CN103243131B - Method for preparing L-glutamic acid by fermentation - Google Patents
Method for preparing L-glutamic acid by fermentation Download PDFInfo
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- CN103243131B CN103243131B CN201310204307.9A CN201310204307A CN103243131B CN 103243131 B CN103243131 B CN 103243131B CN 201310204307 A CN201310204307 A CN 201310204307A CN 103243131 B CN103243131 B CN 103243131B
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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, particularly relate to and a kind ofly optimize the method for responsive to temperature type glutamate producing bacterium fermentation for Pidolidone.
Background technology
Pidolidone all has been widely used in food, medicine, industrial aspect, and especially in food, it is the precursor manufacturing monosodium glutamate.About the preparation method of Pidolidone is more, based on fermentation method.Through the effort of numerous scientific research personnel, constantly select the higher novel bacterial of some activity for production and application, and develop many novel methods, but glucose acid invert ratio is lower.In addition, rise steadily by supplementary material price, the impact that L-glutamic acid production cost improves constantly, produces the declination of profits of L-glutamic acid.Therefore, seek the fermentation condition more optimized, improve glutamic acid yield, reducing costs is the problem needing to solve.Trimethyl-glycine is as the efficient methyl donor of one, and what have alternative methionine(Met) supplies methyl effect.In addition, trimethyl-glycine or a kind of atoxic, osmotic pressure buffer substance to biologically effective, can be used as the osmoprotectant of cell.At various pressures, trimethyl-glycine keeps highly consistent with the function of other enzymes, and the metabolism for stabilized cell plays an important role for various biology and animal tissues.When intracellular osmotic pressure violent change, during as external penetration voltage rise height, cell starts to produce or absorb trimethyl-glycine to maintain the balance of normal osmotic pressure, prevents cellular water from distributing the invasion with salinity simultaneously, and effect of sodium [potassium can be improved, guarantee the normal function of cell.Therefore, trimethyl-glycine is widely used in responsive to temperature type bacterial strain forced fermentation as fermentation assistant and produces in the production of Pidolidone, and addition manner generally adopts disposable interpolation in initial medium.Research shows, in fermented liquid, the trimethyl-glycine of lower concentration is obviously conducive to Product formation.In the thalli growth stage, the trimethyl-glycine excessive concentration in initial medium, then easily cause a large amount of trimethyl-glycines to be decomposed the synthesis of metabolism for thalline, cause the increase of trimethyl-glycine consumption.In the Product formation phase, the trimethyl-glycine of high density is unfavorable for product accumulation, and the trimethyl-glycine of lower concentration is conducive to intracellular product to the release outside born of the same parents increases synthesis, and it is relevant that this and trimethyl-glycine molecule have both sexes.But trimethyl-glycine concentration is not more low better, and the needs that beet paper mill wastewater should control meeting methyl donor are conducive to again in the scope of Product formation.During the fermentation, trimethyl-glycine has mitigation to the osmotic pressure respiration inhibition that bacterium is subject to, thus stimulates breathing, improves the oxygen consumption rate of thalline, the accumulation of lactic acid bacteria inhibiting, improves biomass growth rate and rate of producing acid.Owing to adding phosphoric acid salt trimethyl-glycine in the fermentation medium, must containing the composition of phosphorus in obtained Pidolidone, in the higher protective foods of security requirement and pharmaceutical industries, often directly can not apply, limit 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 low concentration trimethyl-glycine improves the method for responsive to temperature type glutamate producing bacterium fermentation production rate.
The invention provides the method for a kind of fermentation for Pidolidone.
