CN105441499A - Method for extracting gamma-polyglutamic acid from fermentation liquor - Google Patents
Method for extracting gamma-polyglutamic acid from fermentation liquor Download PDFInfo
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- CN105441499A CN105441499A CN201510884983.4A CN201510884983A CN105441499A CN 105441499 A CN105441499 A CN 105441499A CN 201510884983 A CN201510884983 A CN 201510884983A CN 105441499 A CN105441499 A CN 105441499A
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- massfraction
- fermented liquid
- polyglutamic acid
- gamma
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Abstract
The invention provides a method for extracting gamma-polyglutamic acid from fermentation liquor. According to the method, first, a mixed solution is prepared from PEG1000, (NH4)2SO4, fermentation liquor and water, and the fermentation liquor is prepared from bacillus subtilis by means of liquid state fermentation; next, the mixed solution is subjected to natural phase splitting, the environment temperature is controlled between 20 DEG C and 40 DEG C, the upper phase is taken, an ultrafiltration membrane with the molecular weight cut off of 30 KDa is adopted to penetrate PEG1000, and cut-off gamma-polyglutamic acid concentrated liquor is frozen at the temperature between -10 DEG C and -20 DEG C and then subjected to vacuum freeze-drying at the temperature between -40 DEG C and -60 DEG C for 10-30 hours to obtain a pure product. The method solves the problems that gamma-polyglutamic acid concentrated liquor is sticky and pretreatment cost is too high. A follow-up two-aqueous-phase system is moderate in reaction, so that damage to the molecular weight of gamma-polyglutamic acid through a traditional precipitation method is avoided.
Description
Technical field
The invention belongs to bioengineering field, relate to a kind of method extracting gamma-polyglutamic acid-, specifically a kind of method extracting gamma-polyglutamic acid-from fermented liquid.
Background technology
Gamma-polyglutamic acid-is by the outer high-molecular polypeptide of the water-soluble born of the same parents of the one of Microbe synthesis, relative molecular weight is between 100kD-1000kD, excellent water-soluble owing to having, superpower adsorptivity and biodegradability, degraded product is non-harmful L-glutamic acid, it is a kind of excellent environment-friendly type macromolecule material, can be used as water-holding agent, adsorbent for heavy metal, flocculation agent, sustained release dosage and pharmaceutical carrier etc., at makeup, environment protection, food, medicine, agricultural, the industries such as desertification control all have very large commercial value and social value, but gamma-polyglutamic acid-fermented liquid thickness, pre-treatment cost is excessive, and traditional precipitator method can damage gamma-polyglutamic acid-molecular weight.Double-aqueous phase system because its preparation technology is simple, cost is low, reaction temperature and, be widely used in the extraction purification of the bioactive ingredients such as protein, enzyme.
Summary of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of method extracting gamma-polyglutamic acid-from fermented liquid, the described this method extracting gamma-polyglutamic acid-from fermented liquid will solve the technical problem that excessive, the traditional precipitator method of gamma-polyglutamic acid-fermented liquid thickness of the prior art, pre-treatment cost can damage gamma-polyglutamic acid-molecular weight.
The invention provides a kind of method extracting gamma-polyglutamic acid-from fermented liquid, comprise the steps:
1) step of mixing solutions is prepared for one; Described mixing solutions is by PEG1000, (NH
4)
2sO
4, fermented liquid and water composition, described fermented liquid is obtained through liquid state fermentation by subtilis, and the pH value of fermented liquid is 3.75-8.46, and in described mixing solutions, the massfraction of PEG1000 is 10%-30%, (NH
4)
2sO
4massfraction be 5%-25%, the massfraction of fermented liquid is 8-12%, surplus is water;
2) step of a natural phase-splitting, leaves standstill nature phase-splitting by the mixing solutions made, and control temperature is 20 ~ 40 DEG C;
3) the upper phase of step (2) mixing solutions is got, molecular weight cut-off is adopted to be that the ultra-filtration membrane of 30KDa is through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-10 ~-20 DEG C, then in-40 ~-60 DEG C of vacuum lyophilization 10-30h, obtain sterling.
