CN1067434C - Glutamine fermentation production process - Google Patents
Glutamine fermentation production process Download PDFInfo
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- CN1067434C CN1067434C CN98122156A CN98122156A CN1067434C CN 1067434 C CN1067434 C CN 1067434C CN 98122156 A CN98122156 A CN 98122156A CN 98122156 A CN98122156 A CN 98122156A CN 1067434 C CN1067434 C CN 1067434C
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Abstract
The present invention discloses a glutamine fermentation production process which effectively solves the problems that the shake flask fermentation result can not be reproduced on the scale of a fermentation tank, and the strain potentiality can not be fully brought into play in the existing glutamine production study. The present invention uses the processes of the sectional control of pH value, oxygen dissolution, the control of sugar concentration in fermentation liquid, the replenishment of potassium chloride and corn steep liquor, etc. for the fermentation production of glutamine. The method of the present invention has the advantages of novelty and high fermentation level of glutamine and is suitable for the mass production of glutamine.
Description
The invention belongs to the amino acid fermentation field, relate in particular to a kind of glutamine fermentation production process.
The understanding of people for glutamine has been corrected in trophology and new discovery medically gradually, and glutamine has been used as the essential amino acid of a kind of condition now to be treated, and the industrialization of glutamine will have great economic benefit and social benefit.Although there are some research groups that many research work (microbiology circular, 4 (5): 211-212 (1984) have been done in its generation both at home and abroad; 16 (2): 73-76 (1989); JOURNAL OF MICROBIOLOGY, 6 (3): 35-37 (1986); Industrial microorganism, 21 (5): 11-17 (1988); Food and fermentation industries, No.2:21-25 (1992); The scientific and technological communication of fermenting, 19 (2): 16-18 (1990); Agri.Biol.Chem., 51 (8): 2089-2094 (1987), the clear 62-186796 of Japan's special permission communique; Clear 63-21379), but most researchs concentrate on the culture medium prescription and the fermentation condition optimization of strain improvement and shake flask fermentation, then there are not document or patent to deliver to the process optimization on the vital fermentor tank level of scale production, consequently the shake flask fermentation level can not be reappeared on the fermentor tank scale, and the bacterial classification potentiality are difficult to performance.
The present invention is directed to above-mentioned deficiency, the glutamine fermentation production process of a whole set of optimization is proposed, provide the control method of all factors that influence the glutamine generation, thereby on the fermentor tank scale, further reappear and improve the result of shake flask fermentation, give full play to the bacterial classification potentiality.
Fermentation manufacturing technique of the present invention comprises the step of following order:
(1) selection of fermentation strain: one of brevibacterium flavum (Brevibacterium flavum), Corynebacterium glutamicum (Corynebacterium glutamicum), Beijing rod bacillus (Corynebacterium pekinense), Corynebacterium crenatum (Corynebacterium crenatum).
(2) female inclined-plane preparation: with above-mentioned fermentation strain inoculation inclined-plane, 29-37 ℃ constant temperature culture 8-20 hour, make female inclined-plane, the refrigeration of female inclined-plane is stand-by.
(3) sub-inclined-plane: sub-inclined-plane is inoculated on female inclined-plane that step (2) obtains, the same step of cultural method (2).
(4) first order seed is cultivated: the inclined-plane lawn that step (3) is obtained is inoculated in sterilized the shaking in the bottle of seed culture medium of being equipped with, and cultivates 8-20 hour at 29-37 ℃ of following shaking table.
(5) secondary seed is cultivated: the cultured first order seed of step (4) is inoculated in the sterilized seeding tank that seed culture medium is housed by 2%-10% (volume ratio) begins to cultivate; Culture condition: air flow 0.5-1.5VVM, tank pressure 0.01-0.05Mpa, temperature 29-37 ℃, dissolved oxygen 3%-80% saturation ratio; Incubation time 6-18 hour.
