CN102174193B - Method for efficiently extracting gamma-polyglutamic acid - Google Patents
Method for efficiently extracting gamma-polyglutamic acid Download PDFInfo
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- CN102174193B CN102174193B CN201010608782A CN201010608782A CN102174193B CN 102174193 B CN102174193 B CN 102174193B CN 201010608782 A CN201010608782 A CN 201010608782A CN 201010608782 A CN201010608782 A CN 201010608782A CN 102174193 B CN102174193 B CN 102174193B
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Abstract
The invention relates to a method for efficiently extracting gamma-polyglutamic acid, belonging to the method for extracting gamma-polyglutamic acid and aiming to solve the problems of complex separation and purification processes, high cost, long time consumption and low product purity of the prior method for extracting gamma-polyglutamic acid. The method comprises the following steps: (1) putting dry sephadex G-200 power into water, stirring to obtain sephadex G-200 suspension, filling the sephadex G-200 suspension into a chromatographic column, adding fermentation broth containing gamma-polyglutamic acid; and (2) washing fermentation broth containing the gamma-polyglutamic acid at the periphery of the chromatographic column and left on the surface of the sephadex G-200 with water, detecting on line by using an ultraviolet detector, and collecting eluate with the wavelength of 210mm to complete extracting gamma-polyglutamic acid. Due to the adoption of the method for efficiently extracting the gamma-polyglutamic acid, the separation time is shortened by 6-9 times, the product yield is improved by more than 1 time, the amount of ethanol used in the process for extracting is reduced, the process is simple, the cost is lowered, the light-colored product is milk-white and has good toughness and high purity.
Description
Technical field
The present invention relates to a kind of process for extracting of gamma-polyglutamic acid-.
Background technology
(γ-PGA) is found in nineteen thirty-seven to gamma-polyglutamic acid-the earliest; The researchist finds γ-PGA in the cell pod membrane of Bacillus anthracis (Bacillus anthracis) and in the cell pod membrane of amylomyces (Bacillus mesentericus), in subtilis (Bacillus.subtilis) and bacillus natto (Bacillus natto), also found gamma-polyglutamic acid-afterwards.Gamma-polyglutamic acid-is a kind of peptide molecule that is formed with the form condensation of peptide bond with γ-carboxyl and alpha-amino group by D-L-glutamic acid (D-GLu) and L-L-glutamic acid (L-GLu) monomer; Has the higher side chain carboxyl group of a large amount of activity on the molecular chain; Have high moisture retention and water-absorbent; Being easy to combine to generate stabilized complex with some macromolecular substance, is one type of ideal biodegradable polymer.The gamma-polyglutamic acid-environmentally safe; Be the green bio product; Physics and chemistry and biological characteristics with many uniquenesses such as splendid biodegradability, film-forming properties, moisture retentions; In today of paying attention to environmental protection, emphasical Sustainable development; This biosynthetic degradable type functional materials receives people's favor, little by little is applied to many fields such as antifreeze of medical manufacturing, food-processing and vegetables, fruit, sea-food, is a kind of Multifucntional biological products with very big exploitation value and bright prospects.
In the production process of gamma-polyglutamic acid-; Because the resulting fermented liquid of solution fermentation has very big viscosity; Bring big difficulty for the separation and purification in downstream, common separating and extracting method all can not get ideal effect, therefore; The separation efficiency that improves gamma-polyglutamic acid-is another key problem in technology of this material of fermentative prodn, and the research in this respect of present stage China still is in the starting stage.At present, this process generally adopts the traditional organic solvent precipitator method or the membrane sepn precipitator method.
The conventional organic solvents precipitator method are about to comprise the at first centrifugal mikrobe of removing wherein of fermented liquid of gamma-polyglutamic acid-; To methyl alcohol that wherein adds some volumes or ethanol, placing spends the night obtains throw out, and throw out through centrifugal collection, is obtained head product through lyophilize again; Head product is dissolved in the zero(ppm) water, and through centrifugal removal insoluble substance, the gamma-polyglutamic acid-in the aqueous solution obtains the higher gamma-polyglutamic acid-of purity through osmosis and lyophilize.There is repeatedly freeze dried process in this traditional gamma-polyglutamic acid-process for separating and purifying, cause separation costs too high, and the gamma-polyglutamic acid-product gas purity that obtains by this method is not high, often needs purification repeatedly, makes the yield of product very low.Simultaneously, separate a large amount of organic alcoholic solution of use in the purification process in tradition, this has not only increased gamma-polyglutamic acid-greatly and has separated the expense of purifying, and environment is also produced potentially dangerous.
