CN107974472A - A kind of preparation method added methionine and improve gamma-polyglutamic acid yield - Google Patents
A kind of preparation method added methionine and improve gamma-polyglutamic acid yield Download PDFInfo
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- CN107974472A CN107974472A CN201711430802.6A CN201711430802A CN107974472A CN 107974472 A CN107974472 A CN 107974472A CN 201711430802 A CN201711430802 A CN 201711430802A CN 107974472 A CN107974472 A CN 107974472A
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Abstract
The invention discloses a kind of preparation method added methionine and improve γ polyglutamic acid yield, the preparation process includes：Actication of culture：Bacterial strain CGMCC No.3336 are seeded in slant medium；Seed liquor culture：The activated seed of slant medium is inoculated in seed liquid culture medium；Fermentation tank culture, according to 10% inoculum concentration of fermentation medium stereometer, the seed liquor is inoculated in fermentation tank；Fermentation liquid culture medium includes glucose, NH4Cl, MgSO47H2O, anhydrous calcium chloride g/L, FeSO4·7H2Og/L, yeast extract, peptone, monosodium glutamate, growth factor；Separation and Extraction.This method can improve the yield of polyglutamic acid, improve substrate conversion efficiency, not only easy to operate, but also while yield is improved and cost that the later stage of which reducing polyglutamic acid extracts.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of methionine that adds to improve gamma-polyglutamic acid yield
Preparation method.
Background technology
γ-polyglutamic acid is to pass through the amido link between α-amino and γ-carboxyl by D- glutamic acid and L- glutamic acid
A kind of multifunctional bio degradable high polymer material being combined into.Molecular weight distribution is between 100kDa to 10000kDa.
Gamma-polyglutamic acid has excellent water-soluble, superpower adsorptivity and biodegradability, and catabolite is non-harmful paddy ammonia
Acid, is a kind of excellent environment-friendly type macromolecule material, can be used as water-retaining agent, adsorbent for heavy metal, flocculant, sustained release agent with
And pharmaceutical carrier etc., very high business is respectively provided with industries such as cosmetics, environmental protection, food, medicine, agricultural, control of desert
Value and social value.
Only has the history of decades so far from the discovery of polyglutamic acid, the research of polyglutamic acid is mainly in laboratory
Stage, only a small number of enterprises have carried out industrialized production.Laboratory stage mainly includes to its property Quality Research, the improvement of producing strains
And gene studies, fermentation process research and extraction purification process study, and production and the property Quality Research of derivative.In recent years
Come, due to the enhancing of people's environmental consciousness and the requirement of National Sustainable Development Strategies, develop environment amenable material and open
Hair improves the product of environmental problem as the trend in a kind of industry, it has also promoted polyglutamic acid study on the industrialization and exploration
Process.Into this century, indivedual world renowned companies proceed by the research of production and the application of polyglutamic acid, and Some Domestic is big
Learn and research institute has also actively developed relevant research, domestic Geng Youshuojia enterprises start to plan the extensive raw of polyglutamic acid
Production.Due to the follow-up of these study on the industrialization so that polyglutamic acid becomes one of biological products being most concerned by people at this stage.
Growth factor refers to necessary in microorganism growth process, it is necessary to the nutritional ingredient directly provided by the external world, it is common
Growth factor have vitamin, amino acid, purine pyrimidine etc..Growth factor can provide the important chemical substance of microorganism, auxiliary
The factor（Coenzyme and prothetic group）Component and participate in be metabolized.Belong to secondary metabolite, logarithmic phase later stage, stationary phase are in thalline
Start early period to produce.
Mainly optimized at present among document on polyglutamic acid output increased by induction mutation of bacterium, medium component, it is past
It is less obvious toward effect, fermentation period length, and the conversion ratio of substrate is not high, causes polyglutamic acid cost to remain high.
The content of the invention
In order to solve the deficiency in polyglutamic acid tradition optimization process, the yield of polyglutamic acid is targetedly improved, is carried
High substrate conversion efficiency, the present invention provides a kind of addition growth factor methionine to improve the method for gamma-polyglutamic acid yield,
It is not only easy to operate, and while yield is improved and the later stage of which reducing polyglutamic acid extraction cost.