(6) ferment tank: step (5) cultured seed is inoculated in the sterilized fermentor tank that fermention medium is housed by 2%-10% (volume ratio) begins fermentation, control condition is: temperature 29-37 ℃, air flow 0.5-1.5VVM, tank pressure 0.01-0.05MPa, dissolved oxygen 0.5%-90% saturation ratio, pH6.5-7.5, and with the defoamer froth breaking; Fermented 8-24 hour, and mended the Repone K of initial medium 0.5-1.5% (weightmeasurement ratio) of going into to ferment; Fermented 14-26 hour, stop pH control, make pH be reduced to 6.2-6.6 naturally, control pH afterwards in this scope, dissolved oxygen is between the 20-80% saturation ratio, and Continuous Flow adds the sugar soln of 30-80% (weightmeasurement ratio), perhaps sugar soln criticized by each every liter of fermented liquid 10-30 gram sugar and mended, and sugared concentration is between 5-40g/L in the control fermentor tank; Add after the Repone K, every interval 8-24 hour benefit gone into the corn steep liquor of fermented liquid cumulative volume 0.05%-0.3% (volume ratio); Fermenting stopped to mend sugar in 50-68 hour, fermentation ends when treating that sugared concentration is reduced to 1-10g/L.
In above-mentioned fermentation manufacturing technique process, brevibacterium flavum in the step (1) is brevibacterium flavum (Brevibacterium flavum) ATCC 14067, AS 1.495, one of AS 1.582 and their mutant strain, Corynebacterium glutamicum is meant Corynebacterium glutamicum (Corynebacterium glutamicum) ATCC13286, ATCC 14751, ATCC 14752, ATCC 13032, AS 1.805, ACCC11067, ACCC 11063, IFFI 10056, IFFI 10054, IFFI 10131, IFFI 10039, IFFI10031, IFFI 10051, IFFI 10052, one of IFFI 10111 and their mutant strain, Beijing rod bacillus is meant one of Beijing rod bacillus (Corynebacterium pekinense) AS 1.299 and its mutant strain, and Corynebacterium crenatum is meant one of Corynebacterium crenatum (Corynebacterium crenatum) AS 1.452 and its mutant strain.
The time of preserving bacterial strain in the step (2) is to be no more than one month for good.
The Control of Dissolved Oxygen is by adjusting mixing speed, air flow, tank pressure realization in the step (6).
PH is by mending the ammoniacal liquor adjusting that concentration is 10%-30% (weightmeasurement ratio) in the step (6).
Defoamer is one of polyethers or silicone defoamer in the step (6).
Corn steep liquor is dense corn steep liquor or rare corn steep liquor in the step (6), and dense corn steep liquor dry matter content is 50%-90% (weight ratio), and rare corn steep liquor dry matter content is 5%-50% (weight ratio).
It is better with the 0.10%-0.30% (volume ratio) of fermented liquid cumulative volume that the benefit of rare corn steep liquor is gone into amount in the step (6); Dense corn steep liquor is mended into amount better with the 0.05%-0.15% (volume ratio) of fermented liquid cumulative volume.
Sugar soln is meant one of glucose, amylum hydrolysate of the sugar in the step (6).
After adopting above-mentioned technology on the fermentor tank, fermentation level is much higher than the shake flask fermentation result, and the bacterial classification potentiality are not fully exerted, and its technology provides foundation for the scale operation glutamine.Following table has been showed the effect comparison of shake flask fermentation experiment with 50 liters of ferment tank experiments.
Table: shake flask fermentation and ferment tank produced glutamine concentration contrast (unit: g/L) in 72 hours
| Strain name | Brevibacterium flavum ATCC 14067 | Corynebacterium glutamicum ATCC 14751 | Corynebacterium glutamicum ATCC 14752 | Beijing rod bacillus AS 1.299 | Corynebacterium crenatum AS 1.452 |
| A shakes bottle | 22.4 | 13.5 | 16.2 | 8.4 | 11.5 |
| The B fermentor tank | 56 | 45 | 35.7 | 22.2 | 31.6 |
| B increases than A | 150.0％ | 233.3％ | 120.4％ | 164.3％ | 174.8％ |
The invention will be further described below in conjunction with embodiment.
