CN105837815A - Technology for efficiently extracting gama-polyglutamic acid from fermentation liquor - Google Patents
Technology for efficiently extracting gama-polyglutamic acid from fermentation liquor Download PDFInfo
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- CN105837815A CN105837815A CN201610329177.5A CN201610329177A CN105837815A CN 105837815 A CN105837815 A CN 105837815A CN 201610329177 A CN201610329177 A CN 201610329177A CN 105837815 A CN105837815 A CN 105837815A
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- pga
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a technology for efficiently extracting gama-polyglutamic acid from fermentation liquor. The technology selectively precipitates gama-PGA in the fermentation liquor through cetyl trimethyl ammonium bromide (CTAB), and then performs dissociation dissolution, alcohol precipitation, solid-liquid separation and drying on precipitate in a NaCl solution with certain concentration, so as to obtain gama-PGA with high purity. Compared with the prior art, the technology has the advantages that the technology can not only improve the extraction efficiency and product purity of gama-PGA, but also greatly reduce the dosage of an organic solvent used for alcohol precipitation, the production cost is greatly reduced, and the technology has a good application prospect.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of from fermentation liquid high efficiency extraction γ-
The technique of polyglutamic acid.
Background technology
Gamma-polyglutamic acid-(poly-γ-glutamic acid, be called for short γ-PGA) be by Pidolidone and
D-Glu combines a kind of peptide molecule formed through γ-amido link, is a kind of water solublity high score
Sub-amino acid polymer.Due to γ-PGA have biodegradability, good biocompatibility,
Water-retaining property, to the unique physics of human non-toxic's evil and environmentally safe etc., chemistry and biology
Character so that γ-PGA be widely used in agricultural, food, medicine, cosmetic technology and
Multiple field such as environmental protection.
At present, the method extracting separating gamma-PGA from high viscosity fermentation liquid mainly has three kinds:
Organic solvent precipitation method (generally using separating out alcohol method), chemical precipitation method and the membrance separation sedimentation method.
Organic solvent deposit refers to utilize centrifugal or cohesion thalline method to remove the thalline in fermentation liquid,
Lower alcohols (such as methanol, ethanol, the isopropanol etc.) precipitable γ-PGA that obtains is added in supernatant,
Freeze-dried obtain faint yellow color jelly.Chemical precipitation is to use saturated CuSO4、NaCl
Solution replaces lower alcohols salt precipitation γ-PGA.Full-bodied fermentation liquid also can be taked film divide
From the sedimentation method.Alcohol analysis first or the γ-PGA crude product color saltouing prepared are generally yellow, need
After being dissolved in distilled water, again carry out fine straining, decolouring, desalination etc. and process, through low pressure
After lyophilizing, just can obtain white γ-PGA highly finished product.Owing to chemical precipitation method and membrance separation precipitate
The extraction process of method is complicated, extraction cost is high and purity is low, therefore current most common method
It it is organic solvent precipitation method.Organic solvent precipitation method (separating out alcohol method) equally exists a lot of problem.
Because γ-PGA fermentation liquid very thickness, carry out pretreatment and go the removal of impurity to need suitably to dilute ability
Smooth operation.When in fermentation liquid, γ-PGA concentration is relatively low, not only alcohol analysis effect is bad, γ-PGA
The response rate is the lowest, and consumes a large amount of organic solvent.The use of a large amount of organic solvents not only increases
Production cost, and bring bigger potential safety hazard.In order to reduce solvent consumption, fermentation liquid is located in advance
Typically it is concentrated by ultrafiltration after reason, but ultra-filtration process γ-PGA loss is very big, and ultrafilter membrane holds
The most contaminated, treatment effeciency step-down.Isolated and purified difficulty and extraction recovery are lowly
The major reason that γ-PGA production cost is high.
In view of this, the commercial production of γ-PGA is needed a set of economically viable separation badly pure
Metallization processes.
Summary of the invention
It is an object of the invention to: for the deficiencies in the prior art, and provide a kind of from fermentation liquid
The new technology of middle high efficiency extraction γ-PGA, to improve the yield of γ-PGA, purity and reduction
Production cost.
