CN102492746A - Method for co-producing gamma-polyglutamic acid and glutamic acid by Bacillus licheniformis fermentation - Google Patents
Method for co-producing gamma-polyglutamic acid and glutamic acid by Bacillus licheniformis fermentation Download PDFInfo
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Abstract
The invention discloses a method for co-producing gamma-polyglutamic acid and glutamic acid by Bacillus licheniformis fermentation. According to a technical scheme of the invention, the method comprises the following steps: 1) activating bacterium, 2) preparing a seed liquid, and 3) fermenting in a fermentation cylinder. The method is characterized in that the optimized Bacillus licheniformis cultured by co-produced medium produces the bacterium, so that the co-production of gamma-polyglutamic acid and glutamic acid can be realized, the utilization rate of the raw materials can be increased, and the production cost can be substantially reduced.
Description
Technical field:
The present invention relates to a kind of method of producing gamma-polyglutamic acid-and L-glutamic acid, belong to the biological fermentation engineering field through Bacillus licheniformis.
Background technology:
Gamma-polyglutamic acid-and L-glutamic acid are important chemical material; Be the most general method with fermentative Production gamma-polyglutamic acid-or L-glutamic acid both at home and abroad at present, promptly the bacterial classification through cultivating and producing polyglutamic acid or L-glutamic acid obtains product, produces the level of product in order to improve this method; People are through the strong bacterial strain of seed selection synthesis capability; Screening and optimization culture medium prescription, foundation and optimization fermentation control, many-sides such as improvement and raising downstream engineering technology improve productive rate and quality.As: Nanjing University of Technology screen a strain gamma-polyglutamic acid-superior strain Bacillus subtilis NX-2, and liquid fermenting is produced gamma-polyglutamic acid-has done, and applied for patent than more comprehensively research, patent publication No. is CN1346891.Hua Zhong Agriculture University has also carried out gamma-polyglutamic acid-and has produced the screening of bacterium and the work of process exploitation, and its bacterium and liquid prodn technology have also been applied for patent, and patent publication No. is CN1536071.The Korea S researchist adopts stream to add the high-density cultured method to Bacillus licheniformis ATCC9945a in 2.5 liters of fermentor tanks; The whole output (Yoon S H, DO J H, the Lee S Y.Biotechnol.Lett that ferment and to reach 35g/L after 35 hours; 2000,22:585-588).Kubota separates a strain Bacillus subtilis F201 who obtains in Osaka City University soil; This bacterium can reach the production peak of 50g/L under best fermentative prodn condition; This be bibliographical information production peak (Kubota H.Biosci Biotec Biochem, 1993:1212-1213).
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; Fierce further with market competition of rising steadily along with the prices of raw and semifnished materials; Improve glutamic acid yield and the purpose of transformation efficiency in order to reach simultaneously to reduce production costs; Still have in a large number and report, mainly concentrate on the aspects such as exploitation of utilization 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.
In sum, what existing technology was done all is to utilize the production bacterial classification to produce gamma-polyglutamic acid-separately or produce a kind of product of L-glutamic acid separately, has problems such as raw material availability is low, cost height.
Summary of the invention:
Technical problem to be solved by this invention provides the method for a kind of coproduction gamma-polyglutamic acid-and L-glutamic acid
Technical scheme of the present invention is summarized as follows:
The present invention cultivates Bacillus licheniformis with the coproduction fermention medium of optimizing, and (this bacterial classification of Bacillus licheniformis has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center at present; Bacterium numbering CGMCC3336), realize the coproduction of gamma-polyglutamic acid-and L-glutamic acid.
Described coproduction fermention medium is formed (g/L) as follows: glucose 60-100, Sodium Glutamate 60-100, an ammonium nitrate 10-30, NaCl 5.0-25, MgSO
46H
2O 0.1-2.0, CaCl
26H
2O 0.5-2.5, FeSO
46H
2O 0.005-0.025, l-arginine 0.1-1.5, choline 0.05-0.25.
The coproduction fermention medium consists of (g/L): glucose 80, and Sodium Glutamate 80, an ammonium nitrate 18, NaCl 10, MgSO
46H
2O 0.5, CaCl
26H
2O 1.0, FeSO
46H
2O 0.01, l-arginine 0.44, choline 0.1.
The coproduction fermention medium consists of (g/L): glucose 70, and Sodium Glutamate 60, an ammonium nitrate 10, NaCl 15, MgSO
46H
2O 1.0, CaCl
26H
2O 0.5, FeSO
46H
2O 0.02, l-arginine 0.88, choline 0.05.
The coproduction fermention medium consists of (g/L): glucose 100, and Sodium Glutamate 80, an ammonium nitrate 30, NaCl 20, MgSO
46H
2O 1.5, CaCl
26H
2O 1.5, FeSO
46H
2O 0.005, l-arginine 1.5, choline 0.15.
