CN103184247A - Method for co-producing L-lactic acid with L-lysine - Google Patents
Method for co-producing L-lactic acid with L-lysine Download PDFInfo
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- CN103184247A CN103184247A CN2013101196607A CN201310119660A CN103184247A CN 103184247 A CN103184247 A CN 103184247A CN 2013101196607 A CN2013101196607 A CN 2013101196607A CN 201310119660 A CN201310119660 A CN 201310119660A CN 103184247 A CN103184247 A CN 103184247A
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- lactic acid
- methionin
- coproduction
- lysine
- fermentation
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Abstract
The invention discloses a method for co-producing L-lactic acid with L-lysine, which comprises the following steps: (1) performing aerobic culturing for L-lysine production strains for 48 to 72 h to produce L-lysine by fermentation; (2) collecting strains obtained from step 1 by centrifugation, allowing fermentation medium to be subjected to anaerobic fermentation to suspend strain sludge again and then transporting into a fermentation cylinder, blowing N2 gas to keep positive pressure inside the cylinder, stirring and culturing for 24 to 40 h at the temperature of 28 to 34 DEG C and maintaining the PH of the anaerobic fermentation broth between 6.5 and 7.5, so as to produce L-lactic acid by fermentation. Through the analysis of the fermented products, L-lysine can be well accumulated by corynebacterium glutamicum in aerobiotic condition and L-lactic acid can be well accumulated by corynebacterium glutamicum in anaerobic condition.
Description
Technical field
The organic acid fermentation process is produced in the amino acid series connection that the present invention relates in the biological chemical field, specifically, relates to a kind of method of L-Methionin coproduction L-lactic acid.
Background technology
The main force that Corynebacterium glutamicum produces as industrial amino acid (as L-glutamic acid, Methionin, Threonine etc.), wherein Methionin is as a kind of alkaline indispensable amino acid, be widely used in food, feed and the medicine industry, play a part aspect the balance amino acid composition very important.At present, the Methionin industrial production is based on the Corynebacterium glutamicum fermentation method, and a large amount of thalline that produce after aerobic fermentation finishes generally are used to protein fodder processing or directly go out of use, and exist certain resource utilization low, and added value is not high.If the discarded thalline of aerobic production L-Methionin can be handled through simple, be directly used in anaerobism and produce L-lactic acid, this aerobic, serially anerobic training mode can the fully efficient utilization of resourcesization, solve the high value added utilization of thalline after the production of L-Methionin effectively, and anaerobism is produced the problem such as expensive of L-lactic acid yeast culture.
L-lactic acid is mainly used in fields such as foodstuffs industry, medicine, makeup, also is one of monomer of Biodegradable material poly(lactic acid).The production of poly(lactic acid) and application and development are polluted the generation epoch-making significance for eliminating " white ", and market potential is huge.Fermentative Production L-lactic acid is according to the pathways metabolism of synthesizing lactic acid and the difference of generation product, lactic fermentation can be divided into homotype, abnormal shape and mixed type, but the general character in the fermenting process all is to be prerequisite with highdensity yeast culture, this need consume a large amount of energy consumptions, and particularly the manufacturing cost for poly(lactic acid) replacement conventional plastic becomes very big restriction.
Summary of the invention
Problem to be solved by this invention provides a kind of method of L-Methionin coproduction L-lactic acid.This method reclaims the thalline of producing L-Methionin, and anaerobically fermenting is produced L-lactic acid again, and is simple to operate, and equipment requirements is not high, is easy to automatization control, has good economic benefits.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is as follows:
A kind of method of L-Methionin coproduction L-lactic acid, this method comprises the steps:
(1) L-Methionin is produced the preferred 60h of the aerobic cultivation 48～72h(of bacterium) fermentation production of L-lysine;
(2) thalline after the aerobic cultivation of centrifugal collection, resuspended this bacterium mud of anaerobically fermenting substratum changes in the fermentor tank then, feeds N
2Gas is kept a jar interior malleation, under 28～34 ℃ (preferred 30 ℃), and stir culture 24～40 hours, and the pH that keeps anaerobic fermented liquid is 6.5～7.5, fermentation production of L-lactic acid.