To achieve these goals, the present invention is by the following technical solutions:
A kind of technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid, comprises the following steps:
Step (1): the γ-PGA fermentation liquid obtained by fermentable is 1～50 μm by aperture
Filter bag and aperture be 0.25～1 μm microfilter filter, or in high speed centrifuge from
The heart separates, and removes the substantial amounts of remaining nutrient of insoluble fermentation and thalline in fermentation liquid；
Step (2): add trichloroacetic acid regulation pH in the fermentation liquid after step (1) is centrifugal
Value is 3～5, removes the foreign protein in fermentation liquid；
Step (3): add 1～30g/L diatomite adsorption fermentation in step (2) fermentation liquid
Remaining thalline in liquid, filters or centrifugation goes out insoluble matter, adds 0.5～20g/L activity
Pigment in charcoal absorption fermentation liquid and other impurity, through filtering or centrifugation acquisition supernatant；
Step (4): sample in the fermentation liquid after step (3) processes, i.e. in step (3)
In supernatant sampling, measure and estimate the content of γ-PGA in fermentation liquid；
Step (5): according to the content of γ-PGA in the fermentation liquid that step (4) is calculated, adds
The CTAB of 1～5 times of the content of γ-PGA, is 7～13 with NaOH solution regulation pH value,
Mix and blend 1～12h so that CTAB fully reacts with γ-PGA in fermentation liquid, is then centrifuged for
Isolated insoluble CTAB/ γ-PGA complex；
Step (6): step (5) is collected the CTAB/ γ-PGA complex obtained and is placed in NaCl
In solution, regulation pH value is 6～9, dissociates 1～12h, obtains the clarification containing γ-PGA molten
Liquid；
Step (7): add γ-PGA settled solution in step (6) γ-PGA settled solution
The ethanol of 2～5 times of volume, under conditions of temperature is 4～10 DEG C, cooling stands 12～24h,
It is centrifuged to obtain precipitate, then precipitate lyophilization is obtained highly purified γ-PGA white knot
Brilliant.
It should be noted that in the basic conditions, CTAB with γ-PGA defines and can not dissociate
Ionic compound precipitate, and the neutrallty condition of pH 6～8 and high ionic strength (from
Sub-intensity ＞ 150mmol/L) solution in, this ionic compound can dissociate again dissolving again,
It is to say, CTAB has the feature of selective precipitation γ-PGA from LISS.
As described a kind of improvement of high efficiency extraction gamma-polyglutamic acid-technique, institute from fermentation liquid
Stating the γ-PGA fermentation liquid in step (1) is the γ-PGA obtained by microbial liquid fermentation
Fermentation liquid.
As described a kind of improvement of high efficiency extraction gamma-polyglutamic acid-technique, institute from fermentation liquid
State the γ-PGA fermentation liquid in step (1) be microbial solid fermentation obtain containing γ-PGA
Solid matrix in add the distilled water of 8～12 times of solid matrix volume and be stirred extraction
After 0.5～2h, the γ-PGA lixiviating solution obtained by 100 eye mesh screen filter and remove residues.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
Centrifugation in described step (1), rotating speed is 5000～15000rpm, and centrifugation time is
5～30min.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
Filtration in described step (3) is
The microfilter of 0.25-1 μm filters..
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
Centrifugation in described step (3), rotating speed is 5000～15000rpm, and centrifugation time is
5～30min.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
The concentration of the NaCl solution in described step (6) is 0.1～1.0mol/L.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
The concentration of the ethanol in described step (7) is more than 95%.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
Lyophilization in described step (7) refers under the condition of high vacuum degree that pressure is 10Pa～30Pa
Sublimation drying is to ambient temperature, after 1～5h, pressure be 15Pa～30Pa, temperature is less than 40 DEG C
Under, it is dried 4～10h.
As described a kind of improvement of the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid,
γ-PGA white crystals in described step (7) is Na-type γ-PGA and H-type γ-PGA
Mixture.
