CN102268465B - Method for massively producing gamma-poly-glutamic acid - Google Patents

Method for massively producing gamma-poly-glutamic acid Download PDF

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CN102268465B
CN102268465B CN 201110216717 CN201110216717A CN102268465B CN 102268465 B CN102268465 B CN 102268465B CN 201110216717 CN201110216717 CN 201110216717 CN 201110216717 A CN201110216717 A CN 201110216717A CN 102268465 B CN102268465 B CN 102268465B
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fed
glucose
polyglutamic acid
gamma
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CN102268465A (en
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乔长昇
兰玲芳
李雪
张帅
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Jinlin Hanggai Tongbo Biotechnology Co ltd
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Tianjin Peiyang Biotrans Biotech Co Ltd
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Abstract

The invention discloses a method for producing gamma-poly-glutamic acid, and in particular relates to a method for massively producing high-concentration gamma-poly-glutamic acid. The method comprises the following steps of: 1, activating strains; 2, preparing a seed solution; and 3, fermenting in a fermentation tank, and is characterized in that: in the fermentation process, a medium consisting of glucose, ammonium nitrate, CaCl2.6H2O and FeCl3.6H2O is fed in a flowing mode, so that the fermentation yield reaches 1.18g/l.h; therefore, the aim of massively preparing the gamma-poly-glutamic acid at high efficiency is fulfilled.

