CN103243131A - Method for preparing L-glutamic acid by fermentation - Google Patents
Method for preparing L-glutamic acid by fermentation Download PDFInfo
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- CN103243131A CN103243131A CN2013102043079A CN201310204307A CN103243131A CN 103243131 A CN103243131 A CN 103243131A CN 2013102043079 A CN2013102043079 A CN 2013102043079A CN 201310204307 A CN201310204307 A CN 201310204307A CN 103243131 A CN103243131 A CN 103243131A
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Abstract
The invention provides a method for preparing L-glutamic acid by fermentation. According to the method provided by the invention, a temperature-sensitive type glutamic acid producing strain is adopted during the fermentation process of the L-glutamic acid, and different low-concentration betaines are added into a culture medium by stages for stimulating cell respiration, so that the oxygen consumption rate of thalli is improved, the accumulation of lactic acid bacteria is inhibited, the fermentation culture temperature is simultaneously regulated and controlled, and then the acid yield and glucose-acid inversion rate of the glutamic acid are improved. The glutamic acid prepared by the method provided by the invention does not contain phosphorus components, can be directly used for industries of health care foods and medicines, and has greater application values.
Description
Technical field
The present invention relates to biotechnology, be specifically related to amino acid whose preparation method, relate in particular to a kind of method of optimizing responsive to temperature type glutamate producing bacterium fermentative preparation L-L-glutamic acid.
Background technology
L-L-glutamic acid all has been widely used in food, medicine, industrial aspect, and especially aspect food, it is the precursor of making monosodium glutamate.The preparation method of relevant L-L-glutamic acid is more, based on fermentation method.Through numerous scientific research personnel's effort, constantly select some active higher novel bacterials and use for producing, and develop many novel methods, yet glucose acid invert ratio is lower.In addition, risen steadily by the supplementary material price, the influence that the L-glutamic acid production cost improves constantly, the profit of producing L-glutamic acid glides.Therefore, the fermentation condition of seeking more to optimize improves glutamic acid yield, and reducing cost is the problem that needs solution.Trimethyl-glycine has the confession methyl effect of alternative methionine(Met) as a kind of methyl donor efficiently.In addition, trimethyl-glycine still be a kind of atoxic, to the effective osmotic pressure buffer substance of biology, can be used as the osmotic pressure protective material of cell.Various biologies and animal tissues are under different pressures, and it is highly consistent that the function of trimethyl-glycine and other enzymes keeps, and play an important role for the metabolism of stabilized cell.When in the cell during osmotic pressure violent change, when high as the external penetration voltage rise, cell begins to produce or absorbs trimethyl-glycine to keep the balance of normal osmotic pressure, prevents that simultaneously cellular water from distributing the invasion with salinity, and can improve the effect of sodium [potassium, guarantee the normal function of cell.Therefore, trimethyl-glycine is widely used in responsive to temperature type bacterial strain forced fermentation as fermentation assistant to be produced in the production of L-L-glutamic acid, and addition manner generally adopts disposable interpolation in initial medium.Studies show that it is synthetic that the trimethyl-glycine of lower concentration obviously is conducive to product in the fermented liquid.In the thalli growth stage, the trimethyl-glycine excessive concentration in the initial medium then causes the metabolism that is decomposed of a large amount of trimethyl-glycines to be used for the synthetic of thalline easily, causes the increase of trimethyl-glycine consumption.In the product synthesis phase, the trimethyl-glycine of high density is unfavorable for the product accumulation, increases and synthesizes thereby the trimethyl-glycine of lower concentration is conducive to the release of intracellular product to born of the same parents outside, and to have both sexes relevant with the trimethyl-glycine molecule for this.But trimethyl-glycine concentration is not more low more good, and the concentration of trimethyl-glycine should be controlled in the needs that can satisfy methyl donor are conducive to the synthetic scope of product again.During the fermentation, trimethyl-glycine has mitigation to the osmotic pressure respiration inhibition that bacterium is subjected to, and breathes thereby stimulate, and improves the oxygen consumption rate of thalline, and the accumulation of lactic acid bacteria inhibiting improves biomass growth rate and rate of producing acid.Owing in fermention medium, add the phosphoric acid salt trimethyl-glycine, must contain the composition of phosphorus in the L-L-glutamic acid that makes, in security requirement higher protective foods and pharmaceutical industries, often can not directly use, limited its use.