The beneficial effects of the present invention is: the present invention utilizes CTAB and the γ-PGA in fermentation liquid
There is chemical reaction under proper condition, form insoluble ionic compound, then pass through solid-liquid
Separate and collect precipitation, precipitation is dissolved in certain density NaCl solution, add 3～5 times
Organic solvent (lower alcohol) in this solution, through dissociating dissolvings, alcohol analyse, solid-liquid separation
And be dried wait operate obtain the γ-PGA that purity is higher.Relative to prior art, this technique by
In utilizing CTAB selective precipitation γ-PGA feature more completely, reach efficient concentration
γ-PGA also goes deimpurity purpose, not only increases γ-PGA extraction efficiency and product purity,
Also substantially reduce the number the organic solvent for alcohol analysis, make production cost be substantially reduced, have good
Application prospect.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention and beneficial effect thereof are made the most in detail
Illustrate, but, the detailed description of the invention of the present invention is not limited thereto.
Embodiment 1
A kind of technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid, comprises the following steps:
Step (1): the γ-PGA fermentation liquid obtained by 100L fermentable in rotating speed is
In 5000rpm centrifugal separator, centrifugal 30min, is separated off in fermentation liquid substantial amounts of insoluble
The remaining nutrient of fermentation and thalline；
Step (2): add trichloroacetic acid regulation pH in the fermentation liquid after step (1) is centrifugal
It is 3, removes the foreign protein in fermentation liquid, reduce the viscosity of fermentation liquid, it is simple to follow-up simultaneously
Purification process；
Step (3): add 20g/L diatomite adsorption in every liter of step (2) fermentation liquid and send out
Remaining thalline in ferment liquid, filters or insoluble matter is removed in centrifugation, adds 5g/L activated carbon
Pigment in absorption fermentation liquid and other impurity, centrifugal in the centrifuge that rotating speed is 5000rpm
30min, separates and obtains supernatant；
Step (4): sample in the fermentation liquid after step (3) processes, measures in fermentation liquid
The content of γ-PGA；
Step (5): according to the content of γ-PGA in the fermentation liquid that step (4) is calculated, adds
The CTAB of 2 times of the content of γ-PGA, adding NaOH regulation pH is 10, mix and blend
4h makes CTAB fully react with γ-PGA in fermentation liquid, is then 5000rpm at rotating speed
Centrifuge in centrifugal 30min, isolated insoluble CTAB/ γ-PGA complex；
Step (6): step (5) is collected the CTAB/ γ-PGA complex obtained and is placed in dense
In the degree NaCl solution for 0.6mol/L, regulation pH value is 7, and dissociate 3h, is contained
The settled solution of γ-PGA；
Step (7): add γ-PGA settled solution in step (6) γ-PGA settled solution
The dehydrated alcohol of 4 times of volume, under conditions of temperature is 4 DEG C, cooling stands 12h, is turning
Speed be 5000rpm centrifuge in centrifugal 30min, separate to obtain precipitate, then by precipitate
Lyophilization, obtains γ-PGA white crystals.
Embodiment 2
A kind of technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid, comprises the following steps:
Step (1): the solid matrix 10kg containing γ-PGA that microbial solid fermentation is obtained
The distilled water adding 10 times of volumes first passes through 100 eye mesh screens in 40 DEG C of stirring and leaching 1h, lixiviating solution
Filter and remove residue, filtrate is the most respectively by 5 μm aperture sock filtration and 0.5 μm microfilter
Filter, collect lixiviating solution.
