CN105837815B - A kind of technique of the high efficiency extraction γ polyglutamic acids from zymotic fluid - Google Patents
A kind of technique of the high efficiency extraction γ polyglutamic acids from zymotic fluid Download PDFInfo
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- CN105837815B CN105837815B CN201610329177.5A CN201610329177A CN105837815B CN 105837815 B CN105837815 B CN 105837815B CN 201610329177 A CN201610329177 A CN 201610329177A CN 105837815 B CN105837815 B CN 105837815B
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- pga
- zymotic fluid
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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
Abstract
The invention belongs to biological technical field, a kind of more particularly to technique of the high efficiency extraction γ polyglutamic acids from zymotic fluid, using the γ PGA in cetyl trimethylammonium bromide (CTAB) selective precipitation zymotic fluid, then sediment is dissociated to the operations such as dissolving, alcohol analysis, separation of solid and liquid and drying in certain density NaCl solution obtain the higher γ PGA of purity.Relative to prior art, the technique can not only improve γ PGA extraction efficiencies and product purity, can also largely reduce the organic solvent for alcohol analysis, substantially reduce production cost, have a good application prospect.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of work of the high efficiency extraction gamma-polyglutamic acid from zymotic fluid
Skill.
Background technology
Gamma-polyglutamic acid (poly- γ-glutamic acid, abbreviation γ-PGA) is by Pidolidone and D-Glu warp
A kind of peptide molecule that γ-amido link combines to form is crossed, is a kind of water soluble polymer amino acid polymer.Because γ-PGA have
Have biodegradability, good biocompatibility, water-retaining property, harmless to the human body and unique physics such as environmentally safe,
Chemistry and biology property so that it is multiple that γ-PGA are widely used in agricultural, food, medicine, cosmetic technology and environmental protection etc.
Field.
At present, the method for extracting separating gamma-PGA from high viscosity fermentation liquid mainly has three kinds：Organic solvent precipitation method (one
As use separating out alcohol method), chemical precipitation method and the UF membrane precipitation method.Organic solvent deposit refers to utilize the side for centrifuging or condensing thalline
Method removes the thalline in zymotic fluid, and addition lower alcohols (such as methanol, ethanol, isopropanol) are precipitable in supernatant obtains
γ-PGA, it is freeze-dried to obtain faint yellow color jelly.Chemical precipitation is to use saturation CuSO4, NaCl solution replace lower alcohol
Class salt precipitation γ-PGA.The UF membrane precipitation method can also be taken to highly viscous zymotic fluid.First alcohol analysis or the obtained γ that saltouts-
PGA crude product colors are generally yellow, after need to being dissolved in distilled water, carry out the processing such as refined filtration, decolouring, desalination, warp again
After lyophilized, white γ-PGA highly finished product can be just obtained.Due to the extraction process of chemical precipitation method and the UF membrane precipitation method is complicated,
Extraction cost is high and purity is low, therefore method the most frequently used at present is organic solvent precipitation method.Organic solvent precipitation method (analyse by alcohol
Method) equally exist many problems.Because γ-PGA zymotic fluids are very sticky, carry out pretreatment and go the removal of impurity to need appropriate dilution ability
Smooth operation.When γ-PGA concentration is relatively low in zymotic fluid, not only alcohol analysis effect is bad, and the γ-PGA rate of recovery is very low, and disappears
Consume a large amount of organic solvents.The use of a large amount of organic solvents not only increases production cost, and brings larger potential safety hazard.In order to subtract
Few solvent consumption, it is concentrated by ultrafiltration as fermentation liquor pretreatment is latter, but ultra-filtration process γ-PGA losses are very big, and milipore filter holds
It is easily contaminated, treatment effeciency step-down.It is lowly that γ-PGA production costs remain high to isolate and purify difficult and extraction recovery
Major reason.
In view of this, a set of economically viable separation purifying technique is needed badly in γ-PGA industrial production.
The content of the invention
It is an object of the invention to：In view of the shortcomings of the prior art, and provide one kind from zymotic fluid high efficiency extraction γ-
PGA new technology, to improve γ-PGA yield, purity and reduce production cost.