Summary of the invention
Technical problem to be solved by this invention is to overcome above-mentioned weak point, and research and design is a kind of in initial medium and fermentation alternating temperature process, and the method for adding the low concentration trimethyl-glycine improves the method for responsive to temperature type glutamate producing bacterium fermentation production rate.
The invention provides a kind of method of fermentative preparation L-L-glutamic acid.
The present invention adopts the responsive to temperature type glutamate producing bacterium, in the L-glutamic acid fermentation process, divides aspire to the different lower concentration trimethyl-glycine of interpolation in the substratum, and regulates and control fermentation culture temperature, improves product acid amount and the glucose acid invert ratio of L-glutamic acid.
The inventive method comprises the following steps:
(1) seed culture
Seed culture medium (/L):
Glucose 15g, corn steep liquor 20g, K2HPO
43g, MgSO47H
2O1g, VB1200 μ g/L, DL-methionine 50 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 2mg/L of O, urea 0.55(branch disappears), pH7.0～7.2,121 ℃ sterilization 15min;
Method: adopt responsive to temperature type glutamate producing bacterium (L-glutamic acid brevibacterium flavum, Corynebacterium glutamicum etc.), inoculum size is 12%; 32 ℃ of culture temperature, 160rpm, shaking culture 14～16h is to logarithmic phase;
(2) fermentation culture
Fermention medium (/L):
Glucose 20g, corn steep liquor 20g, hydrochloride trimethyl-glycine 0.1～0.3g, K
2HPO
47.5g, MgSO
47H
2O1.5g, VB1200 μ g/L, VH600 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 30mg/L of O, urea 0.55(branch disappears), pH7.0～7.2,121 ℃ sterilization 15min;
Method: the inoculum size by 12% inserts seed liquor and contains in the fermention medium full automatic control fermentor tank, control pH value 7.0～7.2; The control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is that 20%, 10～32h is 5%; Control initial incubation temperature is 32 ℃; When the allometry △ OD=0.3 left and right sides (this for 20 times of concentration of fermented liquid dilution in the OD at 620nm place value), conversion temp to 37 ℃ is added the hydrochloride trimethyl-glycine of 0.1～0.3g/L simultaneously; During △ OD=0.6, improve temperature to 38 ℃ continuation fermentation again to finishing.Add 80% glucose strong solution by stream residual sugar control is being 0.1～3g/L.Whole process stream adds liquefied ammonia control pH, fermentation 0～14h, pH is 7.0～7.1,14～25h be 7.2～7.3,25h to fermentation ends be 6.8, produce that acid number no longer changes and be fermentation termination when the rising of pH value is arranged; Fermentation time is 30-32h.
The described step of the inventive method (1) responsive to temperature type glutamate producing bacterium is L-glutamic acid brevibacterium flavum or Corynebacterium glutamicum.Described bacterial strain is that open report uses, as: Chen Ning, Peng Fei, cytogamy breeding and the fermentation condition of Zhang Kexu .L-L-glutamic acid temperature sensitive mutant, the scientific and technological communication of fermenting, 2004,33 (1), 10-14.
Zhang Kexu, Chen Ning. the research of L-glutamic acid temperature sensitive mutant C N1021 fermentation condition, the scientific and technological communication of fermenting, 2003,33 (2): 3-5.
The trimethyl-glycine that the described step of the inventive method (2) is added is one or more the mixture in hydrochloride trimethyl-glycine, BETAINE anhydrous or the water trimethyl-glycine.
The present invention is according in responsive to temperature type glutamate producing bacterium fermenting process, the trimethyl-glycine of lower concentration has the synthetic characteristic of the growth that utilizes thalline and product, respectively in initial and alternating temperature process, in substratum, add trimethyl-glycine methyl is provided, and cellular respiration stimulating, improve the oxygen consumption rate of thalline, the accumulation of lactic acid bacteria inhibiting improves biomass growth rate and rate of producing acid.The L-glutamic acid that makes thus, not phosphorous composition can directly be used in protective foods and pharmaceutical industries, and bigger using value is arranged.