Further, in step (1), in described mixing solutions, the massfraction of PEG1000 is 20%, (NH
4)
2sO
4massfraction be 5%, the pH value of fermented liquid is 3.75 and massfraction is 10%, surplus is water; In step (2), control temperature is 20 DEG C.
Further, in step (1), in described mixing solutions, the massfraction of PEG1000 is 30%, (NH
4)
2sO
4massfraction be 25%, the pH value of fermented liquid is 8.46 and massfraction is 10%, surplus is water; In step (2), control temperature is 40 DEG C.
Further, in step (1), in described mixing solutions, the massfraction of PEG1000 is 20%, (NH
4)
2sO
4massfraction be 15%, fermented liquid pH value is 5.31 and massfraction is 10%, surplus is water; In step (2), control temperature is 30 DEG C.
Further, in step (1), in described mixing solutions, the massfraction of PEG1000 is 15%, (NH
4)
2sO
4massfraction be 10%, fermented liquid pH value is 4.66 and massfraction is 10%, surplus is water; In step (2), control temperature is 25 DEG C.
Further, in step (1), in described mixing solutions, the massfraction of PEG1000 is 25%, (NH
4)
2sO
4massfraction be 20%, fermented liquid pH value is 7.55 and massfraction is 10%, surplus is water; In step (2), control temperature is 40 DEG C.
Further, adopt the gamma-polyglutamic acid-that aforesaid method extracts, its partition ratio is 0.81-2.31, and extraction yield is 45.06%-78.02%.
The present invention utilizes target product in fermentation liquor at two-phase (PEG1000 solution, (NH
4)
2sO
4solution) in the difference of solubleness, under the condition that fermented liquid massfraction is certain in mixing solutions, by changing two matcheds, fermented liquid pH value and outside temperature, Gamma-polyglutamic acid from fermentation broth is enriched in the higher upper phase PEG1000 solution of water content.The gamma-polyglutamic acid-mixing solutions of upper middle PEG1000 and enrichment mutually, adopt the PEG1000 of ultra-filtration membrane through small-molecular-weight of 30kD, in trapped fluid, gamma-polyglutamic acid-is constantly concentrated, concentrated gamma-polyglutamic acid-solution, after cryogenic freezing, adopts vacuum lyophilization to obtain gamma-polyglutamic acid-sterling.
The present invention compares with prior art, and its technical progress is significant.The Two-phase system that the present invention adopts, from fermented liquid, extract gamma-polyglutamic acid-solve gamma-polyglutamic acid-fermented liquid thickness, the problem that pre-treatment cost is excessive, simultaneously, follow-up double-aqueous phase system reaction temperature of the present invention and, avoid the destruction of traditional precipitator method to gamma-polyglutamic acid-molecular weight.
Embodiment
Below by specific embodiment, the present invention is set forth further, but do not limit the present invention.
Two-phase system of the present invention extracts gamma-polyglutamic acid-fermented liquid used and is obtained by subtilis (BacillussubtilisGIM1.286) liquid state fermentation from fermented liquid, culture medium prescription (g/L) in fermenting process and culture condition as follows: peptone 30, glucose 40, Sodium Glutamate 30, sodium-chlor 15, dipotassium hydrogen phosphate 2, potassium primary phosphate 4, magnesium sulfate 0.5, calcium chloride 0.25 surplus is water; Fermented liquid pH value 7.0-7.2,37 DEG C, 250r/min cultivates 48h.
Two-phase system of the present invention extracts gamma-polyglutamic acid-small-sized tangential flow ultra-filtration unit used from fermented liquid, is produced by Millipore company of the U.S..
Embodiment 1
From fermented liquid, extract a method for gamma-polyglutamic acid-, be prepared by a method comprising the following steps and form:
(1), mixing solutions preparation;
In mixing solutions, the massfraction of PEG1000 is 10%, (NH
4)
2sO
4massfraction be 5%, fermented liquid pH value is 3.75 and massfraction is 10%, surplus is water;
(2), natural phase-splitting;
The mixing solutions made in step (1) is left standstill nature phase-splitting, and the temperature that controls environment is 20 DEG C;
(3), get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-15 DEG C, in-50 DEG C of vacuum lyophilization 24h, obtain sterling.