To achieve these goals, the present invention uses following technical scheme：
A kind of technique of the high efficiency extraction gamma-polyglutamic acid from zymotic fluid, comprises the following steps：
Step (1)：By the γ-PGA zymotic fluids that microbial fermentation obtains be 1~50 μm of filter bag by aperture and aperture is
0.25~1 μm of millipore filter filtering, or centrifuged in supercentrifuge, remove substantial amounts of insoluble hair in zymotic fluid
Ferment remnants nutrients and thalline；
Step (2)：It is 3~5 that trichloroacetic acid regulation pH value is added in zymotic fluid after step (1) centrifugation, removes fermentation
Foreign protein in liquid；
Step (3)：The remaining thalline added in step (2) zymotic fluid in 1~30g/L kieselguhr adsorption zymotic fluids, mistake
Filter or centrifuge out insoluble matter, the pigment and other impurity added in 0.5~20g/L charcoal absorption zymotic fluids, pass through
Filter centrifuges acquisition supernatant；
Step (4)：Sampled in zymotic fluid after step (3) processing, i.e., the supernatant sampling in step (3), measurement
And estimate the content of γ-PGA in zymotic fluid；
Step (5)：γ-PGA content in the zymotic fluid calculated according to step (4), adds the 1~5 of γ-PGA content
CTAB again, it is 7~13 with NaOH solution regulation pH value, mixes 1~12h so that CTAB fills with γ-PGA in zymotic fluid
Divide reaction, be then centrifuged for isolated insoluble CTAB/ γ-PGA compounds；
Step (6)：Step (5) is collected to obtained CTAB/ γ-PGA compounds to be placed in NaCl solution, regulation pH value is
6~9,1~12h is dissociated, obtains the settled solution containing γ-PGA；
Step (7)：2~5 times of second of γ-PGA settled solution volumes is added in step (6) γ-PGA settled solutions
Alcohol, 12~24h of cooling and standings under conditions of being 4~10 DEG C in temperature, centrifuges to obtain sediment, is then freeze-dried sediment
To the γ-PGA white crystals of high-purity.
It should be noted that in the basic conditions, CTAB and γ-PGA form the ionic compound that can not be dissociated and precipitated
Get off, and in the solution of pH 6~8 neutrallty condition and high ionic strength (ionic strength ＞ 150mmol/L), this ion
Compound can dissociate dissolving again again, that is to say, that CTAB has the selective precipitation γ-PGA from low ionic strength solution
Feature.
One kind as the high efficiency extraction gamma-polyglutamic acid technique from zymotic fluid is improved, in the step (1)
γ-PGA zymotic fluids are the γ-PGA zymotic fluids obtained that fermented by microbial liquid.
One kind as the high efficiency extraction gamma-polyglutamic acid technique from zymotic fluid is improved, in the step (1)
γ-PGA zymotic fluids in the solid matrix for-PGA containing γ that microbial solid fermentation obtains add solid matrix volume 8~
After 12 times of distilled water is stirred 0.5~2h of extraction, pass through the γ-PGA leaching liquors of 100 eye mesh screen filter and remove residues acquisition.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (1)
Centrifugation, rotating speed is 5000~15000rpm, and centrifugation time is 5~30min.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (3)
Filtering be 1-50 μm by aperture filter bag and aperture be 0.25-1 μm millipore filter filtering..
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (3)
Centrifugation, rotating speed is 5000~15000rpm, and centrifugation time is 5~30min.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (6)
The concentration of NaCl solution be 0.1~1.0mol/L.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (7)
Ethanol concentration be more than 95%.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (7)
Freeze-drying refer to that lyophilization is to environment temperature in the case where pressure is 10Pa~30Pa condition of high vacuum degree, after 1~5h, in pressure
It is less than for 15Pa~30Pa, temperature at 40 DEG C, dries 4~10h.
The a kind of of technique as the high efficiency extraction gamma-polyglutamic acid from zymotic fluid improves, in the step (7)
γ-PGA white crystals be Na- type γ-PGA and H- type γ-PGA mixture.
The beneficial effects of the present invention are：The present invention is occurred under proper condition using the CTAB and γ-PGA in zymotic fluid
Chemical reaction, forms insoluble ionic compound, is then collected and precipitated by separation of solid and liquid, precipitation is dissolved in certain density
In NaCl solution, the organic solvent (lower alcohol) for adding 3~5 times divides in the solution by dissociation dissolving, alcohol analysis, solid-liquid
From and dry etc. operation obtain the higher γ-PGA of purity.Relative to prior art, the technique is due to utilizing CTAB selectivity
γ-PGA more completely features are precipitated, has reached efficient concentration γ-PGA and has gone deimpurity purpose, not only increased γ-PGA
Extraction efficiency and product purity, the organic solvent for alcohol analysis is also substantially reduce the number, substantially reduces production cost, had good
Application prospect.
Embodiment
The present invention and its advantage are described in further detail below in conjunction with embodiment, still, this hair
Bright embodiment is not limited thereto.