Description

A kind of method of a large amount of production gamma-polyglutamic acid-s
Technical field:
The invention belongs to macromolecule material preparation area, the present invention relates to a kind of method of producing gamma-polyglutamic acid-.
Background technology:
Gamma-polyglutamic acid-is a kind of multifunctional bio degradable high polymer material that is combined into by the amido linkage between alpha-amino group and γ-carboxyl by D-Glu and Pidolidone.Because gamma-polyglutamic acid-and derivative thereof have good water-soluble and adsorptivity, can thoroughly be biodegradable, to the humans and animals safety non-toxic, the advantage such as edible, thereby can be used as water-holding agent, thickening material, flocculation agent, heavy metal absorbent, medicine/fertilizer slow-release formulation and pharmaceutical carrier etc., have broad application prospects at agricultural, food, medicine, makeup, environmental protection, synthon and the field such as film.Along with the environmental pollution that the chemical industry synthetic materials causes is day by day serious, exploitation Biodegradable material instead of chemical material has to a certain extent become the active demand of whole international community.
The preparation method of polyglutamic acid has chemosynthesis, extraction and three kinds of methods of microbial fermentation.Microbe fermentation method is lower than the production cost of front two kinds of methods, and production process is little to the pollution of environment, so mainly adopt now the Production by Microorganism Fermentation gamma-polyglutamic acid-.Both at home and abroad the zymotechnique of gamma-polyglutamic acid-conducted extensive research, mainly take the culture medium prescription of liquid submerged fermentation and culture process as main.A strain gamma-polyglutamic acid-superior strain Bcillius subtilis NX-2 screens to get in Nanjing University of Technology, and liquid fermenting is produced gamma-polyglutamic acid-done than more comprehensively research, and has applied for patent, and patent publication No. is CN1346891.Hua Zhong Agriculture University has also carried out the screening of gamma-polyglutamic acid generating bacterium and the work of process exploitation, and its bacterial classification and liquid production technique have also been applied for patent, and patent publication No. is CN1536071.The Korea S researchist adopts stream to add the method that high-density is cultivated to Bacillus licheniformis ATCC9945a in 2.5 liters of fermentor tanks, the whole output (Yoon SH, DO JH, the Lee SY.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 bacterial strain can reach the production peak of 50g/L under best fermentation conditions, this is the production peak (Kubota H.Biosci Biotec Biochem, 1993:1212-1213) of bibliographical information.
Although the fermentative production of gamma-polyglutamic acid-has obtained larger progress, still exist the production cycle longer, production efficiency is low, the problem that production cost is higher.Therefore, efficient, low cost preparing gamma-polyglutamic acid still remains further to be studied.
Summary of the invention:
Technical problem to be solved by this invention is to provide a kind of method of a large amount of production high density gamma-polyglutamic acid-s
Technical scheme of the present invention is summarized as follows:
A kind of method of a large amount of production gamma-polyglutamic acid-s comprises the activation of bacterial classification, preparation and the ferment tank of seed liquor, when in fermention medium, sugared concentration is down to 5-20g/L, adds fed-batch medium to the fermentation ends 2-6 hour with the speed of 0.1-2.0ml/min; In fed-batch medium, each material concentration is respectively: glucose 600-1000g/L, ammonium nitrate 30-60g/L, CaCl 26H 2O 5-20g/L, FeCl 36H 2O 0.05-0.15g/L.
Fermention medium (g/L) composed as follows: glucose 60-100, Sodium Glutamate 60-100, yeast extract paste 10-30, ammonium nitrate 3.0-6.0, NaCl 5.0-15, MgSO 46H 2O 0.1-1.5, CaCl 26H 2O 0.5-2.0, FeCl 36H 2O 0.005-0.015, pH6.0-8.0.
The composition of described fed-batch medium is (g/L): glucose 600-900, ammonium nitrate 30.0-60.0, CaCl 26H 2O 5.0-20.0, FeCl 36H 2O 0.05-0.15;
The composition of fed-batch medium is (g/L): glucose 800, ammonium nitrate 41, CaCl 26H 2O 10, FeCl 36H 2O0.1;
The composition of fed-batch medium is (g/L): glucose 700, ammonium nitrate 30, CaCl 26H 2O 5, FeCl 36H 2O0.05;
The composition of fed-batch medium is (g/L): glucose 900, ammonium nitrate 60, CaCl 26H 2O 20, FeCl 36H 2O0.15;
The composition of fed-batch medium is (g/L): glucose 1000, ammonium nitrate 45, CaCl 26H 2O 15, FeCl 36H 2O 0.1;
The composition of fed-batch medium is (g/L): glucose 600, ammonium nitrate 41, CaCl 26H 2O 10, FeCl 36H 2O0.1;
When in described fermention medium, sugared concentration is down to 7-15g/L, add fed-batch medium;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in above-mentioned fermention medium, the inoculum size of seed liquor is 5.0%-15.0%, leavening temperature 30-40 ℃, fermentation time 24-72 hour, ventilation 1.0-2.0vvm, rotating speed 200-600rpm; Add fed-batch medium in fermenting process to fermentation ends front 6 hours when residual sugar is down to 5-20g/L, flow acceleration is 0.1-2.0ml/min;
Beneficial effect:
(1) the Bacillus licheniformis microorganism strains that uses of the present invention fermentative production gamma-polyglutamic acid-efficiently;