The process that the membrane sepn precipitator method obtain head product is identical with the conventional organic solvents method.Afterwards, head product is dissolved in the zero(ppm) water, centrifugal remove not the capacitive material after, the gamma-polyglutamic acid-aqueous solution is obtained the gamma-polyglutamic acid-liquid concentrator with peristaltic pump is auxiliary through membrane module, go out the gamma-polyglutamic acid-product with organic solvent deposit again.Mainly there is the shortcoming of three aspects in this technology: 1. membrane module and corresponding auxiliary equipment cost are high; 2. the control that should separate purification process is complicated, is unfavorable for that industry controls; 3. film easy blocking and pollution influence separation efficiency, and are unfavorable for the continuous production needs of industry.
Summary of the invention
The objective of the invention is to have the problem that process is complicated, the time is long, cost is high and product purity is low of separation and purifying, and a kind of highly effective extraction method of gamma-polyglutamic acid-is provided in order to solve the existing method of extracting gamma-polyglutamic acid-.
The highly effective extraction method of gamma-polyglutamic acid-carries out according to the following steps: one, 20g sephadex G-200 dry powder is put into 1000～1200ml water, under the boiling water bath condition, stir 1～2h, get gel suspension; Leave standstill 1h, gel suspension is poured in the chromatography column of vertical fixing, open the gate out switch below the chromatography column then along glass stick; Flowing liquid; Make the gel natural subsidence, gel length is 15～20cm, treat gel surface be bordering on drain off after; Close gate out switch, add the fermented liquid that 2～6ml contains gamma-polyglutamic acid-then; Two, open gate out switch, water washs around the chromatography column and remains in the fermented liquid that contains gamma-polyglutamic acid-of gel surface, detects through online UV-detector, and collecting wavelength is the elutriant of 210nm, promptly accomplishes the high efficiency extraction of gamma-polyglutamic acid-.
The highly effective extraction method of gamma-polyglutamic acid-of the present invention and traditional dialysis partition method compare; Adopt GFC that the gamma-polyglutamic acid-fermented liquid is separated, can make disengaging time shorten 6～9 times, the output of product can improve more than 1 times; Also can reduce amount of ethanol used in the purifying technique simultaneously; Technology is easy, practices thrift cost, thus for gamma-polyglutamic acid-subtract depletion row, green production provides theoretical foundation.In addition; The present invention extracts the gamma-polyglutamic acid-paler colour that obtains, and is creamy white, and has good toughness; And do not find tangible impurity peaks among its magnetic resonance detection result, explain that the gel chromatography separation method can make the gamma-polyglutamic acid-product have higher purity.
Description of drawings
Fig. 1 is the nmr spectrum of gained gamma-polyglutamic acid-in the embodiment five; Fig. 2 is the outside drawing of gained gamma-polyglutamic acid-in the embodiment five; Fig. 3 adopts tradition dialysis partition method to extract the outside drawing of gained gamma-polyglutamic acid-in the embodiment five.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the highly effective extraction method of this embodiment gamma-polyglutamic acid-carries out according to the following steps: one, 20g sephadex G-200 dry powder is put into 1000～1200ml water, under the boiling water bath condition, stir 1～2h, get gel suspension; Leave standstill 1h, gel suspension is poured in the chromatography column of vertical fixing, open the gate out switch below the chromatography column then along glass stick; Flowing liquid; Make the gel natural subsidence, gel length is 15～20cm, treat gel surface be bordering on drain off after; Close gate out switch, add the fermented liquid that 2～6ml contains gamma-polyglutamic acid-then; Two, open gate out switch, water washs around the chromatography column and remains in the fermented liquid that contains gamma-polyglutamic acid-of gel surface, detects through online UV-detector, and collecting wavelength is the elutriant of 210nm, promptly accomplishes the high efficiency extraction of gamma-polyglutamic acid-.
The purpose that stirs 2h in this embodiment step 1 is that gel is expanded fully.
The diameter of chromatography column is 3cm in this embodiment step 1, column length 25cm.
Collecting wavelength in this embodiment step 2 is the elutriant of 210nm, and the ethanolic soln deposition with 2 times obtains the throw out thing, places 56 ℃ of drying bakers, obtains the gamma-polyglutamic acid-product behind the dry 48h.
Embodiment two: what this embodiment and embodiment one were different is that sephadex G in the step 1-200 dry powder adopts 20～80 orders, 100～200 orders or 200～400 orders.Other step and parameter are identical with embodiment one.
Embodiment three: what this embodiment and embodiment one were different is in the step 1 20g sephadex G-200 dry powder to be put into 1100ml water, under the boiling water bath condition, stirs 1.5h, gel suspension.Other step and parameter are identical with embodiment one.