Embodiment 1
A kind of technique of the high efficiency extraction gamma-polyglutamic acid from zymotic fluid, comprises the following steps：
Step (1)：By the γ-PGA zymotic fluids that 100L microbial fermentations obtain in rotating speed in 5000rpm centrifugal separators
30min is centrifuged, is separated off in zymotic fluid the remaining nutrients of substantial amounts of insoluble fermentation and thalline；
Step (2)：It is 3 that trichloroacetic acid regulation pH is added in zymotic fluid after step (1) centrifugation, is removed in zymotic fluid
Foreign protein, while the viscosity of zymotic fluid is reduced, it is easy to follow-up purification process；
Step (3)：The remaining thalline added in every liter of step (2) zymotic fluid in 20g/L kieselguhr adsorption zymotic fluids, mistake
Insoluble matter is removed in filter or centrifugation, the pigment and other impurities added in 5g/L charcoal absorption zymotic fluids, is in rotating speed
30min is centrifuged in 5000rpm centrifuge, separation obtains supernatant；
Step (4)：Sampled in zymotic fluid after step (3) processing, determine the content of γ-PGA in zymotic fluid；
Step (5)：γ-PGA content in the zymotic fluid calculated according to step (4), add 2 times of γ-PGA content
CTAB, it is 10 to add NaOH regulations pH, mixes 4h and CTAB is fully reacted with γ-PGA in zymotic fluid, then in rotating speed
To centrifuge 30min, isolated insoluble CTAB/ γ-PGA compounds in 5000rpm centrifuge；
Step (6)：Step (5) is collected to obtained CTAB/ γ-PGA compounds and is placed in the NaCl that concentration is 0.6mol/L
In solution, regulation pH value is 7, dissociates 3h, obtains the settled solution containing γ-PGA；
Step (7)：4 times of anhydrous second of γ-PGA settled solution volumes is added in step (6) γ-PGA settled solutions
Alcohol, cooling and standings 12h under conditions of being 4 DEG C in temperature, centrifuges 30min, separating to sink in the centrifuge that rotating speed is 5000rpm
Starch, then sediment is freeze-dried, obtains γ-PGA white crystals.
Embodiment 2
A kind of technique of the high efficiency extraction gamma-polyglutamic acid from zymotic fluid, comprises the following steps：
Step (1)：The solid matrix 10kg for-PGA containing γ that microbial solid fermentation is obtained adds the distillation of 10 times of volumes
For water in 40 DEG C of stirring and leaching 1h, leaching liquor first passes through 100 eye mesh screen filter and remove residues, and filtrate passes through 5 μm of aperture filter bag mistakes respectively again
Filter and 0.5 μm of millipore filter filter, and collect leaching liquor.
Step (2)：It is 4 that trichloroacetic acid regulation pH value is added in step (1) leaching liquor；
Step (3)：The color added in step (2) leaching liquor in 10g/L diatomite and 8g/L charcoal absorption zymotic fluids
Element and other impurities, centrifuge 25min in the centrifuge that rotating speed is 10000rpm, and separation obtains supernatant；
Step (4)：Sampled in leaching liquor after step (3) processing, determine the content of γ-PGA in leaching liquor；
Step (5)：γ-PGA content in the leaching liquor calculated according to step (4), add 4 times of γ-PGA content
CTAB, it is 11 to add NaOH regulation pH value, mixes 8h and CTAB is fully reacted with γ-PGA in leaching liquor, is then being turned
Speed is to centrifuge 25min, isolated insoluble CTAB/ γ-PGA compounds in 10000rpm centrifuge；
Step (6)：Step (5) is collected to obtained CTAB/ γ-PGA compounds and is placed in the NaCl that concentration is 0.5mol/L
In solution, regulation pH value is 6.0, dissociates 5h, obtains the settled solution containing γ-PGA；
Step (7)：3 times of anhydrous second of γ-PGA settled solution volumes is added in step (6) γ-PGA settled solutions
Alcohol, cooling and standings 15h under conditions of being 4 DEG C in temperature, centrifuges 25min in the centrifuge that rotating speed is 10000rpm, separates
Sediment, then sediment is freeze-dried, obtains γ-PGA white crystals.
Comparative example 1~2
Unlike embodiment 1~2：Comparative example 1~2 is using Ethanol Method to the γ-PGA in pretreated zymotic fluid
Extracted.
Experimental result
To extracting turbidity, protein content and γ-PGA contents after product redissolves in embodiment 1~2 and comparative example 1~2
It is measured, measurement result is as shown in table 1.
Extraction effect of the CTAB methods of table 1 with Ethanol Method to γ-PGA in zymotic fluid
The miscellaneous bacteria in zymotic fluid, pigment and foreign protein after diatomite, Activated Carbon Pretreatment have preferably been gone
Remove, can largely reduce the impurity of wrapped folder in CTAB and γ-PGA precipitation processes.From table 1, Ethanol Method and the extraction of CTAB methods
After product dissolving, Ethanol Method only has 54% or so to the recovery levels of γ-PGA in zymotic fluid, pigment (turbidity OD660) and albumen
Matter content is higher.And the recovery levels of CTAB methods are then higher than 80%, pigment and protein content significantly reduce compared with Ethanol Method.Cause
This, for CTAB methods in zymotic fluid on γ-PGA extraction effect, the rate of recovery and purity are all much better than Ethanol Method.