In the step (1), it is conventional Corynebacterium glutamicum of using that described L-Methionin is produced bacterium, as Corynebacterium glutamicum Corynebacterium glutamicum ATCC21300 and Corynebacterium glutamicum Corynebacterium glutamicum ATCC21513.
In the step (1), described aerobic culture condition is:
Aerobic culture medium comprises following component: glucose 80g/L, K
2HPO
43H
2O1.0g/L, KH
2PO
41.0g/L, MgSO
47H
2O0.25g/L, (NH
4)
2SO
440g/L, Mn
2+, Fe
2+0.02g/L, ZnCl
21mg/L, CuSO
40.2mg/L, vitamin H 100 μ g/L, VB1200 μ g/L, CaCO
330g/L, pH are 7.0;
Culture temperature: 28～34 ℃, preferred 30 ℃;
Ventilation ratio: 0.5～1.5v/vm, preferred 1v/vm.
In the step (1), described anaerobically fermenting substratum comprises following component: glucose 40～80g/L, K
2HPO
43H
2O0.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.5g/L, Mn
2+, Fe
2+0.02g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, initial pH7.0.
In the step (2), the combination of any one or a few in use carbonate, ammoniacal liquor and the alkali lye is kept the pH of anaerobic fermented liquid 6.5～7.5.
L-Methionin of the present invention is produced bacterium and was permitted before aerobic fermentation through conventional seed culture: will cultivate 20～24h for dull and stereotyped last 30 ℃ in solid LB, shake in the bottle to the 500mL that the 30mL seed culture medium is housed with transfering loop picking 3 ring bacterium then, 30 ℃, 200r/min cultivates 12～14h, the thalline OD of this moment
600Value is about 25-28, generally according to 3 (v/v) % inoculum size seed liquor is forwarded to fermention medium then and carries out the aerobic fermentation cultivation.Wherein said seed culture medium comprises following component: glucose 20g/L, K
2HPO
43H
2O1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.4g/L, urea 2.5g/L, Mn
2+, Fe
2+0.02g/L, vitamin H 100 μ g/L, VB1200 μ g/L, pH are 7.0.
Beneficial effect: the present invention compared with prior art has following advantage.
1, the product that the present invention is obtained is analyzed, and Corynebacterium glutamicum accumulates 18～22g/L L-Methionin under aerobic condition; Under the anaerobic condition, can accumulate L-lactic acid significantly, so the economic benefit of its industrialization is tangible.
2, utilize method of the present invention, produce a large amount of thalline when the Methionin aerobic fermentation stage finishes, directly utilize its anaerobism to produce L-lactic acid, saved the process of anaerobic process thalline enrichment, effectively utilized biomass resource, reduced production cost.Discarded thalline behind the anaerobically fermenting L-lactic acid, renewable white feed, the nucleic acid etc. of laying eggs continue to create the high added value profit.
3, the more original simple aerobic product amino acid of production technique of the present invention, anaerobism is produced organic acid, and the thalline high efficiente callback utilizes, and need not to add auxiliary material especially, and environmentally safe is eco-friendly processing method.
Embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand that the described content of embodiment only is used for explanation the present invention, and should also can not limit the present invention described in detail in claims.
In following examples,
Seed culture medium comprises following component: glucose 20g/L, K
2HPO
43H
2O1.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.4g/L, urea 2.5g/L, Mn
2+, Fe
2+0.02g/L, vitamin H 100 μ g/L, VB1200 μ g/L, pH are 7.0.
Aerobic culture medium comprises following component: glucose 80g/L, K
2HPO
43H
2O1.0g/L, KH
2PO
41.0g/L, MgSO
47H
2O0.25g/L, (NH
4)
2SO
440g/L, Mn
2+, Fe
2+0.02g/L, ZnCl
21mg/L, CuSO
40.2mg/L, vitamin H 100 μ g/L, VB1200 μ g/L, CaCO
330g/L, pH are 7.0.
The anaerobically fermenting substratum comprises following component: glucose 40～80g/L, K
2HPO
43H
2O0.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.5g/L, Mn
2+, Fe
2+0.02g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, initial pH7.0.