To achieve these goals, technical scheme is as follows：
The invention discloses it is a kind of add methionine improve gamma-polyglutamic acid yield preparation method,
The gamma-polyglutamic acid is fermented by bacillus licheniformis to be prepared：
The bacillus licheniformis（Bacillus licheniformis）TKPG091, the bacterial strain is on October 14th, 2009
In Chinese microorganism strain preservation management committee of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, its preserving number is CGMCC No.3336；Preparation method includes：
（1）Actication of culture：Bacillus licheniformis CGMCC NO.3336 original strains are seeded on slant medium, in 37 DEG C
Cultivate 16 it is small when, so activation 2-3 time, prepares ripe inclined-plane seed；
（2）The preparation of seed liquor：Inclined-plane seed after activation is inoculated in the 500ml triangular flasks equipped with 50ml fluid nutrient mediums
In, 37 DEG C, shaking speed 220rpm cultivates 16-20 hours to exponential phase, with reference to OD660Value rises to 0.3.
The seed culture medium forms：7 g/L of yeast extract, tryptone 10/L, K2HPO40.5 g/L, MgSO4·
7H20.5 g/L of O, pH value 7.2, surplus are pure water；Glucose 30g/L is individually added after individually sterilizing, glucose and culture
Base sterilizes the conversion and utilization rate that can reduce thalline to glucose together, therefore individually addition so that the conversion ratio of monosodium glutamate improves.
（3）Ferment tank：According to 10% inoculum concentration of fermentation medium stereometer, by step（2）In seed liquor connect
Kind is in fermentation tank, with 10% inoculum concentration, 37-38 DEG C of Preliminary fermentation temperature, tank pressure 0.01-0.05Mpa ventilation quantities 0.5-
1.5vvm, rotating speed 400rpm；The pH value of zymotic fluid is 6.0-7.2, wherein, Preliminary fermentation liquid pH value is adjusted to 7.2.In fermentation process
In preceding 20h control pH value not less than 6.0,20-28h control pH value be not less than 6.5, the later stage do not have to adjust again pH value until fermentation tie
Beam, fermentation time 72h.
The fermentation medium component is：Glucose 78-85g/L, NH4Cl 5.32-6g/L, MgSO4·7H2O 0.45-
0.6g/L, anhydrous calcium chloride 0.45-0.55g/L, FeSO4·7H2O 0.08-0.12g/L, yeast extract 10-15g/L, peptone 5-
10g/L；Monosodium glutamate 75-85g/L, growth factor 0.2-0.6g/L, surplus are water；Sterilize 30-40min at 121 DEG C of temperature, obtains
The fermentation medium；Wherein monosodium glutamate individually sterilizes, individually addition.The growth factor can be pyridoxol, adenine, first
The best results of any one of methyllanthionine, D-Glu, arginine, glutamine, wherein methionine.
Monosodium glutamate and culture medium sterilize the conversion and utilization rate that can reduce thalline to monosodium glutamate together, therefore individually addition so that taste
The conversion ratio of essence improves.
（4）The extraction of gamma-polyglutamic acid：Thalline is removed in centrifugation after fermentation, then with the second of 4-5 times of supernatant volume
Alcohol, alcohol precipitation are separated out polyglutamic acid, then redissolve in the distilled water isometric with supernatant, prepared using spray drying process again
Go out yellow dry powder-shaped polyglutamic acid.Finally by efficient liquid phase（It is ultraviolet or show difference）Detect the polyglutamic acid yield of respective sample.
The optimization process of the addition growth factor：
Gamma-polyglutamic acid belongs to secondary metabolite, starts have small part in microorganism logarithmic phase later stage, early stage stationary phase
Produced in later stage stationary phase.；In order to shorten fermentation period, improve substrate conversion efficiency, Optimal Medium, take fermentation medium its
Remaining components unchanged, fermentation synthesis of the single growth factor to polyglutamic acid has larger facilitation, and cost is relatively low.Laboratory
By numerous studies, and the screening to growth factor species, dosage is optimized, is drawn：Add methionine its γ-poly-
Glutamic acid yield highest, obtained gamma-polyglutamic acid positive effect increase, and the polyglutamic acid that methionine 0.6g/L is obtained
Yield be highest.