The present invention adopts responsive to temperature type glutamate producing bacterium, in Pidolidone fermenting process, divides aspire to adding different lower concentration trimethyl-glycines in substratum, and regulation and control fermentation culture temperature, improve 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
2o 1g, VB1200 μ g, DL-methionine 50 μ g, FeSO
47H
2o, MnSO
4h
2the each 2mg of O, urea 0.55g (divide and disappear), pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Method: adopt responsive to temperature type glutamate producing bacterium (L-glutamic acid brevibacterium flavum, Corynebacterium glutamicum etc.), inoculum size is 12%; Culture temperature 32 DEG C, 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
2o 1.5g, VB1200 μ g, VH 600 μ g, FeSO
47H
2o, MnSO
4h
2the each 30mg of O, urea 0.55g (divide and disappear), pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Method: seed liquor access contains in fermention medium full automatic control fermentor tank by the inoculum size by 12%, control ph 7.0 ~ 7.2; Different oxygen supply Schema control dissolved oxygen: 0 ~ 10h is 20%, 10 ~ 32h is 5%; Controlling initial incubation temperature is 32 DEG C; As allometry △ about OD=0.3 (this dilute the OD value of 20 times of concentration at 620nm place for fermented liquid), conversion temp, to 37 DEG C, adds the hydrochloride trimethyl-glycine of 0.1 ~ 0.3g/L simultaneously; During △ OD=0.6, again improve temperature and continue fermentation to terminating to 38 DEG C.Residual sugar controls be 0.1 ~ 3g/L by the glucose strong solution being added 80% by stream.Whole process stream adds liquefied ammonia control pH, and fermentation 0 ~ 14h, pH be 7.0 ~ 7.1,14 ~ 25h are 7.2 ~ 7.3,25h is 6.8 to fermentation ends, produces when acid number no longer changes and has pH value to rise and is fermentation termination; Fermentation time is 30-32h.
Step described in 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: the cytogamy breeding of Chen Ning, Peng Fei, Zhang Kexu .L-L-glutamic acid temperature sensitive mutant and fermentation condition, 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, scientific and technological communication of fermenting, 2003,33 (2): 3-5.
The trimethyl-glycine that step described in the inventive method (2) is added is one or more the mixture in hydrochloride trimethyl-glycine, BETAINE anhydrous or a 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 characteristic utilizing the growth of thalline and the synthesis of product, respectively in initial and alternating temperature process, add trimethyl-glycine in the medium and methyl is provided, and cellular respiration stimulating, improve the oxygen consumption rate of thalline, the accumulation of lactic acid bacteria inhibiting, improve biomass growth rate and rate of producing acid.L-glutamic acid obtained thus, not phosphorous composition, can directly be used in protective foods and pharmaceutical industries, have larger using value.
Embodiment
Further illustrate the present invention below by embodiment, the cited case does not limit the scope of the invention:
Embodiment 1
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum
Seed culture medium (/L):
Glucose 15g, corn steep liquor 20g, K2HPO
43g, MgSO47H
2o 1g, VB1200 μ g, DL-methionine 50 μ g, FeSO
47H
2o, MnSO
4h
2the each 2mg of O, urea 0.55g (divide and disappear), pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Seed culture method: by under aseptic for slant strains 10ml washing, transfers in the 5000mL kind bottle that 1200mL seed culture medium is housed, culture temperature 32 DEG C, 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
2o 1.5g, VB1200 μ g, VH 600 μ g, FeSO
47H
2o, MnSO
4h
2the each 30mg of O, urea 0.55g (divide and disappear), pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Fermentation culture method: seed liquor all accesses in the 15L full automatic control fermentor tank containing fermention medium by the inoculum size by 12%, and control ph is adjusted to 7.0 ~ 7.2; Different oxygen supply Schema control dissolved oxygen: 0 ~ 10h is 20%, 10 ~ 32h is 5%; Controlling initial incubation temperature is 32 DEG C.As allometry optical density value △ about OD=0.3 (fermented liquid dilutes the OD value of 20 times of concentration at 620nm place), conversion temp, to 37 DEG C, meanwhile, then adds the hydrochloride trimethyl-glycine of 0.1g/L; During △ OD=0.6, again improve temperature and continue fermentation to terminating to 38 DEG C.Residual sugar controls be 1g/L by the glucose strong solution being added 80% by stream.Whole process stream adds liquefied ammonia control pH, and fermentation 0 ~ 14h, pH be 7.0 ~ 7.1,14 ~ 25h are 7.2 ~ 7.3,25h is 6.8 to fermentation ends, produces when acid number no longer changes and has pH value slightly to rise and is fermentation termination (31h).When putting tank, the output of Pidolidone is 216.7g/L, and glucose acid invert ratio is 65.3%.