Embodiment
Further specify the present invention below by embodiment, the cited case does not limit protection scope of the present invention:
Embodiment 1
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum
Seed culture medium (/L):
Glucose 15g, corn steep liquor 20g, K2HPO
43g, MgSO47H
2O1g, VB1200 μ g/L, DL-methionine 50 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 2mg/L of O, urea 0.55(branch disappears), pH7.0～7.2,121 ℃ sterilization 15min;
Seed culture method: slant strains is washed with the 10ml sterilized water, transfer in the 5000mL kind bottle that the 1200mL seed culture medium is housed 32 ℃ of culture temperature, 160rpm, shaking culture 14～16h;
Fermention medium (/L):
Glucose 20g, corn steep liquor 20g, hydrochloride trimethyl-glycine 0.1g, K
2HPO
47.5g, MgSO
47H
2O1.5g, VB1200 μ g/L, VH600 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 30mg/L of O, urea 0.55(branch disappears), pH7.0～7.2,121 ℃ sterilization 15min;
Fermentation culture method: the inoculum size by 12% all inserts seed liquor and contains in the 15L full automatic control fermentor tank of fermention medium, and control pH value transfers to 7.0～7.2; The control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is that 20%, 10～32h is 5%; Control initial incubation temperature is 32 ℃.When the allometry optical density value △ OD=0.3 left and right sides (fermented liquid dilution 20 times of concentration in the OD at 620nm place value), conversion temp to 37 ℃ simultaneously, is added the hydrochloride trimethyl-glycine of 0.1g/L again; During △ OD=0.6, improve temperature to 38 ℃ continuation fermentation again to finishing.Add 80% glucose strong solution by stream residual sugar control is being 1g/L.Whole process stream adds liquefied ammonia control pH, fermentation 0～14h, and pH is 7.0～7.1, and 14～25h is that 7.2～7.3,25h is 6.8 to fermentation ends, and the product acid number no longer changes and has the pH value slightly to be fermentation termination (31h) when rising.When putting jar, the output of L-L-glutamic acid is 216.7g/L, and glucose acid invert ratio is 65.3%.
Comparative Examples 1:
Reference examples 1 is with the difference of embodiment 1: do not add trimethyl-glycine when temperature transition to 37 ℃, and when temperature transition to 38 ℃, add the water trimethyl-glycine of 1.0g/L, other conditions are identical with embodiment 1.The output of L-L-glutamic acid is 212.9g/L during fermentation ends, and glucose acid invert ratio is 63.3%.L-glutamic acid yield and glucose acid invert ratio when comparing result shows embodiment 1 fermentation ends obviously are better than Comparative Examples 1.
Embodiment 2:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 213.1g/L, and glucose acid invert ratio is 62.8%.
Embodiment 3:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 211.8g/L, and glucose acid invert ratio is 61.7%.
Embodiment 4:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; The fermentation initial medium is for adding the hydrochloride trimethyl-glycine of 0.3g/L in fermention medium; Other conditions and cultural method are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 219.4g/L, and glucose acid invert ratio is 68.1%.
Comparative Examples 2:
Reference examples 2 is with the difference of embodiment 4: add 80% glucose strong solution by stream residual sugar control is being 4g/L.Other conditions are identical with embodiment 4.The output of L-L-glutamic acid is 216.8g/L during fermentation ends, and glucose acid invert ratio is 65.5%.L-glutamic acid yield and glucose acid invert ratio when comparing result shows embodiment 4 fermentation ends obviously are better than Comparative Examples 2.
Comparative Examples 3:
Reference examples 3 is with the difference of embodiment 4: add 80% glucose strong solution by stream residual sugar control is being 10g/L.Other conditions are identical with embodiment 4.The output of L-L-glutamic acid is 211.2g/L during fermentation ends, and glucose acid invert ratio is 61.3%.L-glutamic acid yield and glucose acid invert ratio when comparing result shows embodiment 4 fermentation ends obviously are better than Comparative Examples 3.