The announcement and inspiration of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and changed.Therefore, the invention is not limited in above-mentioned embodiment, every those skilled in the art exist
Made any conspicuously improved, replacement or modification belong to protection scope of the present invention on the basis of the present invention.This
Outside, although having used some specific terms in this specification, these terms are merely for convenience of description.
Claims (10)
1. a kind of technique of the high efficiency extraction gamma-polyglutamic acid from zymotic fluid, it is characterised in that comprise the following steps：
Step (1)：By the γ-PGA zymotic fluids that microbial fermentation obtains be 1~50 μm of filter bag by aperture and aperture is 0.25
~1 μm of millipore filter filtering, or centrifuged in supercentrifuge, it is residual to remove substantial amounts of insoluble fermentation in zymotic fluid
Remaining nutrients and thalline；
Step (2)：It is 3~5 that trichloroacetic acid regulation pH value is added in the zymotic fluid that step (1) obtains, and is removed in zymotic fluid
Foreign protein；
Step (3)：In step (2) zymotic fluid add 1~30g/L kieselguhr adsorption zymotic fluids in remaining thalline, filtering or
Centrifuge out insoluble matter, the pigment and other impurity added in 0.5~20g/L charcoal absorption zymotic fluids, it is filtered or
Centrifuge and obtain supernatant；
Step (4)：Sampled in zymotic fluid after step (3) processing, i.e., the supernatant sampling in step (3), measure and estimate
Calculate the content of γ-PGA in zymotic fluid；
Step (5)：γ-PGA content in the zymotic fluid calculated according to step (4), add 1~5 times of γ-PGA content
CTAB, it is 7~13 with NaOH solution regulation pH value, mixes 1~12h so that CTAB and γ-PGA in zymotic fluid are fully anti-
Should, it is then centrifuged for isolated insoluble CTAB/ γ-PGA compounds；
Step (6)：Step (5) to be collected into obtained CTAB/ γ-PGA compounds to be placed in NaCl solution, regulation pH value is 6~
9,1~12h is dissociated, obtains the settled solution containing γ-PGA；
Step (7)：2~5 times of ethanol of γ-PGA settled solution volumes is added in step (6) γ-PGA settled solutions,
Temperature is 12~24h of cooling and standings under conditions of 4~10 DEG C, centrifuges to obtain sediment, is then freeze-dried sediment to obtain height
γ-PGA the white crystals of purity.
2. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
γ-PGA zymotic fluids in step (1) are the γ-PGA zymotic fluids obtained that fermented by microbial liquid.
3. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
γ-PGA zymotic fluids in step (1) are to add solid-based in the solid matrix for-PGA containing γ that microbial solid fermentation obtains
Plastid product 8~12 times of distilled water be stirred 0.5~2h of extraction after, by 100 eye mesh screen filter and remove residues obtain γ-
PGA leaching liquors.
4. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
Centrifugation in step (1), rotating speed are 5000~15000rpm, and centrifugation time is 5~30min.
5. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
The millipore filter filtering that the filter bag and aperture that it is 1-50 μm by aperture that filtering in step (3), which is, are 0.25-1 μm.
6. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
Centrifugation in step (3), rotating speed are 5000~15000rpm, and centrifugation time is 5~30min.
7. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
The concentration of NaCl solution in step (6) is 0.1~1.0mol/L.
8. the technique of the high efficiency extraction according to claim 1 from zymotic fluid, it is characterised in that：In the step (7)
The concentration of ethanol is more than 95%.
9. the technique of the high efficiency extraction according to claim 1 from zymotic fluid, it is characterised in that：In the step (7)
Lyophilization after 1~5h, is in pressure to environment temperature under the condition of high vacuum degree for being freeze-dried to be 10Pa~30Pa in pressure
15Pa~30Pa, temperature are less than at 40 DEG C, dry 4~10h.
10. the technique of the high efficiency extraction gamma-polyglutamic acid according to claim 1 from zymotic fluid, it is characterised in that described
The mixture that γ-PGA white crystals in step (7) are Na- type γ-PGA and H- type γ-PGA.
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| CN1038346C (en) * | 1993-12-07 | 1998-05-13 | 中国科学院化工冶金研究所 | Method for purifying glutami acid fermentation liquor |
| CN101948769B (en) * | 2010-08-20 | 2012-01-04 | 南京农业大学 | Bacteria YXY-C1 for preparing gamma-polyglutamic acid by solid fermentation and products thereof |
| 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 |
| CN104804183B (en) * | 2015-04-21 | 2017-05-10 | 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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