Embodiment 1
The substratum that uses comprises slant medium (glucose 0.1, extractum carnis 1.0, peptone 1.0, NaCl0.5, agar 2.0), shake-flask seed substratum (glucose 2.0, (NH
4)
2SO
41.0, KH
2PO
40.1, K
2HPO
43H
2O0.3, MgSO
47H
2O0.01, urea 0.2, corn steep liquor 0.1), fermention medium (glucose 10.0, NH
4Cl4.0, KH
2PO
40.025, urea 0.5, K
2HPO
43H
2O0.025, MgSO
47H
2O0.05, corn slurries 1.0, vitamin H 5 μ g/L, vitamin B12 0 μ g/L, ZnSO
45mg/L, FeSO
45mg/L), the composition of undeclared unit is percent weight in volume more than.
Brevibacterium flavum ATCC14067 bacterial strain is made female inclined-plane in 31 ℃ of following constant temperature culture after 10 hours, be positioned in 4 ℃ of refrigerators and preserved 15 days.The sub-inclined-plane of transferring from female inclined-plane, in 31 ℃ cultivate 10 hours after, connect a ring culture respectively in two sterilized 500ml triangular flasks that the 50ml seed culture medium respectively is housed, 31 ℃ of reciprocating type shaking tables are cultivated reciprocating frequence 110 times, 5.8 centimetres of amplitudes.Cultivate after 15 hours, by 5% volume ratio be inoculated in sterilized the secondary seed jar of 2 liters of seed culture mediums is housed and begins cultivate air flow 1.0VVM in the culturing process, tank pressure 0.03-0.035Mpa, 31 ℃ of temperature are regulated mixing speed control dissolved oxygen between 30%-50%.Behind the incubation time 13 hours, this nutrient solution is inoculated in the sterilized fermentor tank that 40 liters of fermention mediums are housed by 5% volume ratio begins fermentation, 31 ℃ of control leavening temperatures, air flow 1.0VVM, tank pressure 0.035-0.040MPa, with 25% weightmeasurement ratio ammoniacal liquor control pH6.9-7.1,, and be between the 20%-30% saturation ratio by adjusting mixing speed control dissolved oxygen with polyoxyethylene oxypropylene glycerine (claiming the bubble enemy again) defoamer froth breaking; Fermented 13 hours, disposable benefit is gone into 1.35 liters of the Klorvess Liquids that sterilized concentration is 30% (weightmeasurement ratio); Fermented 18 hours, stop to mend ammoniacal liquor, make pH be reduced to 6.40 naturally, continue afterwards with 25% ammoniacal liquor control pH in the 6.35-6.45 scope, dissolved oxygen is between the 35-50% saturation ratio, and Continuous Flow adds the glucose solution of 70% weightmeasurement ratio, and sugared concentration is between 10-15g/L in the control fermentor tank.To 34 hours and 50 hours, mend sterilized dense corn steep liquor 40ml in fermentation respectively.Fermenting stopped to mend sugar in 64 hours, and 72 hours finish fermentation, at this moment, and remaining sugar concentration 2g/L in the substratum, L-glutaminate concentration 56g/L.
Embodiment 2
Used various culture medium prescription is with embodiment 1.