The gamma-polyglutamic acid-double-aqueous phase system of above-mentioned gained, partition ratio is 0.93, and extraction yield is 51.25%.
Embodiment 2
From fermented liquid, extract a method for gamma-polyglutamic acid-, be prepared by a method comprising the following steps and form:
(1), mixing solutions preparation;
In mixing solutions, the massfraction of PEG1000 is 30%, (NH
4)
2sO
4massfraction be 25%, fermented liquid pH value is 8.46 and massfraction is 10%, surplus is water;
(2), natural phase-splitting;
The mixing solutions made in step (1) is left standstill nature phase-splitting, and the temperature that controls environment is 40 DEG C;
(3), get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-20 DEG C, in-50 DEG C of vacuum lyophilization 24h, obtain sterling.
The gamma-polyglutamic acid-double-aqueous phase system of above-mentioned gained, partition ratio is 0.81, and extraction yield is 45.06%.
Embodiment 3
From fermented liquid, extract a method for gamma-polyglutamic acid-, be prepared by a method comprising the following steps and form:
(1), mixing solutions preparation;
In mixing solutions, the massfraction of PEG1000 is 20%, (NH
4)
2sO
4massfraction be 15%, fermented liquid pH value is 5.31 and massfraction is 10%, surplus is water;
(2), natural phase-splitting;
The mixing solutions made in step (1) is left standstill nature phase-splitting, and the temperature that controls environment is 30 DEG C;
(3), get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-10 DEG C, in-50 DEG C of vacuum lyophilization 24h, obtain sterling.
The gamma-polyglutamic acid-double-aqueous phase system of above-mentioned gained, partition ratio is 1.21, and extraction yield is 67.59%.
Embodiment 4
From fermented liquid, extract a method for gamma-polyglutamic acid-, be prepared by a method comprising the following steps and form:
(1), mixing solutions preparation;
In mixing solutions, the massfraction of PEG1000 is 15%, (NH
4)
2sO
4massfraction be 10%, fermented liquid pH value is 4.66 and massfraction is 10%, surplus is water;
(2), natural phase-splitting;
The mixing solutions made in step (1) is left standstill nature phase-splitting, and the temperature that controls environment is 25 DEG C;
(3), get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-13 DEG C, in-50 DEG C of vacuum lyophilization 24h, obtain sterling.
The gamma-polyglutamic acid-double-aqueous phase system of above-mentioned gained, partition ratio is 2.31, and extraction yield is 78.02%.
Embodiment 5
From fermented liquid, extract a method for gamma-polyglutamic acid-, be prepared by a method comprising the following steps and form:
(1), mixing solutions preparation;
In mixing solutions, the massfraction of PEG1000 is 25%, (NH
4)
2sO
4massfraction be 20%, fermented liquid pH value is 7.55 and massfraction is 10%, surplus is water;
(2), natural phase-splitting;
The mixing solutions made in step (1) is left standstill nature phase-splitting, and the temperature that controls environment is 35 DEG C;
(3), get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-18 DEG C, in-50 DEG C of vacuum lyophilization 24h, obtain sterling.
The gamma-polyglutamic acid-double-aqueous phase system of above-mentioned gained, partition ratio is 1.52, and extraction yield is 73.25%.
In sum, a kind of method adopting Two-phase system to extract gamma-polyglutamic acid-from fermented liquid of the present invention, technique is simple, and cost is low, and its partition ratio is 0.81-2.31, and extraction yield is 45.06%-78.02%.
Above embodiment is only for illustration of content of the present invention; in addition; the present invention also has other embodiments, as long as those skilled in the art are because of technology involved in the present invention enlightenment, and the technical scheme adopting equivalent replacement or equivalent deformation mode to be formed all drops in protection scope of the present invention.