(2) by the optimization to the lichem bacillus strain culture condition, particularly add during the fermentation glucose, ammonium nitrate, CaCl 26H 2O and FeCl 36H 2O makes the content of the gamma-polyglutamic acid-in fermented liquid up to 40-50g/L, and makes the productive rate of gamma-polyglutamic acid-reach 1.0-1.2g/Lh, thereby provides a kind of efficient cheapness to prepare the method for gamma-polyglutamic acid-.
Embodiment:
Production method below by polyglutamic acid in specific embodiment narration the present invention.Unless stated otherwise, in the present invention, technique means used is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention are only limited by 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 domain of the present utility model.
Embodiment 1 controlled trial
1. the activation of bacterial classification: cultivated 16 hours being rich on the culture medium slant of nutrition in 37 ℃, the inclined-plane seed that preparation is ripe, the composition of solid medium comprises: peptone 10g/L, yeast powder 5g/L, NaCl10g/L, agar 20g/L, pH7.2;
2. the preparation of seed liquor:
The one above-mentioned seed of ring is changed in the triangular flask that contains the liquid nutrient medium that is rich in nutrition, 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 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, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O 1.0, FeCl 36H 2O 0.01, pH7.0;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 48 hours, ventilation 1.5vvm, rotating speed 400rpm; Fermentation ends is 22.5g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 0.47g/lh;
Embodiment 2
The activation of bacterial classification and the preparation of seed liquor are substantially with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O 1.0, FeCl 36H 2O 0.01, pH7.0;
Fed-batch medium: the composition of fed-batch medium is (g/L): glucose 800, ammonium nitrate 41, CaCl 26H 2O10, FeCl 36H 2O 0.1;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 36 hours, ventilation 1.5vvm, rotating speed 400rpm; Add fed-batch medium in fermenting process to fermentation ends front 6 hours when residual sugar is down to 10g/L, flow acceleration is 0.1ml/min; Fermentation ends is 22.0g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 0.61g/lh;
Embodiment 3
The activation of bacterial classification and the preparation of seed liquor are substantially with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O 1.0, FeCl 36H 2O 0.01, pH7.0;
Fed-batch medium: the composition of fed-batch medium is (g/L): glucose 700, ammonium nitrate 30, CaCl 26H 2O5, FeCl 36H 2O 0.05;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and by the formulated fermention medium shown in above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 36 hours, ventilation 1.5vvm, rotating speed 400rpm; Add fed-batch medium in fermenting process to fermentation ends front 2 hours when residual sugar is down to 20g/L, flow acceleration is 0.5ml/min; Fermentation ends is 30g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 0.83g/lh;
Embodiment 4
The activation of bacterial classification and the preparation of seed liquor are substantially with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O1.0, FeCl 36H 2O 0.01, pH7.0;
Fed-batch medium: the composition of fed-batch medium is (g/L): glucose 900, ammonium nitrate 60, CaCl 26H 2O20, FeCl 36H 2O 0.15;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and with embodiment 1 preparation fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 36 hours, ventilation 1.5vvm, rotating speed 400rpm; Add fed-batch medium in fermenting process to fermentation ends front 4 hours when residual sugar is down to 5g/L, flow acceleration is 1ml/min; Fermentation ends is 42.5g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 1.18g/lh;
Embodiment 5
The activation of bacterial classification and the preparation of seed liquor are substantially with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O 1.0, FeCl 36H 2O 0.01, pH7.0;
Fed-batch medium: the composition of fed-batch medium is (g/L): glucose 1000, ammonium nitrate 45, CaCl 26H 2O15, FeCl 36H 2O 0.1;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and with embodiment 1 preparation fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 36 hours, ventilation 1.5vvm, rotating speed 400rpm; Add fed-batch medium when in fermenting process, residual sugar is down to 7g/L to fermentation ends front 6 hours, flow acceleration is 1.5ml/min; Fermentation ends is 34.5g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 0.96g/lh;
Embodiment 6
The activation of bacterial classification and the preparation of seed liquor are substantially with embodiment 1;
3. ferment tank:
Fermention medium: the fermentation culture based component is (g/L): glucose 80, and Sodium Glutamate 80, yeast extract paste 20, ammonium nitrate 4.1, NaCl 10, MgSO 46H 2O 0.5, CaCl 26H 2O 1.0, FeCl 36H 2O 0.01, pH7.0;
Fed-batch medium: the composition of fed-batch medium is (g/L): glucose 600, ammonium nitrate 41, CaCl 26H 2O10, FeCl 36H 2O 0.1;
Fermentation condition: 30L fermentor tank liquid amount is 15L, and with embodiment 1 preparation fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 36 hours, ventilation 1.5vvm, rotating speed 400rpm; Add fed-batch medium when in fermenting process, residual sugar is down to 15g/L to fermentation ends front 6 hours, flow acceleration is 2.0ml/min; Fermentation ends is 18.7g/L by the output that detects gamma-polyglutamic acid-, and the productive rate of gamma-polyglutamic acid-is 0.52g/lh;

