present invention and positively effect:
Compare with existing technology, method of the present invention has following characteristics:
1, the present invention's glucose solution of adding higher concentration by proper flow is controlled at appropriate level with remaining sugar concentration, make and cause because of glucose concn is too high neither in the whole fermentation process that osmotic pressure is excessive and produce " glucose effect " and check catabolite, do not cross the low substrate restriction that constitutes because of glucose concn yet, residual sugar in the fermented liquid is exhausted rapidly, cause bacterial classification throughput not brought into play to greatest extent;
2, the present invention is controlled at oxyty on the appropriate level by suitable adjusting fermentor tank mixing speed and air quantity, making in the whole fermentation process neither causes TCA cyclic metabolism flow to reduce because of oxyty is low excessively, be not enough to balance glucose glycolysis speed, thereby stimulated the enzyme of serum lactic dehydrogenase to live, the metabolism circulation is generated to lactic acid, cause the lactic acid accumulation, also do not cause the glutamate dehydrogenase enzyme to be lived because of oxyty is too high and obviously reduce, the TCA circular flow strengthens and generates a large amount of CO
2And cause a large amount of losses of carbon source;
3, the present invention is comparing under the situation that does not increase any extras and human input with existing production technique, shortened whole fermentation period, seed culture foreshortens to 5～6h, and the output (more than the 150g/L) and the transformation efficiency (more than 60%) of L-L-glutamic acid have significantly been improved, whole simple operation of process, production cost is lower, and remarkable in economical benefits very is suitable for suitability for industrialized production.
[embodiment]:
Embodiment 1:
The microbial strains of using is existing glutamate producing bacterium.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: dense 2%, the K of glucose 2.5%, corn steep liquor 3%, beans
2HPO
43H
2O 0.2%, MgSO
47H
2O 0.1%, urea 0.025%.Transfer pH to 7.0～7.2,115 ℃ sterilization 15min with NaOH and hydrochloric acid.
(2) fermention medium: glucose 8%, cane molasses 0.1%, corn steep liquor 0.4%, MgSO
47H
2O0.15%, Na
2HPO
412H
2O 0.2%, KCl 0.1%, MnSO
40.0001%, FeSO
40.0001%, V
B10.00001%.Transfer pH to 7.0～7.2,115 ℃ sterilization 15min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 5%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 6h to logarithmic phase, inoculum size by 10% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature employing order temperature raising pattern: 0～5h is 34 ℃, 5～10h is 35 ℃, 10～18h is 36 ℃, 18～26h is 37 ℃, 26～32h is 38 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is 20%, 10～32h is 5%, 7.0～7.2, adds an amount of bubble enemy froth breaking by stream by auto-feeding ammoniacal liquor control pH, and to add concentration by stream be that the glucose solution of 500g/L is controlled at 0.5% with residual sugar, ferments to 32h to stop.The output of L-L-glutamic acid is 150g/L, and glucose acid invert ratio is 60%.
Embodiment 2:
The microbial strains of using is existing glutamate producing bacterium.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: with embodiment 1.
(2) fermention medium: glucose 10%, cane molasses 0.15%, corn steep liquor 0.6%, MgSO
47H
2O 0.18%, Na
2HPO
412H
2O 0.25%, KCl 0.13%, MnSO
40.0002%, FeSO
40.0002%, V
B10.00002%.Transfer pH to 7.0～7.2,115 ℃ sterilization 15min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 5%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 6h to logarithmic phase, inoculum size by 15% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature employing order temperature raising pattern: 0～5h is 34 ℃, 5～10h is 35 ℃, 10～18h is 36 ℃, 18～26h is 37 ℃, 26～32h is 38 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is 20%, 10～32h is 5%, 7.0～7.2, adds an amount of bubble enemy froth breaking by stream by auto-feeding ammoniacal liquor control pH, and to add concentration by stream be that the glucose solution of 800g/L is controlled at 0.5～2.0% with residual sugar, ferments to 32h to stop.The output of L-L-glutamic acid is 151g/L, and glucose acid invert ratio is 61%.
Embodiment 3:
The microbial strains of using is existing glutamate producing bacterium.Efficiently ferment with above-mentioned bacterial strains, used substratum has two kinds:
(1) seed culture medium: with embodiment 1.
(2) fermention medium: glucose 12%, cane molasses 0.2%, corn steep liquor 0.8%, MgSO
47H
2O 0.2%, Na
2HPO
412H
2O 0.3%, KCl 0.15%, MnSO
40.0003%, FeSO
40.0003%, V
B10.00003%.Transfer pH to 7.0～7.2,115 ℃ sterilization 15min with NaOH and hydrochloric acid.