The coproduction fermention medium consists of (g/L): glucose 80, and Sodium Glutamate 60, an ammonium nitrate 18, NaCl 25, MgSO
46H
2O 0.1, CaCl
26H
2O 2.5, FeSO
46H
2O 0.025, l-arginine 0.1, choline 0.2.
The coproduction fermention medium consists of (g/L): glucose 60, and Sodium Glutamate 100, an ammonium nitrate 27, NaCl 5, MgSO
46H
2O 2.0, CaCl
26H
2O 1.5, FeSO
46H
2O 0.02, l-arginine 0.44, choline 0.25.
Fermentation process: 30L fermentor tank liquid amount is 15L, uses the coproduction fermention medium, and the inoculum size of seed liquor is 1.0%-15.0%, leavening temperature 30-40 ℃, and fermentation time 24-72 hour, ventilation 0.5-2.0vvm, dissolved oxygen 1-50%, rotating speed 200-400rpm.
Invent used bacterial classification and see that Tianjin Peiyang Biotrans Biotech Co., Ltd applies for a patent the process patent that name is called producing gamma-polyglutamic acid by inert carrier solid state fermentation method, application number: 200910228297.This bacterial classification is that this bacterial classification of Bacillus licheniformis (Bacillus licheniformis) has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, bacterium numbering CGMCC3336 at present.
Beneficial effect:
The present invention cultivates Bacillus licheniformis with the coproduction fermention medium of optimizing, and realizes the coproduction of gamma-polyglutamic acid-and L-glutamic acid, thereby has improved raw material availability, lowers production cost greatly.
Embodiment:
Working method through coproduction gamma-polyglutamic acid-and L-glutamic acid among concrete embodiment narration the present invention below.Unless stated otherwise, used technique means is method known in those skilled in the art among the present invention.In addition, embodiment is interpreted as illustrative, and unrestricted scope of the present invention, essence of the present invention and scope only are defined by the claims.To those skilled in the art, under the prerequisite that does not deviate from essence of the present invention and scope, various changes that the material component in these embodiments and consumption are carried out or change and also belong to protection scope of the present invention.
Embodiment 1
1.CGMCC3336 the activation of bacterial classification: cultivated 16 hours being rich on the culture medium slant of nutrition in 37 ℃, prepare sophisticated inclined-plane seed, the composition of solid medium comprises: peptone 10g/L; Yeast powder 5g/L; NaCl 10g/L, agar 20g/L, pH7.2;
2. the preparation of seed liquor:
The above-mentioned seed of one ring is changed in the triangular flask that contains the liquid nutrient medium that is rich in nutritive substance, and 37 ℃, 220rpm cultivates 16 hours to logarithmic phase;
Add-on preparation seed culture medium according to every liter of substratum: glucose 30; Yeast extract paste 7; Tryptones 10; K
2HPO
40.5; MgSO
46H
2O 0.5, pH7.2 before the sterilization; The substratum of packing 50ml in the triangular flask of 500ml;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, an ammonium nitrate 18, NaCl 10, MgSO
46H
2O 0.5, CaCl
26H
2O 1.0, FeSO
46H
2O 0.01, l-arginine 0.44, choline 0.1, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in the above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 72 hours, ventilation 1.0vvm, dissolved oxygen 10%, rotating speed 200-400rpm; Fermentation ends is after the output of detection γ polyglutamic acid is 5g/L, and the output of L-glutamic acid is 10g/L;
Embodiment 2
The activation of bacterial classification and the preparation of seed liquor are basically with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 70, and Sodium Glutamate 60, an ammonium nitrate 10, NaCl 15, MgSO
46H
2O 1.0, CaCl
26H
2O 0.5, FeSO
46H
2O 0.02, l-arginine 0.88, choline 0.05, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in the above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 72 hours, ventilation 1.0vvm, dissolved oxygen 10%, rotating speed 200-400rpm; Fermentation ends is after the output of detection γ polyglutamic acid is 15g/L, and the output of L-glutamic acid is 30g/L;
Embodiment 3
The activation of bacterial classification and the preparation of seed liquor are basically with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 100, and Sodium Glutamate 80, an ammonium nitrate 30, NaCl 20, MgSO
46H
2O 1.5, CaCl
26H
2O 1.5, FeSO
46H
2O 0.005, l-arginine 1.5, choline 0.15, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in the above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 72 hours, ventilation 1.0vvm, dissolved oxygen 10%, rotating speed 200-400rpm; Fermentation ends is after the output of detection γ polyglutamic acid is 7.5g/L, and the output of L-glutamic acid is 20g/L;
Embodiment 4
The activation of bacterial classification and the preparation of seed liquor are basically with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 60, an ammonium nitrate 18, NaCl 25, MgSO
46H
2O 0.1, CaCl
26H
2O 2.5, FeSO
46H
2O 0.025, l-arginine 0.1, choline 0.2, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in the above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 72 hours, ventilation 1.0vvm, dissolved oxygen 10%, rotating speed 200-400rpm; Fermentation ends is after the output of detection γ polyglutamic acid is 10g/L, and the output of L-glutamic acid is 25g/L;
Embodiment 5
The activation of bacterial classification and the preparation of seed liquor are basically with embodiment 1;
Fermention medium: the fermentation culture based component is (g/L): glucose 60, and Sodium Glutamate 100, an ammonium nitrate 27, NaCl 5, MgSO
46H
2O 2.0, CaCl
26H
2O 1.5, FeSO
46H
2O 0.02, l-arginine 0.44, choline 0.25, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in the above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 72 hours, ventilation 1.0vvm, dissolved oxygen 10%, rotating speed 200-400rpm; Fermentation ends is after the output of detection γ polyglutamic acid is 12.5g/L, and the output of L-glutamic acid is 15g/L.