Described Methionin produces bacterium Corynebacterium glutamicum ATCC21300 and Corynebacterium glutamicum ATCC21513 is conventional Corynebacterium glutamicum of using, and is commercially available.
Embodiment 1:
L-Methionin is produced bacterium (Corynebacterium glutamicum ATCC21513) cultivate 20h for dull and stereotyped last 30 ℃ in solid LB, encircle bacterium to the 30mL seed culture medium with transfering loop picking 3,30 ℃, 200r/min cultivates 12h, % is transferred to fermention medium with seed liquor by inoculum size 3 (v/v), and 30 ℃, 500r/min, ventilation is cultivated 60h than being 1v/vm.After aerobic fermentation finished, the centrifugal 10min of 5000r/min collected thalline, with anaerobically fermenting substratum (wherein containing 40g/L glucose) washing once, was resuspended in then in the anaerobically fermenting substratum, changed in the fermentor tank, fed N
20.2v/vm rotating speed 200r/min cultivates 24h for 30 ℃.The fermentation result, aerobic stage L-lysine production is 18.6g/L, anaerobic stages L-lactic acid production is 30.2g/L.
Embodiment 2:
L-Methionin is produced bacterium (Corynebacterium glutamicum ATCC21300) cultivate 20h for dull and stereotyped last 30 ℃ in solid LB, encircle bacterium to the 30mL seed culture medium with transfering loop picking 3,30 ℃, 200r/min cultivates 12h, % is transferred to fermention medium with seed liquor by inoculum size 3 (v/v), and 30 ℃, 200r/min, ventilation is cultivated 60h than being 1v/vm.After aerobic fermentation finished, the centrifugal 10min of 5000r/min collected thalline, with anaerobically fermenting substratum (wherein containing 80g/L glucose) washing once, was resuspended in then in the anaerobically fermenting substratum, changed in the fermentor tank, fed N
20.2v/vm rotating speed 200r/min cultivates 40h for 30 ℃.Measure tunning, aerobic stage L-lysine production is 18.2g/L, and anaerobic stages L-lactic acid production is 64.8g/L.
Embodiment 3:
L-Methionin is produced bacterium (Corynebacterium glutamicum ATCC21513) cultivate 20h for dull and stereotyped last 30 ℃ in solid LB, encircle the bacterium mud to the 30mL seed culture medium with transfering loop picking 3,30 ℃, 200r/min cultivates 12h, and % is transferred to fermention medium with seed liquor by inoculum size 3 (v/v), 30 ℃, 500r/min, ventilation is cultivated 72h than being 1v/vm, and ventilation is than being 1v/vm.After aerobic fermentation finished, the centrifugal 10min of 5000r/min collected thalline, with anaerobically fermenting substratum (wherein containing 60g/L glucose) washing once, was resuspended in then in the anaerobically fermenting substratum, changed in the fermentor tank, fed N
20.2v/vm 200r/min cultivates 40h for 30 ℃.Measure tunning, aerobic stage L-lysine production is 21.5g/L, and anaerobic stages L-lactic acid production is 51.3g/L.
Embodiment 4:
L-Methionin is produced bacterium (Corynebacterium glutamicum ATCC21300) cultivate 20h for dull and stereotyped last 30 ℃ in solid LB, encircle the bacterium mud to the 30mL seed culture medium with transfering loop picking 3,30 ℃, 200r/min cultivates 12h, % is transferred to fermention medium with seed liquor by inoculum size 3 (v/v), and 30 ℃, 500r/min, ventilation is cultivated 72h than being 1v/vm.After aerobic fermentation finished, the centrifugal 10min of 5000r/min collected thalline, with anaerobically fermenting substratum (wherein containing 40g/L glucose) washing once, was resuspended in then in the anaerobically fermenting substratum, changed in the fermentor tank, fed N
20.2v/vm 200r/min cultivates 40h for 30 ℃.Measure tunning, aerobic stage L-lysine production is 20.5g/L, anaerobic stages L-lactic acid 32.7g/L.