Bacterial classification is inserted in the seed culture medium, and inoculum size is 5%; At 32 ℃, pH be 7.0 and dissolved oxygen be to control automatically in the fermentor tank in 5L under 20% condition to cultivate 6h to logarithmic phase, inoculum size by 20% inserts the 5L that contains fermention medium and controls in the fermentor tank automatically, control fermented liquid temperature employing order temperature raising pattern: 0～5h is 34 ℃, 5～10h is 35 ℃, 10～18h is 36 ℃, 18～26h is 37 ℃, 26～32h is 38 ℃, feed suitable air, regulate the agitation as appropriate rotating speed, adopting the control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is 20%, 10～32h is 5%, 7.0～7.2, adds an amount of bubble enemy froth breaking by stream by auto-feeding ammoniacal liquor control pH, and to add concentration by stream be that the glucose solution of 800g/L is controlled at 2.0% with residual sugar, ferments to 32h to stop.The output of L-L-glutamic acid is 153g/L, and glucose acid invert ratio is 63%.
Claims (4)
1. the method for an efficient fermentation production of L-glutamic acid, comprise seed culture and carried out the hair care ferment, it is characterized in that: in the fermenting process, by regulating fermentor tank mixing speed and air quantity dissolved oxygen is controlled on the proper level, make whole fermentation process neither produce more lactic acid, also do not cause generating a large amount of CO because the TCA circular flow strengthens
2And cause carbon source to run off in a large number, by stream ammonification water management pH, add an amount of bubble enemy froth breaking by stream, and add certain density glucose solution by stream residual sugar is controlled at lower level, make neither to produce glucose in the whole fermentation process and suppress, also do not constitute the substrate restriction, fermenting to 32h stops.
2. method according to claim 1 is characterized in that: the temperature of described fermented liquid is that sequential control method of temperature: 0～5h is 34 ℃, and 5～10h is 35 ℃, and 10～18h is 36 ℃, and 18～26h is 37 ℃, and 26～32h is 38 ℃.
3. method according to claim 1 is characterized in that: described dissolved oxygen of fermentation liquid concentration is control stage by stage: 0～10h is that 20%, 10～32h is 5%.
4. method according to claim 1 is characterized in that: the per-cent of each ingredients constitute total amount is in the described fermented liquid: inoculum size 10～20%, glucose 8～12%, cane molasses 0.1～0.2%, corn steep liquor 0.4～0.8%, MgSO
47H
2O 0.15～0.2%, Na
2HPO
412H
2O 0.2～0.3%, KCl0.1～0.15%, MnSO
40.0001～0.0003%, FeSO
40.0001～0.0003%, V
B100001～0.00003%.
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| CNA2008101528832A CN101402979A (en) | 2008-11-07 | 2008-11-07 | High-efficiency method for fermentation production of L-glutamic acid |
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|---|---|---|---|
| CNA2008101528832A CN101402979A (en) | 2008-11-07 | 2008-11-07 | High-efficiency method for fermentation production of L-glutamic acid |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154397A (en) * | 2010-12-22 | 2011-08-17 | 菱花集团有限公司 | Method for producing glutamic acid from corn immersing water in biotin-suboptimal fermentation process |
| CN102329897A (en) * | 2011-10-09 | 2012-01-25 | 江西新瑞丰生化有限公司 | Method for controlling pH value in biological fermentation |
| CN103205390A (en) * | 2013-04-12 | 2013-07-17 | 北京轻发生物技术中心 | L-glutamic acid gene engineering bacterium and application thereof |
| CN103215323A (en) * | 2013-04-12 | 2013-07-24 | 北京轻发生物技术中心 | Method for producing L-glutamic acid by fermentation in staged gradient oxygen supply manner |
| CN104561162A (en) * | 2013-10-23 | 2015-04-29 | 中粮营养健康研究院有限公司 | Method for preparing lysine by fermentation |
| CN104673853A (en) * | 2015-03-23 | 2015-06-03 | 中粮生化能源（龙江）有限公司 | Fermentation medium for fermenting and producing glutamic acid from thermo-sensitive type strain and fermenting method for producing glutamic acid by using fermentation medium and application |
| CN104694591A (en) * | 2015-03-23 | 2015-06-10 | 中粮生化能源（龙江）有限公司 | Fermentation medium for biotin defined amount fermentation production glutamic acid, fermentation method for producing glutamic acid by using fermentation medium and application |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN107227324A (en) * | 2017-08-07 | 2017-10-03 | 天津科技大学 | A kind of glutamic acid fermentation technique |