Corynebacterium glutamicum ATCC13286 bacterial strain is made female inclined-plane in 29 ℃ of following constant temperature culture after 20 hours, be positioned in 4 ℃ of refrigerators and preserved 3 days.The sub-inclined-plane of transferring from female inclined-plane, in 29 ℃ cultivate 20 hours after, connect a ring culture respectively in two sterilized 500ml triangular flasks that the 50ml seed culture medium respectively is housed, 29 ℃ of shaking tables are cultivated, reciprocating frequence 110 times, 5.8 centimetres of amplitudes, cultivate after 20 hours, by 2% volume ratio be inoculated in sterilized the secondary seed jar of 2 liters of seed culture mediums is housed and begins cultivate, air flow 1.5VVM in the culturing process, tank pressure 0.01-0.03Mpa, 29 ℃ of temperature are regulated mixing speed control dissolved oxygen between the 40%-90% saturation ratio.Behind the incubation time 18 hours, this nutrient solution is inoculated in the sterilized fermentor tank that 40 liters of fermention mediums are housed by 2% volume ratio begins fermentation, 29 ℃ of control leavening temperatures, air flow 1.5VVM, tank pressure 0.01-0.03MPa, with 29% weightmeasurement ratio ammoniacal liquor control pH7.2-7.5, oppose the defoamer froth breaking with bubble, and be between the 30%-80% saturation ratio by adjusting mixing speed control dissolved oxygen; Fermented 24 hours, disposable benefit is gone into 3.0 liters of the Klorvess Liquids that sterilized concentration is 20% (weightmeasurement ratio); Fermented 26 hours, stop to mend ammoniacal liquor, make pH be reduced to 6.45 naturally, continue afterwards with 29% ammoniacal liquor control pH in the 6.5-6.6 scope, dissolved oxygen is between the 40-80% saturation ratio, and Continuous Flow adds the glucose solution of 80% weightmeasurement ratio, and sugared concentration is between 24-40g/L in the control fermentor tank.To 32 hours, 42 hours, 54 hours, mend sterilized rare corn steep liquor 40ml in fermentation respectively.Fermenting stopped to mend sugar in 68 hours, and 80 hours finish fermentation, at this moment, and remaining sugar concentration 1g/L in the substratum, L-glutaminate concentration 42g/L.
Embodiment 3
Used various culture medium prescription is with embodiment 1.
Beijing rod bacillus AS1.299 bacterial strain is made female inclined-plane in 37 ℃ of following constant temperature culture after 8 hours, be positioned in 4 ℃ of refrigerators and preserved 30 days.The sub-inclined-plane of transferring from female inclined-plane, in 37 ℃ cultivate 8 hours after, connect a ring culture respectively in two sterilized 500ml triangular flasks that the 50ml seed culture medium respectively is housed, 37 ℃ of shaking tables are cultivated, reciprocating frequence 110 times, 5.8 centimetres of amplitudes, cultivate after 8 hours, by 10% volume ratio be inoculated in sterilized the secondary seed jar of 2 liters of seed culture mediums is housed and begins cultivate, air flow 0.5VVM in the culturing process, tank pressure 0.04-0.05Mpa, 37 ℃ of temperature are regulated mixing speed control dissolved oxygen between 3%-40%.Behind the incubation time 8 hours, this nutrient solution is inoculated in the sterilized fermentor tank that 40 liters of fermention mediums are housed by 10% volume ratio begins fermentation, 34 ℃ of control leavening temperatures, air flow 0.5VVM, tank pressure 0.04-0.05MPa, with 10% ammoniacal liquor (weightmeasurement ratio) control pH6.5-6.9,, and be between the 0.5%-40% saturation ratio by adjusting mixing speed control dissolved oxygen with silicone defoaming emulsion froth breaking; Fermented 8 hours, disposable benefit is gone into 0.66 liter of the Klorvess Liquid that sterilized concentration is 30% (weightmeasurement ratio); Fermented 14 hours, stop to mend ammoniacal liquor, make pH be reduced to 6.2 naturally, continue afterwards with 10% (weightmeasurement ratio) ammoniacal liquor control pH in the 6.2-6.3 scope, dissolved oxygen is between the 20-40% saturation ratio, and be that the starch liquefacation sugar soln of 40% (weightmeasurement ratio) is mended by each 2 liters in batches with sterilized concentration, each was mended once in fermentation 22 hours, 34 hours, 50 hours.To 30 hours and 54 hours, mend sterilized rare corn steep liquor 40ml in fermentation respectively.Finished fermentation in 64 hours, at this moment, remaining sugar concentration 10g/L in the substratum, L-glutaminate concentration 19g/L.