Claims (5)
1. the method for a L-Methionin coproduction L-lactic acid is characterized in that this method comprises the steps:
(1) L-Methionin is produced the aerobic cultivation 48～72h of bacterium fermentation production of L-lysine;
(2) thalline after the aerobic cultivation of centrifugal collection, resuspended this bacterium mud of anaerobically fermenting substratum changes in the fermentor tank then, feeds N
2Gas is kept a jar interior malleation, under 28～34 ℃, and stir culture 24～40 hours, and the pH that keeps anaerobic fermented liquid is 6.5～7.5, fermentation production of L-lactic acid.
2. the method for L-Methionin coproduction L-lactic acid according to claim 1 is characterized in that, in the step (1), it is conventional Corynebacterium glutamicum of using that described L-Methionin is produced bacterium.
3. the method for L-Methionin coproduction L-lactic acid according to claim 1 is characterized in that, in the step (1), described aerobic culture condition is:
Aerobic culture medium comprises following component: glucose 80g/L, K
2HPO
43H
2O1.0g/L, KH
2PO
41.0g/L, MgSO
47H
2O0.25g/L, (NH
4)
2SO
440g/L, Mn
2+, Fe
2+0.02g/L, ZnCl
21mg/L, CuSO
40.2mg/L, vitamin H 100 μ g/L, VB1200 μ g/L, CaCO
330g/L, pH are 7.0;
Culture temperature: 28～34 ℃;
Ventilation ratio: 0.5～1.5v/vm.
4. the method for L-Methionin coproduction L-lactic acid according to claim 1 is characterized in that, in the step (1), described anaerobically fermenting substratum comprises following component: glucose 40～80g/L, K
2HPO
43H
2O0.5g/L, KH
2PO
40.5g/L, MgSO
47H
2O0.5g/L, Mn
2+, Fe
2+0.02g/L, vitamin H 100 μ g/L, vitamin B12 00 μ g/L, initial pH7.0.
5. the method for L-Methionin coproduction L-lactic acid according to claim 1 is characterized in that, in the step (2), the combination of any one or a few in use carbonate, ammoniacal liquor and the alkali lye is kept the pH of anaerobic fermented liquid 6.5～7.5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219810B (en) * | 2015-11-09 | 2019-01-22 | 山东寿光巨能金玉米开发有限公司 | A kind of method of D-ALPHA-Hydroxypropionic acid and L-lysine Joint Production |
| WO2023283821A1 (en) * | 2021-07-14 | 2023-01-19 | 万华化学集团股份有限公司 | Preparation method for and application of lactic acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266905A (en) * | 1999-02-20 | 2000-09-20 | 德古萨－于尔斯股份公司 | Method for producing L-amino acid by fermentation with corynebacteria |
| CN1415742A (en) * | 2002-10-29 | 2003-05-07 | 上海新立工业微生物科技有限公司 | Clavibacter and method for fermentation production of L-lysine |
| CN101756010A (en) * | 2008-10-08 | 2010-06-30 | 天津生机集团股份有限公司 | Method for producing lysine fortified feed by soy sauce residue |
| CN101942486A (en) * | 2010-09-07 | 2011-01-12 | 南京工业大学 | Method for producing organic acid by fermenting abandoned thallus with monosodium glutamate |
-
2013
- 2013-04-08 CN CN2013101196607A patent/CN103184247A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1266905A (en) * | 1999-02-20 | 2000-09-20 | 德古萨－于尔斯股份公司 | Method for producing L-amino acid by fermentation with corynebacteria |
| CN1415742A (en) * | 2002-10-29 | 2003-05-07 | 上海新立工业微生物科技有限公司 | Clavibacter and method for fermentation production of L-lysine |
| CN101756010A (en) * | 2008-10-08 | 2010-06-30 | 天津生机集团股份有限公司 | Method for producing lysine fortified feed by soy sauce residue |
| CN101942486A (en) * | 2010-09-07 | 2011-01-12 | 南京工业大学 | Method for producing organic acid by fermenting abandoned thallus with monosodium glutamate |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105219810B (en) * | 2015-11-09 | 2019-01-22 | 山东寿光巨能金玉米开发有限公司 | A kind of method of D-ALPHA-Hydroxypropionic acid and L-lysine Joint Production |
| WO2023283821A1 (en) * | 2021-07-14 | 2023-01-19 | 万华化学集团股份有限公司 | Preparation method for and application of lactic acid |
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Application publication date: 20130703 |