Claims (7)
1. from fermented liquid, extract a method for gamma-polyglutamic acid-, it is characterized in that comprising the steps:
1) step of mixing solutions is prepared for one; Described mixing solutions is by PEG1000, (NH
4)
2sO
4, fermented liquid and water composition, described fermented liquid is obtained through liquid state fermentation by subtilis, and the pH value of fermented liquid is 3.75-8.46, and in described mixing solutions, the massfraction of PEG1000 is 10%-30%, (NH
4)
2sO
4massfraction be 5%-25%, the massfraction of fermented liquid is 8-12%, surplus is water;
2) step of a natural phase-splitting, leaves standstill nature phase-splitting by the mixing solutions made, and control temperature is 20 ~ 40 DEG C;
3) get the upper phase of step (2) mixing solutions, adopt molecular weight cut-off be the ultra-filtration membrane of 30KDa through PEG1000, after the gamma-polyglutamic acid-concentrated solution retained is freezing through-10 ~-20 DEG C, then in-40 ~-60 DEG C of vacuum lyophilization 10-30h, obtain sterling.
2. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: in step (1), in described mixing solutions, and the massfraction of PEG1000 is 20%, (NH
4)
2sO
4massfraction be 5%, the pH value of fermented liquid is 3.75 and massfraction is 10%, surplus is water; In step (2), control temperature is 20 DEG C.
3. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: in step (1), in described mixing solutions, and the massfraction of PEG1000 is 30%, (NH
4)
2sO
4massfraction be 25%, the pH value of fermented liquid is 8.46 and massfraction is 10%, surplus is water; In step (2), control temperature is 40 DEG C.
4. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: in step (1), in described mixing solutions, and the massfraction of PEG1000 is 20%, (NH
4)
2sO
4massfraction be 15%, fermented liquid pH value is 5.31 and massfraction is 10%, surplus is water; In step (2), control temperature is 30 DEG C.
5. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: in step (1), in described mixing solutions, and the massfraction of PEG1000 is 15%, (NH
4)
2sO
4massfraction be 10%, fermented liquid pH value is 4.66 and massfraction is 10%, surplus is water; In step (2), control temperature is 25 DEG C.
6. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: in step (1), in described mixing solutions, and the massfraction of PEG1000 is 25%, (NH
4)
2sO
4massfraction be 20%, fermented liquid pH value is 7.55 and massfraction is 10%, surplus is water; In step (2), control temperature is 40 DEG C.
7. a kind of method extracting gamma-polyglutamic acid-from fermented liquid according to claim 1, is characterized in that: adopt the gamma-polyglutamic acid-that aforesaid method extracts, its partition ratio is 0.81-2.31, and extraction yield is 45.06%-78.02%.
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| CN201510884983.4A CN105441499A (en) | 2015-12-04 | 2015-12-04 | Method for extracting gamma-polyglutamic acid from fermentation liquor |
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Citations (3)
| 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 |
| CN102154395A (en) * | 2010-12-30 | 2011-08-17 | 天津北洋百川生物技术有限公司 | Method for extracting gamma-polyglutamic acid by inorganic salt/organic solvent coprecipitation effect |
| CN102952266A (en) * | 2012-10-18 | 2013-03-06 | 山东鲁北药业有限公司 | Separation and purification method of gamma-polyglutamic acid |
-
2015
- 2015-12-04 CN CN201510884983.4A patent/CN105441499A/en active Pending
Patent Citations (3)
| 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 |
| CN102154395A (en) * | 2010-12-30 | 2011-08-17 | 天津北洋百川生物技术有限公司 | Method for extracting gamma-polyglutamic acid by inorganic salt/organic solvent coprecipitation effect |
| CN102952266A (en) * | 2012-10-18 | 2013-03-06 | 山东鲁北药业有限公司 | Separation and purification method of gamma-polyglutamic acid |
Non-Patent Citations (4)
| Title |
|---|
| GOJA 等: "Aqueous Two-phase extraction advances for bioseparation", 《BIOPROCESSING & BIOTECHNIQUES》 * |
| 王雷 等: "采用双水相系统从发酵液中提取聚谷氨酸", 《食品研究与开发》 * |
| 金谷 编著: "《表面活性剂化学 第2版》", 31 August 2013 * |
| 马霞等: "γ-聚谷氨酸提取条件的优化", 《食品工业科技》 * |
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Application publication date: 20160330 |