Claims (9)
1. glutamine fermentation production process, its sequence of steps is as follows:
(1) selection of fermentation strain: one of brevibacterium flavum (Brevibacterium flavum), Corynebacterium glutamicum (Corynebacterium glutamicum), Beijing rod bacillus (Corynebacterium pekinense), Corynebacterium crenatum (Corynebacterium crenatum);
(2) female inclined-plane preparation: with above-mentioned fermentation strain inoculation inclined-plane, 29-37 ℃ constant temperature culture 8-20 hour, make female inclined-plane, the refrigeration of female inclined-plane is stand-by;
(3) sub-inclined-plane: sub-inclined-plane is inoculated on female inclined-plane that step (2) obtains, the same step of cultural method (2);
(4) first order seed is cultivated: the inclined-plane lawn that step (3) is obtained is inoculated in sterilized the shaking in the bottle of seed culture medium of being equipped with, and cultivates 8-20 hour at 29-37 ℃ of following shaking table;
(5) secondary seed is cultivated: the cultured first order seed of step (4) is inoculated in the sterilized seeding tank that seed culture medium is housed by 2%-10% (volume ratio) begins to cultivate; Culture condition: air flow 0.5-1.5VVM, tank pressure 0.01-0.05Mpa, temperature 29-37 ℃, dissolved oxygen 3%-80% saturation ratio; Incubation time 6-18 hour;
(6) ferment tank: step (5) cultured seed is inoculated in the sterilized fermentor tank that fermention medium is housed by 2%-10% (volume ratio) begins fermentation, control condition is: temperature 29-37 ℃, air flow 0.5-1.5VVM, tank pressure 0.01-0.05MPa, dissolved oxygen 0.5%-90% saturation ratio, pH6.5-7.5, and with the defoamer froth breaking; Fermented 8-24 hour, and mended the Repone K of initial medium 0.5-1.5% (weightmeasurement ratio) of going into to ferment; Fermented 14-26 hour, stop pH control, make pH be reduced to 6.2-6.6 naturally, control pH afterwards in this scope, dissolved oxygen is between the 20-80% saturation ratio, and Continuous Flow adds the sugar soln of 30-80% (weightmeasurement ratio), perhaps sugar soln criticized by each every liter of fermented liquid 10-30 gram sugar and mended, and sugared concentration is between 5-40g/L in the control fermentor tank; Add after the Repone K, every interval 8-24 hour benefit gone into the corn steep liquor of fermented liquid cumulative volume 0.05%-0.3% (volume ratio); Fermenting stopped to mend sugar in 50-68 hour, fermentation ends when treating that sugared concentration is reduced to 1-10g/L.
2. glutamine fermentation production process as claimed in claim 1, it is characterized in that, brevibacterium flavum described in the step (1) is brevibacterium flavum (Brevibacterium flavum) ATCC14067, AS1.495, one of AS 1.582, Corynebacterium glutamicum is meant Corynebacterium glutamicum (Corynebacteriumglutamicum) ATCC13286, ATCC 14751, ATCC 14752, ATCC 13032, AS1.805, ACCC 11067, ACCC 11063, IFFI 10056, IFFI 10054, IFFI10131, IFFI 10039, IFFI 10031, IFFI 10051, IFFI 10052, one of IFFI 10111, Beijing rod bacillus is meant Beijing rod bacillus (Corynebacterium pekinense) AS 1.299, and Corynebacterium crenatum is meant Corynebacterium crenatum (Corynebacterium crenatum) AS 1.452.
3. glutamine fermentation production process as claimed in claim 1 is characterized in that, the time of preserving bacterial strain in the step (2) is no more than one month.
4. glutamine fermentation production process as claimed in claim 1 is characterized in that, The Control of Dissolved Oxygen is by adjusting mixing speed, air flow, tank pressure realization in the step (6).
5. glutamine fermentation production process as claimed in claim 1 is characterized in that, pH is by mending the ammoniacal liquor adjusting that concentration is 10%-30% (weightmeasurement ratio) in the step (6).