Embodiment four: this embodiment and embodiment one are different is that the flow velocity of elutriant washing in the step 2 is 0.5ml/min, 1.0ml/min or 1.5ml/min, adopts constant flow pump to control.Other step and parameter are identical with embodiment one.
Embodiment five: the highly effective extraction method of this embodiment gamma-polyglutamic acid-carries out according to the following steps: one, 20g sephadex G-200 dry powder is put into 1000ml water, under the boiling water bath condition, stir 2h, get gel suspension; Leave standstill 1h, gel suspension is poured in the chromatography column of vertical fixing, open the gate out switch below the chromatography column then along glass stick; Flowing liquid; Make the gel natural subsidence, gel length is 20cm, treat gel surface be bordering on drain off after; Close gate out switch, add the fermented liquid that 4ml contains gamma-polyglutamic acid-then; Two, open gate out switch, water washs around the chromatography column and remains in the fermented liquid that contains gamma-polyglutamic acid-of gel surface, detects through online UV-detector, and collecting wavelength is the elutriant of 210nm, promptly accomplishes the high efficiency extraction of gamma-polyglutamic acid-.
Sephadex G in this embodiment step 1-200 dry powder adopts 100～200 orders; The flow velocity of elutriant washing is 1.0ml/min in the step 2.
The RT of this embodiment gamma-polyglutamic acid-is merely 19min; The content of the corresponding gamma-polyglutamic acid-of obtained the maximum absorption is up to 5.7g/L; Compare with traditional dialysis method; This extraction method output has improved more than 1 times, and impurity peaks do not occur in its nmr spectrum (see figure 1), explains that gamma-polyglutamic acid-purity is higher in the gel separation product.
This embodiment extracts the gamma-polyglutamic acid-paler colour (see figure 2) that obtains, and is creamy white, and purity is high, has good toughness; And it is very poor to adopt tradition dialysis partition method to extract the gamma-polyglutamic acid-product purity that obtains, the dark (see figure 3) of product color, gray; And product has certain rigidity, explains that product purity is not high, in addition; Because product purity is poor, can't carry out magnetic resonance detection to it.
Claims (4)
1. the highly effective extraction method of a gamma-polyglutamic acid-is characterized in that the highly effective extraction method of gamma-polyglutamic acid-carries out according to the following steps: one, 20g sephadex G-200 dry powder is put into 1000～1200ml water, under the boiling water bath condition, stir 1～2h; Get gel suspension, leave standstill 1h, gel suspension is poured in the chromatography column of vertical fixing along glass stick; Open the gate out switch below the chromatography column then, flowing liquid makes the gel natural subsidence; Gel length is 15～20cm; Treat gel surface be bordering on drain off after, close gate out switch, add the fermented liquid that 2～6ml contains gamma-polyglutamic acid-then; Two, open gate out switch, water washs around the chromatography column and remains in the fermented liquid that contains gamma-polyglutamic acid-of gel surface, detects through online UV-detector, and collecting wavelength is the elutriant of 210nm, promptly accomplishes the high efficiency extraction of gamma-polyglutamic acid-.
2. the highly effective extraction method of a kind of gamma-polyglutamic acid-according to claim 1 is characterized in that sephadex G in the step 1-200 dry powder adopts 20～80 orders, 100～200 orders or 200～400 orders.
3. the highly effective extraction method of a kind of gamma-polyglutamic acid-according to claim 1 is characterized in that in the step 1 20g sephadex G-200 dry powder being put into 1100ml water, under the boiling water bath condition, stirs 1.5h, gets gel suspension.
4. the highly effective extraction method of a kind of gamma-polyglutamic acid-according to claim 1 is characterized in that the flow velocity of elutriant washing in the step 2 is 0.5ml/min, 1.0ml/min or 1.5ml/min, adopts constant flow pump to control.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503511A (en) * | 2009-03-06 | 2009-08-12 | 天津商业大学 | Method for extracting polyglutamic acid from original fermentation liquor by double aqueous phase system |
| CN101580587A (en) * | 2009-03-30 | 2009-11-18 | 山东阜丰生物科技开发有限公司 | Novel technology for extracting polyglutamic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101503511A (en) * | 2009-03-06 | 2009-08-12 | 天津商业大学 | Method for extracting polyglutamic acid from original fermentation liquor by double aqueous phase system |
| CN101580587A (en) * | 2009-03-30 | 2009-11-18 | 山东阜丰生物科技开发有限公司 | Novel technology for extracting polyglutamic acid |
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