Embodiment 4 and Comparative Examples 2,3 be contrast table (table 1) as a result
Embodiment 5:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 4.When putting jar, the output of L-L-glutamic acid is 214.3g/L, and glucose acid invert ratio is 63.4%.
Embodiment 6:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 4.When putting jar, the output of L-L-glutamic acid is 209.7g/L, and glucose acid invert ratio is 61.2%.
Embodiment 7:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; The fermentation initial medium is for adding the hydrochloride trimethyl-glycine of 1.5g/L in fermention medium; Other conditions and cultural method are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 216.2g/L, and glucose acid invert ratio is 65.1%.
Embodiment 8:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 7.When putting jar, the output of L-L-glutamic acid is 213.8g/L, and glucose acid invert ratio is 63.1%.
Embodiment 9:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; When conversion temp to 37 ℃, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 7.When putting jar, the output of L-L-glutamic acid is 210.2g/L, and glucose acid invert ratio is 61.4%.
Embodiment 1～9 is contrast table (table 2) as a result
Embodiment 10:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; Use the existing trimethyl-glycine addition manner disposable adding hydrochloride trimethyl-glycine 0.4g/L in the initial medium that namely ferments, the later stage is no longer added trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 213.3g/L, and glucose acid invert ratio is 62.6%.
Embodiment 11:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; Use the existing trimethyl-glycine addition manner disposable adding hydrochloride trimethyl-glycine 1.6g/L in the initial medium that namely ferments, the later stage is no longer added trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 215.6g/L, and glucose acid invert ratio is 64.1%.
Embodiment 12:
The bacterial strain that adopts is the responsive to temperature type glutamate producing bacterium: the L-glutamic acid brevibacterium flavum; Use the existing trimethyl-glycine addition manner disposable adding hydrochloride trimethyl-glycine 3.0g/L in the initial medium that namely ferments, the later stage is no longer added trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting jar, the output of L-L-glutamic acid is 213.1g/L, and glucose acid invert ratio is 62.9%.
Embodiment 10～12 and embodiment 4 be contrast table (table 3) as a result
Claims (5)
1. the method for fermentative preparation L-L-glutamic acid is characterized in that, this method comprises the following steps:
(1) seed culture
Seed culture medium/L:
Glucose 15g, corn steep liquor 20g, K2HPO
43g, MgSO47H
2O1g, VB1200 μ g/L, DL-methionine 50 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 2mg/L of O, urea disappeared in 0.55 minute, pH7.0～7.2,121 ℃ sterilization 15min;
Method: adopt the responsive to temperature type glutamate producing bacterium, inoculum size is 12%; 32 ℃ of culture temperature, 160rpm, shaking culture 14～16h is to logarithmic phase;
(2) fermentation culture
Fermention medium/L:
Glucose 20g, corn steep liquor 20g, hydrochloride trimethyl-glycine 0.1～0.3g, K
2HPO
47.5g, MgSO
47H
2O1.5g, VB1200 μ g/L, VH600 μ g/L, FeSO
47H
2O, MnSO
4H
2Each 30mg/L of O, urea disappeared in 0.55 minute, pH7.0～7.2,121 ℃ sterilization 15min;
Method: the inoculum size by 12% inserts seed liquor and contains in the fermention medium full automatic control fermentor tank, control pH value 7.0～7.2; The control of oxygen supply pattern stage by stage dissolved oxygen: 0～10h is that 20%, 10～32h is 5%; Control initial incubation temperature is 32 ℃; When allometry △ OD=0.3 this for 20 times of concentration of fermented liquid dilution in the OD at 620nm place value, conversion temp to 37 ℃ is added the hydrochloride trimethyl-glycine of 0.1～0.3g/L simultaneously; During △ OD=0.6, improve temperature to 38 ℃ continuation fermentation again to finishing; Add 80% glucose strong solution by stream residual sugar control is being 0.1～3g/L; Whole process stream adds liquefied ammonia control pH, fermentation 0～14h, pH is 7.0～7.1,14～25h be 7.2～7.3,25h to fermentation ends be 6.8, produce that acid number no longer changes and be fermentation termination when the rising of pH value is arranged; Fermentation time is 30-32h.