PH can be reduced in fermentation process, using sodium hydroxide solution regulate and control pH, Preliminary fermentation liquid pH value 7.2 and fermentation before
28 it is small when pH value adjust in 6.0-6.5, be to facilitate thalline in the range of optimal pH, enable thalline quick after lag phase is entered
Into logarithmic phase, during phase thalline enters stationary phase after fermentation, thalline mainly produces the purpose product of gamma-polyglutamic acid, therefore not
Need to adjust pH value again.Ventilation quantity, which crosses conference, makes the molten foster to abundance of thalline, so that the entrance logarithmic phase that thalline is too fast, disappears
The culture substrate such as glucose is consumed so that maintained in stationary phase thalline without sufficient substrate, and then influence polyglutamic acid mesh
Product amount, so ventilation quantity and tank pressure must be adjusted in suitable scope.
Compared with prior art, beneficial effects of the present invention are：
（1）The present invention provides the additive amount of addition methionine growth factor and the growth factor to improve gamma-polyglutamic acid production
Highest method is measured, is targetedly compared according to different amount features is added in different growth factor species and fermentation process
The facilitation of its yield to cosmetics-stage gamma-polyglutamic acid.
（2）The present invention is directed to bacillus licheniformis CGMCC NO.3336 fermenting and producing cosmetics-stage gamma-polyglutamic acid processes
In there are low output, substrate conversion efficiency is low the defects of.By the optimizing research of culture medium, targetedly add methionine and promote
The generation of end-product gamma-polyglutamic acid.The cycle of medium optimization is highly shortened, improves the yield of polyglutamic acid, is improved
Substrate conversion efficiency, it is not only easy to operate, but also while yield is improved and the later stage of which reducing polyglutamic acid extraction cost.
（3）The preparation method of the present invention is easy, and feasibility is high, can be used as industrialized production cosmetics-stage gamma-polyglutamic acid,
Its conversion ratio is adapted to industrialized production, improves the utilization ratio of substrate, and fermentation period is shorter, greatly reduces the damage of equipment
Consumption, and the cost such as utilization rate of equipment and installations.
Specific embodiment
Embodiment 1：
Actication of culture：Bacillus licheniformis CGMCC NO.3336 original strains are seeded on slant medium, in 37 DEG C of cultures
18 it is small when, prepare maturation inclined-plane seed；Slant medium forms：Tryptone 10g/L, yeast extract 5g/L, NaCl
10g/L, agar powder 20g/L, surplus are water.
Bacillus licheniformis（Bacillus licheniformis）TKPG091, the bacterial strain is on October 14th, 2019
In Chinese microorganism strain preservation management committee of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, its preserving number is CGMCC No.3336；
(1) seed culture
Inclined-plane seed after one ring is activated is inoculated in the 500ml triangular flasks equipped with 50ml fluid nutrient mediums, 37 DEG C, and shaking table turns
Fast 220rpm cultivates 18 hours to exponential phase, can at 0.3 with reference to being grown with OD660 values
(2) fermented and cultured
The bottled liquid measure 50mL of 500mL baffles, inoculum concentration are 10% (V/V), and 37 DEG C of temperature, shaking speed is that 220rpm is trained
Support.Remaining components unchanged in the medium, the methionine growth factor for adding 0.2g/L carry out fermenting and producing γ-polyglutamic
Acid, when i.e. thalli growth is in early stage stationary phase after fermentation carries out 72h, product starts largely to accumulate.
(3) separation and Extraction of gamma-polyglutamic acid and detection
After zymotic fluid centrifugal filtration removes thalline, take supernatant 5ml to add 4 times of ethanol alcohol precipitations and obtain polyglutamic acid crude product twice, then it is pure
Water purification redissolution and constant volume obtains sample after 25ml by hydrolysis process.Its gamma-polyglutamic acid yield is detected to obtain by efficient liquid phase
For 25g/L.
Embodiment 2
Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, when 30 DEG C of cultures 16 are small, is prepared
Inclined-plane seed after activation；Slant medium forms：10 g/L of tryptone, yeast extract 5g/L, NaCl 10g/L, fine jade
Cosmetics 20g/L, surplus are water.