| CN109371072A (en) * | 2018-10-18 | 2019-02-22 | 许传高 | A kind of technique for reducing glutamic acid fermentation microbiological contamination and improving fermentation efficiency |
| CN109504719A (en) * | 2018-12-19 | 2019-03-22 | 呼伦贝尔东北阜丰生物科技有限公司 | A method of improving glutamic acid acid production rate and recovery rate |
| CN109628513A (en) * | 2018-12-19 | 2019-04-16 | 呼伦贝尔东北阜丰生物科技有限公司 | A kind of amino acid fermentation culture medium and preparation method thereof |
| CN112251474A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid |
| CN112251475A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving L-glutamine fermentation yield and sugar-acid conversion rate |
| CN116113333A (en) * | 2020-07-17 | 2023-05-12 | 国际香料和香精公司 | Taste enhancing composition |
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2008
- 2008-11-07 CN CNA2008101528832A patent/CN101402979A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154397A (en) * | 2010-12-22 | 2011-08-17 | 菱花集团有限公司 | Method for producing glutamic acid from corn immersing water in biotin-suboptimal fermentation process |
| CN102154397B (en) * | 2010-12-22 | 2013-08-14 | 菱花集团有限公司 | Method for producing glutamic acid from corn immersing water in biotin-suboptimal fermentation process |
| CN102329897A (en) * | 2011-10-09 | 2012-01-25 | 江西新瑞丰生化有限公司 | Method for controlling pH value in biological fermentation |
| CN103205390A (en) * | 2013-04-12 | 2013-07-17 | 北京轻发生物技术中心 | L-glutamic acid gene engineering bacterium and application thereof |
| CN103215323A (en) * | 2013-04-12 | 2013-07-24 | 北京轻发生物技术中心 | Method for producing L-glutamic acid by fermentation in staged gradient oxygen supply manner |
| CN104561162A (en) * | 2013-10-23 | 2015-04-29 | 中粮营养健康研究院有限公司 | Method for preparing lysine by fermentation |
| CN104673853B (en) * | 2015-03-23 | 2019-02-15 | 中粮生化能源（龙江）有限公司 | A kind of fermentation process and application producing glutamic acid for the fermentation medium of temperature sensitive type strain fermentation production glutamic acid and using it |
| CN104694591A (en) * | 2015-03-23 | 2015-06-10 | 中粮生化能源（龙江）有限公司 | Fermentation medium for biotin defined amount fermentation production glutamic acid, fermentation method for producing glutamic acid by using fermentation medium and application |
| CN104673853A (en) * | 2015-03-23 | 2015-06-03 | 中粮生化能源（龙江）有限公司 | Fermentation medium for fermenting and producing glutamic acid from thermo-sensitive type strain and fermenting method for producing glutamic acid by using fermentation medium and application |
| CN106086099A (en) * | 2016-08-27 | 2016-11-09 | 河北圣雪大成制药有限责任公司 | L valine fermentation medium and the fermentation process with its production L valine |
| CN106086099B (en) * | 2016-08-27 | 2019-12-13 | 河北圣雪大成制药有限责任公司 | l-valine fermentation medium and fermentation method for producing L-valine by using same |
| CN107227324A (en) * | 2017-08-07 | 2017-10-03 | 天津科技大学 | A kind of glutamic acid fermentation technique |
| CN109371072A (en) * | 2018-10-18 | 2019-02-22 | 许传高 | A kind of technique for reducing glutamic acid fermentation microbiological contamination and improving fermentation efficiency |
| CN109628513A (en) * | 2018-12-19 | 2019-04-16 | 呼伦贝尔东北阜丰生物科技有限公司 | A kind of amino acid fermentation culture medium and preparation method thereof |
| CN109504719A (en) * | 2018-12-19 | 2019-03-22 | 呼伦贝尔东北阜丰生物科技有限公司 | A method of improving glutamic acid acid production rate and recovery rate |
| CN109504719B (en) * | 2018-12-19 | 2021-09-21 | 呼伦贝尔东北阜丰生物科技有限公司 | Method for improving acid production rate and extraction rate of glutamic acid |
| CN109628513B (en) * | 2018-12-19 | 2022-01-04 | 呼伦贝尔东北阜丰生物科技有限公司 | Amino acid fermentation medium and preparation method thereof |
| CN116113333A (en) * | 2020-07-17 | 2023-05-12 | 国际香料和香精公司 | Taste enhancing composition |
| CN112251474A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving fermentation yield and saccharic acid conversion rate of L-glutamic acid |
| CN112251475A (en) * | 2020-11-19 | 2021-01-22 | 乐康珍泰（天津）生物技术有限公司 | Method for improving L-glutamine fermentation yield and sugar-acid conversion rate |
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