Claims (7)

1. an a large amount of method of producing gamma-polyglutamic acid-, by the optimization to the lichem bacillus strain culture condition, particularly add fed-batch medium during the fermentation, can effectively improve gamma-polyglutamic acid-output; Described method comprises the steps:
(1) activation of bacterial classification: cultivated 16 hours being rich on the culture medium slant of nutrition in 37 ℃, the inclined-plane seed that preparation is ripe, the composition of solid medium comprises: peptone 10g/L, yeast powder 5g/L, NaCl10g/L, agar 20g/L, pH7.2;
(2) preparation of seed liquor:
The one above-mentioned seed of ring is changed in the triangular flask of the seed liquid nutrient medium that is rich in nutrition, and 37 ℃, 220rpm cultivates 16 hours to logarithmic phase;
Add-on preparation seed liquid nutrient medium according to every liter of substratum: glucose 30; Yeast extract paste 7; Tryptones 10; K 2HPO 40.5; MgSO 46H 2O0.5, pH7.2 before sterilization; The seed liquid nutrient medium of packing 50ml in the triangular flask of 500ml;
(3) ferment tank:
Fermention medium is composed as follows: glucose 60-100, Sodium Glutamate 60-100, yeast extract paste 10-30, ammonium nitrate 3.0-6.0, NaCl5.0-15, MgSO 46H 2O0.1-1.5, CaCl 26H 2O0.5-2.0, FeCl 36H 2O0.005-0.015, pH6.0-8.0, above composition unit is g/L;
The canned fermention medium amount of fermentation condition: 30L fermentation is 15L, and by the formulated fermention medium shown in above-mentioned fermention medium, the inoculum size of seed liquor is 10%, 37 ℃ of leavening temperatures, fermentation time 48 hours, ventilation 1.5vvm, rotating speed 400rpm;
Add fed-batch medium: when in fermention medium, sugared concentration is down to 5-20g/L, add fed-batch medium to the fermentation ends 2-6 hour with the speed of 0.1-2.0ml/min; In fed-batch medium, each material concentration is respectively: glucose 600-1000g/L, ammonium nitrate 30-60g/L, CaCl 26H 2O5-20g/L, FeCl 36H 2O0.05-0.15g/L.
2. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 600-900, ammonium nitrate 30.0-60.0, CaCl 26H 2O5.0-20.0, FeCl 36H 2O0.05-0.15, above composition unit is g/L.
3. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 800, ammonium nitrate 41, CaCl 26H 2O10, FeCl 36H 2O0.1, above composition unit is g/L.
4. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 700, ammonium nitrate 30, CaCl 26H 2O5, FeCl 36H 2O0.05, above composition unit is g/L.
5. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 900, ammonium nitrate 60, CaCl 26H 2O20, FeCl 36H 2O0.15, above composition unit is g/L.
6. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 1000, ammonium nitrate 45, CaCl 26H 2O15, FeCl 36H 2O0.1, above composition unit is g/L.
7. a kind of method of a large amount of production gamma-polyglutamic acid-s as claimed in claim 1, it is characterized in that: in described step (3), the composition of fed-batch medium is: glucose 600, ammonium nitrate 41, CaCl 26H 2O10, FeCl 36H 2O0.1, above composition unit is g/L.
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CN103613753B (en) * 2013-11-14 2015-12-30 天津北洋百川生物技术有限公司 A kind of method without adding organic separated from solvent purification polyglutamic acid
CN103695485B (en) * 2013-12-27 2017-02-15 天津北洋百川生物技术有限公司 High-efficiency production method of gamma-polyglutamic acid
CN104694437B (en) * 2015-03-23 2018-04-27 领先生物农业股份有限公司 One bacillus licheniformis and its purposes in gamma-polyglutamic acid is produced
CN107335414B (en) * 2015-07-31 2019-12-17 邵素英 Biological purification carrier for wastewater treatment
CN108395059B (en) * 2015-09-24 2020-11-17 天津中天精科科技有限公司 Biological reaction and adsorption water purification device for sewage treatment
CN107974472A (en) * 2017-12-26 2018-05-01 天津北洋百川生物技术有限公司 A kind of preparation method added methionine and improve gamma-polyglutamic acid yield

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Inventor after: Qiao Changcheng

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