Step (2): adding trichloroacetic acid regulation pH value in step (1) lixiviating solution is 4；
Step (3): add 10g/L kieselguhr and 8g/L activity in step (2) lixiviating solution
Pigment in charcoal absorption fermentation liquid and other impurity, in the centrifuge that rotating speed is 10000rpm
Centrifugal 25min, separates and obtains supernatant；
Step (4): sample in the lixiviating solution after step (3) processes, measures in lixiviating solution
The content of γ-PGA；
Step (5): according to the content of γ-PGA in the lixiviating solution that step (4) is calculated, adds
The CTAB of 4 times of the content of γ-PGA, adding NaOH regulation pH value is 11, and mixing is stirred
Mixing 8h makes CTAB fully react with γ-PGA in lixiviating solution, then at rotating speed is
Centrifugal 25min in the centrifuge of 10000rpm, insoluble CTAB/ γ-PGA is multiple for isolated
Compound；
Step (6): step (5) is collected the CTAB/ γ-PGA complex obtained and is placed in dense
In the degree NaCl solution for 0.5mol/L, regulation pH value is 6.0, and dissociate 5h, is contained
There is the settled solution of γ-PGA；
Step (7): add γ-PGA settled solution in step (6) γ-PGA settled solution
The dehydrated alcohol of 3 times of volume, under conditions of temperature is 4 DEG C, cooling stands 15h, is turning
Speed be 10000rpm centrifuge in centrifugal 25min, separate to obtain precipitate, then will precipitation
Thing lyophilization, obtains γ-PGA white crystals.
Comparative example 1～2
Unlike embodiment 1～2: comparative example 1～2 uses Ethanol Method to pretreated
γ-PGA in ferment liquid extracts.
Experimental result
To extracting the turbidity after product redissolves in embodiment 1～2 and comparative example 1～2, protein contains
Amount and γ-PGA content are measured, and measurement result is as shown in table 1.
Table 1 CTAB method and Ethanol Method are to the extraction effect of γ-PGA in fermentation liquid
The miscellaneous bacteria in fermentation liquid, pigment and foreign protein after kieselguhr, Activated Carbon Pretreatment are
Through preferably being removed, it is possible to wrapped folder in a large amount of minimizing CTAB and γ-PGA precipitation processes
Impurity.After being extracted product dissolving from table 1, Ethanol Method and CTAB method, Ethanol Method pair
In fermentation liquid, the recovery levels of γ-PGA only has about 54%, pigment (turbidity OD660) and
Protein content is higher.The recovery levels of CTAB method is then higher than 80%, pigment and albumen
Matter content relatively Ethanol Method significantly reduces.Therefore, CTAB method γ-PGA in fermentation liquid carries
Taking in effect, the response rate and purity are all much better than Ethanol Method.
The announcement of book and inspiration according to the above description, those skilled in the art in the invention can also
Enough above-mentioned embodiment is changed and revises.Therefore, the invention is not limited in above-mentioned
Detailed description of the invention, every those skilled in the art are done any on the basis of the present invention
Conspicuously improved, replace or modification belong to protection scope of the present invention.Although additionally,
This specification employs some specific terms, but these terms are merely for convenience of description.
Claims (10)
1. the technique of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid, it is characterised in that
Comprise the following steps:
Step (1): the γ-PGA fermentation liquid obtained by fermentable is 1～50 μm by aperture
Filter bag and aperture be 0.25～1 μm microfilter filter, or in high speed centrifuge from
The heart separates, and removes the substantial amounts of remaining nutrient of insoluble fermentation and thalline in fermentation liquid；
Step (2): add trichloroacetic acid regulation pH in the fermentation liquid after step (1) is centrifugal
Value is 3～5, removes the foreign protein in fermentation liquid；
Step (3): add 1～30g/L diatomite adsorption fermentation in step (2) fermentation liquid
Remaining thalline in liquid, filters or centrifugation goes out insoluble matter, adds 0.5～20g/L activity
Pigment in charcoal absorption fermentation liquid and other impurity, through filtering or centrifugation acquisition supernatant；
Step (4): sample in the fermentation liquid after step (3) processes, i.e. in step (3)
In supernatant sampling, measure and estimate the content of γ-PGA in fermentation liquid；
Step (5): according to the content of γ-PGA in the fermentation liquid that step (4) is calculated, adds
The CTAB of 1～5 times of the content of γ-PGA, is 7～13 with NaOH solution regulation pH value,