6. glutamine fermentation production process as claimed in claim 1 is characterized in that, defoamer is polyethers or silicone defoamer in the step (6).
7. glutamine fermentation production process as claimed in claim 1, it is characterized in that, corn steep liquor is dense corn steep liquor or rare corn steep liquor in the step (6), and dense corn steep liquor dry matter content is 50%-90% (weight ratio), and rare corn steep liquor dry matter content is 5%-50% (weight ratio).
8. glutamine fermentation production process as claimed in claim 1 is characterized in that, the benefit of rare corn steep liquor is gone into the 0.10%-0.30% (volume ratio) that amount accounts for the fermented liquid cumulative volume in the step (6); Dense corn steep liquor is mended the 0.05%-0.15% (volume ratio) that accounts for the fermented liquid cumulative volume into amount.
9. glutamine fermentation production process as claimed in claim 1 is characterized in that, sugar soln is meant glucose solution in the step (6).
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|---|---|---|---|
| CN98122156A CN1067434C (en) | 1998-12-28 | 1998-12-28 | Glutamine fermentation production process |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98122156A CN1067434C (en) | 1998-12-28 | 1998-12-28 | Glutamine fermentation production process |
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| CN1225946A CN1225946A (en) | 1999-08-18 |
| CN1067434C true CN1067434C (en) | 2001-06-20 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100457894C (en) * | 2001-02-05 | 2009-02-04 | 味之素株式会社 | Fermentation process of proudcing L-glutamine and bacteria of producing L-glutamine |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1332037C (en) * | 2003-12-12 | 2007-08-15 | 上海化工研究院 | Ferment technique for producing 15N stable isotope labeled L-glutamine |
| CN101974603B (en) * | 2010-09-30 | 2013-04-24 | 山东大学 | Method for producing D-alpha-hydroxybutyric acid |
| CN102660482B (en) * | 2012-05-17 | 2013-08-14 | 通辽梅花生物科技有限公司 | Corynebacterium pekinense and application thereof |
| CN103755586B (en) * | 2013-12-24 | 2016-04-06 | 山东民强生物科技股份有限公司 | A kind of preparation method of L-glutaminate |
| CN103667382B (en) * | 2013-12-24 | 2015-09-02 | 山东民强生物科技股份有限公司 | A kind of fermentable produces the method for L-glutaminate |
| CN103667383B (en) * | 2013-12-24 | 2015-09-02 | 山东民强生物科技股份有限公司 | The preparation method of L-glutaminate |
| CN103695491B (en) * | 2013-12-24 | 2016-08-24 | 山东民强生物科技股份有限公司 | The process for purification of L-glutaminate |
| CN103695492B (en) * | 2013-12-24 | 2016-04-06 | 山东民强生物科技股份有限公司 | A kind of method improving L-glutamine yield |
| CN104404097B (en) * | 2014-11-24 | 2018-04-27 | 河南巨龙生物工程股份有限公司 | A kind of fermentation method for producing of high-yield glutamine |
| CN104830941A (en) * | 2015-04-29 | 2015-08-12 | 宁夏诚志万胜生物工程有限公司 | Microbial conversion method utilizing mixed bacteria to efficiently synthesize L-theanine |
| CN105349590B (en) * | 2015-08-10 | 2021-05-25 | 安徽丰原发酵技术工程研究有限公司 | Method for producing glutamine by microbial fermentation feeding |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1128295A (en) * | 1994-01-10 | 1996-08-07 | 味之素株式会社 | Method of producing L-glutamic acid by fermentation |
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1998
- 1998-12-28 CN CN98122156A patent/CN1067434C/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1128295A (en) * | 1994-01-10 | 1996-08-07 | 味之素株式会社 | Method of producing L-glutamic acid by fermentation |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100457894C (en) * | 2001-02-05 | 2009-02-04 | 味之素株式会社 | Fermentation process of proudcing L-glutamine and bacteria of producing L-glutamine |
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