Comparative example 1:
Reference examples 1 is with the difference of embodiment 1: do not add trimethyl-glycine when temperature transition to 37 DEG C, and when temperature transition to 38 DEG C, adds a water trimethyl-glycine of 1.0g/L, and other conditions are identical with embodiment 1.During fermentation ends, the output of Pidolidone is 212.9g/L, and glucose acid invert ratio is 63.3%.Pidolidone output when comparing result shows embodiment 1 fermentation ends and glucose acid invert ratio are obviously better than comparative example 1.
Embodiment 2:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 1.When putting tank, the output of Pidolidone is 213.1g/L, and glucose acid invert ratio is 62.8%.
Embodiment 3:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 1.When putting tank, the output of Pidolidone is 211.8g/L, and glucose acid invert ratio is 61.7%.
Embodiment 4:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; Fermentation initial medium is the hydrochloride trimethyl-glycine adding 0.3g/L in the fermentation medium; Other conditions and cultural method are with embodiment 1.When putting tank, the output of Pidolidone is 219.4g/L, and glucose acid invert ratio is 68.1%.
Comparative example 2:
Reference examples 2 is with the difference of embodiment 4: residual sugar controls be 4g/L by the glucose strong solution being added 80% by stream.Other conditions are identical with embodiment 4.During fermentation ends, the output of Pidolidone is 216.8g/L, and glucose acid invert ratio is 65.5%.Pidolidone output when comparing result shows embodiment 4 fermentation ends and glucose acid invert ratio are obviously better than comparative example 2.
Comparative example 3:
Reference examples 3 is with the difference of embodiment 4: residual sugar controls be 10g/L by the glucose strong solution being added 80% by stream.Other conditions are identical with embodiment 4.During fermentation ends, the output of Pidolidone is 211.2g/L, and glucose acid invert ratio is 61.3%.Pidolidone output when comparing result shows embodiment 4 fermentation ends and glucose acid invert ratio are obviously better than comparative example 3.
Embodiment 4 and comparative example 2,3 Comparative result table (table 1)
Embodiment 5:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 4.When putting tank, the output of Pidolidone is 214.3g/L, and glucose acid invert ratio is 63.4%.
Embodiment 6:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 4.When putting tank, the output of Pidolidone is 209.7g/L, and glucose acid invert ratio is 61.2%.
Embodiment 7:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; Fermentation initial medium is the hydrochloride trimethyl-glycine adding 1.5g/L in the fermentation medium; Other conditions and cultural method are with embodiment 1.When putting tank, the output of Pidolidone is 216.2g/L, and glucose acid invert ratio is 65.1%.
Embodiment 8:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1g/L simultaneously; Other conditions and cultural method are with embodiment 7.When putting tank, the output of Pidolidone is 213.8g/L, and glucose acid invert ratio is 63.1%.
Embodiment 9:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; When conversion temp is to 37 DEG C, add the hydrochloride trimethyl-glycine of 1.5g/L simultaneously; Other conditions and cultural method are with embodiment 7.When putting tank, the output of Pidolidone is 210.2g/L, and glucose acid invert ratio is 61.4%.
Embodiment 1 ~ 9 Comparative result table (table 2)
Embodiment 10:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: Corynebacterium glutamicum; Apply existing trimethyl-glycine addition manner namely to ferment and disposablely in initial medium add hydrochloride trimethyl-glycine 0.4g/L, the later stage no longer adds trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting tank, the output of Pidolidone is 213.3g/L, and glucose acid invert ratio is 62.6%.
Embodiment 11:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; Apply existing trimethyl-glycine addition manner namely to ferment and disposablely in initial medium add hydrochloride trimethyl-glycine 1.6g/L, the later stage no longer adds trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting tank, the output of Pidolidone is 215.6g/L, and glucose acid invert ratio is 64.1%.
Embodiment 12:
The bacterial strain adopted is responsive to temperature type glutamate producing bacterium: L-glutamic acid brevibacterium flavum; Apply existing trimethyl-glycine addition manner namely to ferment and disposablely in initial medium add hydrochloride trimethyl-glycine 3.0g/L, the later stage no longer adds trimethyl-glycine.Other fermentation conditions are with embodiment 1.When putting tank, the output of Pidolidone is 213.1g/L, and glucose acid invert ratio is 62.9%.