2. the method for fermentative preparation L-L-glutamic acid according to claim 1 is characterized in that, described step (1) responsive to temperature type glutamate producing bacterium is L-glutamic acid brevibacterium flavum or Corynebacterium glutamicum.
3. the method for fermentative preparation L-L-glutamic acid according to claim 2 is characterized in that, described step (1) responsive to temperature type glutamate producing bacterium is the L-glutamic acid brevibacterium flavum.
4. the method for fermentative preparation L-L-glutamic acid according to claim 1 is characterized in that, the trimethyl-glycine that described step (2) is added is one or more the mixture in hydrochloride trimethyl-glycine, BETAINE anhydrous or the water trimethyl-glycine.
5. the method for fermentative preparation L-L-glutamic acid according to claim 4 is characterized in that, the trimethyl-glycine that described step (2) is added is the hydrochloride trimethyl-glycine.
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| CN105039271A (en) * | 2015-06-25 | 2015-11-11 | 山东祥维斯生物科技有限公司 | Method for increasing yield of various enzyme preparations |
| CN105296562A (en) * | 2015-10-31 | 2016-02-03 | 梁山菱花生物科技有限公司 | Glutamic acid fermentation culture medium using betaine phosphate to substitute betaine hydrochloride |
| CN105602881A (en) * | 2016-03-11 | 2016-05-25 | 天津科技大学 | Temperature-sensitive recombinant corynebacterium glutamicum producing glutamic acid and application thereof |
| CN106191153A (en) * | 2016-08-31 | 2016-12-07 | 菱花集团有限公司 | The method that glutamic acid fermentation produces |
| CN106191169A (en) * | 2016-07-25 | 2016-12-07 | 安徽丰原发酵技术工程研究有限公司 | A kind of fermentation process improving N acetylglucosamine yield |
| CN112195205A (en) * | 2020-10-20 | 2021-01-08 | 内蒙古阜丰生物科技有限公司 | Method for improving glutamic acid fermentation acid production |
| 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 |
| CN112322673A (en) * | 2020-11-11 | 2021-02-05 | 廊坊梅花生物技术开发有限公司 | Fermentation method of glutamic acid |
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| CN101319239A (en) * | 2008-07-16 | 2008-12-10 | 广州大学 | Application of dimethyl-ethyl-betaine in glutamic acid fermentation |
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| CN105039271A (en) * | 2015-06-25 | 2015-11-11 | 山东祥维斯生物科技有限公司 | Method for increasing yield of various enzyme preparations |
| CN105296562A (en) * | 2015-10-31 | 2016-02-03 | 梁山菱花生物科技有限公司 | Glutamic acid fermentation culture medium using betaine phosphate to substitute betaine hydrochloride |
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| CN106191169B (en) * | 2016-07-25 | 2019-09-13 | 安徽丰原发酵技术工程研究有限公司 | A kind of fermentation process improving N-acetylglucosamine yield |
| CN106191153A (en) * | 2016-08-31 | 2016-12-07 | 菱花集团有限公司 | The method that glutamic acid fermentation produces |
| CN112195205A (en) * | 2020-10-20 | 2021-01-08 | 内蒙古阜丰生物科技有限公司 | Method for improving glutamic acid fermentation acid production |
| CN112322673A (en) * | 2020-11-11 | 2021-02-05 | 廊坊梅花生物技术开发有限公司 | Fermentation method of 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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Address after: 262737 Shandong city of Weifang province Lingang Industrial Park, Binhai Economic Development Zone, Binhai Avenue No. 0199 Patentee after: SHANDONG SUNWIN BIOTECHNOLOGY CO., LTD. Address before: 262736, Shandong, Weifang Binhai Economic Development Zone, Weifang Lingang Industrial Park, west of the North Ring Road Patentee before: Shandong Sunwin Biotechology Co., Ltd. |