Bacillus licheniformis（Bacillus licheniformis）TKPG091, the bacterial strain is on October 14th, 2019
In Chinese microorganism strain preservation management committee of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, its preserving number is CGMCC No.3336；
(1) seed culture
Inclined-plane seed after one ring is activated is inoculated in the 500ml triangular flasks equipped with 50ml fluid nutrient mediums, 37 DEG C, and shaking table turns
Fast 220rpm cultivates 18 hours to exponential phase, can at 0.3 with reference to being grown with OD660 values.
(2) fermented and cultured
The bottled liquid measure 50mL of 500mL baffles, inoculum concentration are 10% (V/V), and 37 DEG C of temperature, shaking speed is that 220rpm is trained
Support.Remaining components unchanged in the medium, the methionine for adding 0.4g/L carry out fermenting and producing gamma-polyglutamic acid, work as fermentation
Product starts largely to accumulate when i.e. thalli growth is in early stage stationary phase after progress 72h.
(3) separation and Extraction of gamma-polyglutamic acid and detection
After fermentation, by zymotic fluid centrifugal filtration remove thalline after, take supernatant 5ml add 4 times of ethanol alcohol precipitations twice poly- paddy
Propylhomoserin crude product, then pure water redissolves and constant volume obtains sample after 25ml by hydrolysis process.By efficient liquid phase detect its γ-
Polyglutamic acid yield is 31g/L.
Embodiment 3
Actication of culture：The original strain of bacillus licheniformis is seeded on slant medium, when 30 DEG C of cultures 16 are small, is prepared
Inclined-plane seed after activation；Slant medium forms：10 g/L of tryptone, yeast extract 5g/L, NaCl 10g/L, fine jade
Cosmetics 20g/L, surplus are water.
Bacillus licheniformis（Bacillus licheniformis）TKPG091, the bacterial strain is on October 14th, 2019
In Chinese microorganism strain preservation management committee of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica
Member's meeting common micro-organisms center preservation, its preserving number is CGMCC No.3336；
(1) seed culture
Inclined-plane seed after one ring is activated is inoculated in the 500ml triangular flasks equipped with 50ml fluid nutrient mediums, 37 DEG C, and shaking table turns
Fast 220rpm cultivates 18 hours to exponential phase, can at 0.3 with reference to being grown with OD660 values
(2) fermented and cultured
The bottled liquid measure 50mL of 500mL baffles, inoculum concentration are 10% (V/V), and 37 DEG C of temperature, shaking speed is that 220rpm is trained
Support.Remaining components unchanged in the medium, adds the methionine of 0.6g/L, carries out fermenting and producing gamma-polyglutamic acid, works as fermentation
Product starts largely to accumulate when i.e. thalli growth is in early stage stationary phase after progress 72h.
(3) separation and Extraction of gamma-polyglutamic acid and detection
After zymotic fluid centrifugal filtration removes thalline, take supernatant 5ml to add 4 times of ethanol alcohol precipitations and obtain polyglutamic acid crude product twice, then it is pure
Water purification redissolution and constant volume obtains sample after 25ml by hydrolysis process.Its gamma-polyglutamic acid yield is detected to obtain by efficient liquid phase
For 35g/L.
Above-described embodiment is in the art the purpose is to be to allow simply to illustrate that the technical concepts and features of the present invention
Those of ordinary skill can understand present disclosure and implement according to this, and it is not intended to limit the scope of the present invention.It is all
It is the equivalent change or modification according to the present invention made by the essence of content, should all covers within the scope of the present invention.
Claims (7)
1. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield, the bacterial strain used is bacillus licheniformis
Bacillus licheniformis, the bacterial strain have been preserved in Chinese microorganism strain preservation management committee on October 14th, 2009
Member's meeting common micro-organisms center preservation, its preserving number is CGMCC No.3336；It is characterized in that, preparation process includes：
（1）Actication of culture：Bacterial strain CGMCC No.3336 are seeded in slant medium；
（2）It is prepared by seed liquor：The strain of activation on slant medium is inoculated in seed liquid culture medium, is cultivated to OD660Value
When rising to 0.3；
（3）Fermentation tank culture：According to 10% inoculum concentration of fermentation medium stereometer, the seed liquor is inoculated in fermentation tank
In, the pH value of zymotic fluid is 6.0-7.2；Fermentation liquid culture medium includes glucose, NH4Cl, MgSO4·7H2O, anhydrous calcium chloride g/
L, FeSO4·7H2Og/L, yeast extract, peptone, monosodium glutamate, methionine；
（4）Separation and Extraction.
2. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 1, its feature
It is, the fermentation medium includes glucose 78-85g/L, NH4Cl5.32-6g/L, MgSO4·7H2O 0.45-0.6g/L,
Anhydrous calcium chloride 0.45-0.55g/L, FeSO4·7H2O 0.08-0.12g/L, yeast extract 10-15g/L, peptone 5-10g/L；
Monosodium glutamate 75-85g/L, methionine 0.2-0.6g/L, surplus are water；Sterilize 30-40min at 121 DEG C of temperature, obtains described
Fermentation medium；Wherein monosodium glutamate individually sterilizes, individually addition.
3. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 1 or 2, its
It is characterized in that, 37-38 DEG C of the Preliminary fermentation temperature of fermentation tank culture, tank pressure 0.01-0.05Mpa ventilation quantity 0.5-1.5vvm, turn
Fast 400rpm, when the fermentation tank culture time is 72 small, Preliminary fermentation liquid pH value is adjusted to 7.2, preceding 20h controls during the fermentation
PH value is not less than 6.5 not less than 6.0,20-28h control ph.
4. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 1, its feature
It is, when the actication of culture process is that 37 DEG C of cultures 16 are small, activates 2-3 times, prepare the inclined-plane seed of maturation.
5. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 1, its feature
It is, the seed liquor preparation process is that the strain that will be activated on slant medium is inoculated in equipped with 50ml seed liquid culture mediums
In 500ml triangular flasks, 37 DEG C, hour when shaking speed 220rpm cultures 16-20 is small.
6. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 5, its feature
It is, seed liquid culture medium composition is：7 g/L of yeast extract, tryptone 10/L, K2HPO40.5 g/L, MgSO4·
7H20.5 g/L of O, glucose 30g/L, pH value 7.2, surplus is pure water；The glucose individually after sterilizing, individually adds,
Mixed with other culture mediums and component.
7. a kind of preparation method added methionine and improve gamma-polyglutamic acid yield according to claim 1, its feature
It is, the separation and Extraction process of gamma-polyglutamic acid is after fermentation, zymotic fluid centrifugation to be gone thalline, then with 4-5 times
The ethanol of supernatant volume, alcohol precipitation separate out polyglutamic acid, then redissolve again in the distilled water isometric with supernatant, dry using spraying
Drying method prepares yellow dry powder-shaped polyglutamic acid.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268465A (en) * | 2011-07-29 | 2011-12-07 | 天津北洋百川生物技术有限公司 | Method for massively producing gamma-poly-glutamic acid |
| CN103710404A (en) * | 2013-12-27 | 2014-04-09 | 天津北洋百川生物技术有限公司 | Production method of gamma-polyglutamic acid with high molecular weight |
-
2017
- 2017-12-26 CN CN201711430802.6A patent/CN107974472A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102268465A (en) * | 2011-07-29 | 2011-12-07 | 天津北洋百川生物技术有限公司 | Method for massively producing gamma-poly-glutamic acid |
| CN103710404A (en) * | 2013-12-27 | 2014-04-09 | 天津北洋百川生物技术有限公司 | Production method of gamma-polyglutamic acid with high molecular weight |
Non-Patent Citations (2)
| Title |
|---|
| CHANGSHENG QIAO, ET AL.: "Reducing Impurities in Fermentation Broth for γ-Polyglutamic Acid Production by Medium Optimization Using Bacillus licheniformis CGMCC 3336", 《PROCEEDINGS OF THE 2012 INTERNATIONAL CONFERENCE ON APPLIED BIOTECHNOLOGY》 * |
| 索晨: "产γ-聚谷氨酸的地衣芽孢杆菌菌株选育及其发酵条件优化和产物分离", 《中国优秀博硕士学位论文全文数据库 (硕士) 工程科技Ⅰ辑》 * |
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