Embodiment 10 ~ 12 and embodiment 4 Comparative result table (table 3)
Claims (5)
1. fermentation is for the method for Pidolidone, it is characterized in that, the method comprises the following steps:
(1) seed culture
Seed culture medium/L:
Glucose 15g, corn steep liquor 20g, K
2hPO
43g, MgSO47H
2o 1g, VB1200 μ g, DL-methionine 50 μ g, FeSO
47H
2o, MnSO
4h
2the each 2mg of O, urea 0.55g divide and disappear, pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Method: adopt responsive to temperature type glutamate producing bacterium, inoculum size is 12%; Culture temperature 32 DEG C, 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
2o 1.5g, VB1200 μ g, VH 600 μ g, FeSO
47H
2o, MnSO
4h
2the each 30mg/L of O, urea 0.55g divide and disappear, pH7.0 ~ 7.2,121 DEG C of sterilizing 15min;
Method: seed liquor access contains in fermention medium full automatic control fermentor tank by the inoculum size by 12%, control ph 7.0 ~ 7.2; Different oxygen supply Schema control dissolved oxygen: 0 ~ 10h is 20%, 10 ~ 32h is 5%; Controlling initial incubation temperature is 32 DEG C; As allometry △ OD=0.3, this dilutes the OD value of 20 times of concentration at 620nm place for fermented liquid, and conversion temp, to 37 DEG C, adds the hydrochloride trimethyl-glycine of 0.1 ~ 0.3g/L simultaneously; During △ OD=0.6, again improve temperature and continue fermentation to terminating to 38 DEG C; Residual sugar controls be 0.1 ~ 3g/L by the glucose strong solution being added 80% by stream; Whole process stream adds liquefied ammonia control pH, and fermentation 0 ~ 14h, pH be 7.0 ~ 7.1,14 ~ 25h are 7.2 ~ 7.3,25h is 6.8 to fermentation ends, produces when acid number no longer changes and has pH value to rise and is fermentation termination; Fermentation time is 30-32h.
2. fermentation according to claim 1 is for the method for Pidolidone, it is characterized in that, described step (1) responsive to temperature type glutamate producing bacterium is L-glutamic acid brevibacterium flavum or Corynebacterium glutamicum.
3. fermentation according to claim 2 is for the method for Pidolidone, it is characterized in that, described step (1) responsive to temperature type glutamate producing bacterium is L-glutamic acid brevibacterium flavum.
4. fermentation according to claim 1 is for the method for Pidolidone, it 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 a water trimethyl-glycine.
5. fermentation according to claim 4 is for the method for Pidolidone, it is characterized in that, the trimethyl-glycine that described step (2) is added is 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 |
| CN105602881B (en) * | 2016-03-11 | 2019-06-25 | 天津科技大学 | A kind of temperature sensitive type recombination corynebacterium glutamicum and its application producing glutamic acid |
| 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 |
| CN112322673B (en) * | 2020-11-11 | 2022-10-18 | 廊坊梅花生物技术开发有限公司 | Fermentation method of glutamic acid |
| CN112251475B (en) * | 2020-11-19 | 2022-09-13 | 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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| CN101319239A (en) * | 2008-07-16 | 2008-12-10 | 广州大学 | Application of dimethyl-ethyl-betaine in glutamic acid fermentation |
| CN101457243A (en) * | 2009-01-06 | 2009-06-17 | 天津科技大学 | Novel process for improving L-glutamic acid fermentation production rate |
| CN101705262A (en) * | 2009-12-03 | 2010-05-12 | 天津科技大学 | New process for improving fermentation and acid production rate of glutamic acid temperature sensitive mutant by utilizing glycine betaine |
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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 |
| CN101457243A (en) * | 2009-01-06 | 2009-06-17 | 天津科技大学 | Novel process for improving L-glutamic acid fermentation production rate |
| CN101705262A (en) * | 2009-12-03 | 2010-05-12 | 天津科技大学 | New process for improving fermentation and acid production rate of glutamic acid temperature sensitive mutant by utilizing glycine betaine |
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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. |