Mix and blend 1～12h so that CTAB fully reacts with γ-PGA in fermentation liquid, is then centrifuged for
Isolated insoluble CTAB/ γ-PGA complex；
Step (6): step (5) is collected the CTAB/ γ-PGA complex obtained and is placed in NaCl
In solution, regulation pH value is 6～9, dissociates 1～12h, obtains the clarification containing γ-PGA molten
Liquid；
Step (7): add γ-PGA settled solution in step (6) γ-PGA settled solution
The ethanol of 2～5 times of volume, under conditions of temperature is 4～10 DEG C, cooling stands 12～24h,
It is centrifuged to obtain precipitate, then precipitate lyophilization is obtained highly purified γ-PGA white knot
Brilliant.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the γ-PGA fermentation liquid in described step (1) is by microorganism liquid
γ-PGA the fermentation liquid that body fermentation obtains.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the γ-PGA fermentation liquid in described step (1) is at microbial solid
The steaming of 8～12 times of addition solid matrix volume in the solid matrix containing γ-PGA that fermentation obtains
After distilled water is stirred extracting 0.5～2h, the γ-PGA obtained by 100 eye mesh screen filter and remove residues
Lixiviating solution.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the centrifugation in described step (1), rotating speed is 5000～15000rpm,
Centrifugation time is 5～30min.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the filtration in described step (3) is the filter by aperture is 1-50 μm
Bag and the microfilter that aperture is 0.25-1 μm filter.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the centrifugation in described step (3), rotating speed is 5000～15000rpm,
Centrifugation time is 5～30min.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the concentration of the NaCl solution in described step (6) is 0.1～1.0mol/L.
The technique of high efficiency extraction from fermentation liquid the most according to claim 1, its feature
It is: the concentration of the ethanol in described step (7) is more than 95%.
The technique of high efficiency extraction from fermentation liquid the most according to claim 1, its feature
It is: the lyophilization in described step (7) is in the fine vacuum that pressure is 10Pa～30Pa
The lower sublimation drying of degree is to ambient temperature, after 1～5h, pressure be 15Pa～30Pa, temperature is less than
At 40 DEG C, it is dried 4～10h.
The work of high efficiency extraction gamma-polyglutamic acid-from fermentation liquid the most according to claim 1
Skill, it is characterised in that the γ-PGA white crystals in described step (7) is Na-type γ-PGA
Mixture with H-type γ-PGA.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112806361A (en) * | 2019-11-18 | 2021-05-18 | 党永富 | Carbon adsorption 4-hydroxycoumarin herbicide safety additive and application thereof in treatment of herbicide side effects |
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| CN103613753A (en) * | 2013-11-14 | 2014-03-05 | 天津北洋百川生物技术有限公司 | Method for separating and purifying polyglutamic acid by using additive-free organic solvent |
| CN104804183A (en) * | 2015-04-21 | 2015-07-29 | 山东福瑞达生物科技有限公司 | Method for purifying and separating gamma-polyglutamic acid from fermentation liquor |
| CN105274181A (en) * | 2015-10-28 | 2016-01-27 | 新疆阜丰生物科技有限公司 | Method for extracting gamma-polyglutamic acid from fermentation liquor |
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| CN1103666A (en) * | 1993-12-07 | 1995-06-14 | 中国科学院化工冶金研究所 | Method for purifying glutami acid fermentation liquor |
| CN101948769A (en) * | 2010-08-20 | 2011-01-19 | 南京农业大学 | Bacteria YXY-C1 for preparing gamma-polyglutamic acid by solid fermentation and products thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112806361A (en) * | 2019-11-18 | 2021-05-18 | 党永富 | Carbon adsorption 4-hydroxycoumarin herbicide safety additive and application thereof in treatment of herbicide side effects |
| CN112806361B (en) * | 2019-11-18 | 2021-12-24 | 党永富 | Carbon adsorption 4-hydroxycoumarin herbicide safety additive and application thereof in treatment of herbicide side effects |
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Application publication date: 20160810 Assignee: Dongguan Xinwang Textile Co.,Ltd. Assignor: DONGGUAN University OF TECHNOLOGY Contract record no.: X2023980034903 Denomination of invention: Efficient extraction from fermentation broth g- Process of Polyglutamic Acid Granted publication date: 20171110 License type: Common License Record date: 20230421 |
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