Claims (7)
1. the method for the lichen bacillus ferments coproduction gamma-polyglutamic acid-and L-glutamic acid, said fermentation process is: 30L fermentor tank liquid amount is 15L, uses the coproduction fermention medium; The inoculum size of seed liquor is 5.0%-15.0%; Leavening temperature 30-40 ℃, fermentation time 24-72 hour, ventilation 0.5-2.0vvm; Dissolved oxygen 5-25%, rotating speed 200-400rpm.
2. coproduction fermention medium as claimed in claim 1 is formed (g/L) as follows: glucose 60-100, Sodium Glutamate 60-100, an ammonium nitrate 10-30, NaCl 5.0-25, MgSO
46H
2O 0.1-2.0, CaCl
26H
2O 0.5-2.5, FeSO
46H
2O 0.005-0.025, l-arginine 0.1-1.5, choline 0.05-0.25.
3. the method for a kind of the lichen bacillus ferments coproduction gamma-polyglutamic acid-as claimed in claim 2 and L-glutamic acid is characterized in that, said coproduction fermention medium consists of (g/L): glucose 80, and Sodium Glutamate 80, an ammonium nitrate 18, NaCl 10, MgSO
46H
2O 0.5, CaCl
26H
2O 1.0, FeSO
46H
2O 0.01, l-arginine 0.44, choline 0.1.
4. coproduction fermention medium as claimed in claim 2 consists of (g/L): glucose 70, and Sodium Glutamate 60, an ammonium nitrate 10, NaCl 15, MgSO
46H
2O 1.0, CaCl
26H
2O 0.5, FeSO
46H
2O 0.02, l-arginine 0.88, choline 0.05.
5. consist of (g/L) like the said coproduction fermention medium of claim 2: glucose 100, Sodium Glutamate 80, an ammonium nitrate 30, NaCl 20, MgSO
46H
2O 1.5, CaCl
26H
2O 1.5, FeSO
46H
2O 0.005, l-arginine 1.5, choline 0.15.
6. consist of (g/L) like the said coproduction fermention medium of claim 2: glucose 80, Sodium Glutamate 60, an ammonium nitrate 18, NaCl 25, MgSO
46H
2O 0.1, CaCl
26H
2O 2.5, FeSO
46H
2O 0.025, l-arginine 0.1, choline 0.2.
7. consist of (g/L) like the said coproduction fermention medium of claim 2: glucose 60, Sodium Glutamate 100, an ammonium nitrate 27, NaCl 5, MgSO
46H
2O 2.0, CaCl
26H
2O 1.5, FeSO
46H
2O 0.02, l-arginine 0.44, choline 0.25.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719501A (en) * | 2012-06-29 | 2012-10-10 | 天津北洋百川生物技术有限公司 | Method for producing polyglutamic acid |
| CN110923275A (en) * | 2019-12-24 | 2020-03-27 | 内蒙古阜丰生物科技有限公司 | Fermentation and extraction process of glutamic acid |
Citations (1)
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| CN101705260A (en) * | 2009-11-16 | 2010-05-12 | 天津北洋百川生物技术有限公司 | Method for producing gamma-polyglutamic acid by inert carrier solid state fermentation method |
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| CN101705260A (en) * | 2009-11-16 | 2010-05-12 | 天津北洋百川生物技术有限公司 | Method for producing gamma-polyglutamic acid by inert carrier solid state fermentation method |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102719501A (en) * | 2012-06-29 | 2012-10-10 | 天津北洋百川生物技术有限公司 | Method for producing polyglutamic acid |
| CN110923275A (en) * | 2019-12-24 | 2020-03-27 | 内蒙古阜丰生物科技有限公司 | Fermentation and extraction process of glutamic acid |
| CN110923275B (en) * | 2019-12-24 | 2024-01-12 | 内蒙古阜丰生物科技有限公司 | Glutamic acid fermentation and extraction process |
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