CN110511374A - A kind of extracting method of Gamma-polyglutamic acid from fermentation broth - Google Patents
A kind of extracting method of Gamma-polyglutamic acid from fermentation broth Download PDFInfo
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Abstract
A kind of presently disclosed extracting method of Gamma-polyglutamic acid from fermentation broth, by the gamma-polyglutamic acid in inverse micelle abstraction fermentation liquid, vacuum freeze drying obtains finished product gamma-polyglutamic acid.Operation of the present invention is simple, process is few, production cost is low, and recovery rate is high, good product quality.
Description
Technical field
The invention belongs to gamma-polyglutamic acid production technical fields, are related to a kind of extracting method of gamma-polyglutamic acid.
Background technique
Gamma-polyglutamic acid is a kind of extracellular polypeptide generated by a variety of bacillus, it is main group of certain micro-organisms pod membrane
Point, it is a kind of water-soluble and biodegradable polymer substance.It is to be connected by D- type or L-type glutamic acid by γ amido bond
It connects.Gamma-polyglutamic acid is found in nineteen thirty-seven earliest, and researcher is in the cell pod membrane of Bacillus anthracis and is saccharified
Gamma-polyglutamic acid is had found in the cell pod membrane of bacterium, and proves that it is one of the main component of certain micro-organisms pod membrane.Exist later
It has also been found that gamma-polyglutamic acid in bacillus subtilis and bacillus natto.Gamma-polyglutamic acid is by D-Glu (D-GLu) and L-
A kind of glutamic acid (L-GLu) monomer peptide molecule made of γ-carboxyl and alpha-amido are condensed in the form of peptide bond.The poly- paddy of γ-
There are a large amount of higher side chain carboxyl groups of activity on the strand of propylhomoserin, there is high moisture retention and water imbibition (1: 3500, m/
v).It is easy to combine with some drugs and generates stable compound, is a kind of ideal biodegradable pharmaceutical macromolecule material
Material.Gamma-polyglutamic acid no pollution to the environment is green bio product.
The application field of gamma-polyglutamic acid can be used as environment protection field such as life by preceding mentioned excellent physicochemical property extensively
Object flocculant, heavy metal absorbent, food industry such as thickener, agriculture field such as moisturizer, medical industry such as pharmaceutical carrier, doctor
With bioadhesive etc., it is considered that it is to the mankind and environment nonhazardous or even edible.Gamma-polyglutamic acid and its derivative
It is a kind of to have the multi-functional of very big Development volue and application prospect that object potential using value, which can greatly develop many specific uses,
New bio product.
Currently, the method extracted mainly has: organic solvent precipitation method, chemical precipitation method, the UF membrane precipitation method obtain γ-and gather
Glutamic acid.But there are high production cost, the technical problem of extraction process complexity for the method that the prior art is extracted.
Summary of the invention
The technical problem to be solved by the present invention is to overcome and extract gamma-polyglutamic acid high production cost in the prior art, mention
The technical problem of taking technique complexity provides the gamma-polyglutamic acid extracting method that a kind of extraction process is simple, production cost is low.
In order to solve the above technical problem, the present invention provides a kind of extracting methods of Gamma-polyglutamic acid from fermentation broth, special
Sign is, comprising the following steps:
Step 1: the preparation of fermentation liquid:
(a) bacillus subtilis is inoculated in slant medium, 37 DEG C of culture 12h in constant incubator, as work
Change strain；
(b) one ring of strain picking after (a) activation is inoculated in seed culture medium, shaking speed 220r/min, 37
DEG C shaken cultivation 15h is as seed liquor；
(c) (b) resulting seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed is
220r/min, 37 DEG C of shaken cultivations obtain the fermentation liquid containing product for 24 hours；
Step 2: fermentation liquor pretreatment:
The fermentation liquid that step 1 obtains is centrifuged 20min in 5000r/min to remove thallus, takes supernatant；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 100~150mmol/L cetyl trimethylammonium bromide
(CTAB)/80% (v/v) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: contain 0.5mol/L
The acetate buffer solution that the p H of KBr is 4.2；
(b) supernatant pre-processes: 0.1mol/L is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained
NaCl adjusts pH to 6~8 with the dilute hydrochloric acid of 6mol/L；
(c) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time be 5~
15min；Then stripping workshop is added, 5~15min of mixing is obtained after mutually separating (25 DEG C) containing gamma-polyglutamic acid
Water phase；
Step 4: dialysing to the water phase that step 3 obtains, the concentrate that dialysis is obtained carries out vacuum freeze drying, obtains
To gamma-polyglutamic acid crude product；
Step 5: the gamma-polyglutamic acid crude product that step 4 obtains is dissolved in distilled water, is dialysed again, concentrate into
Row vacuum freeze drying obtains gamma-polyglutamic acid sterling.
Preferably, bacillus subtilis is Bacillus subtilis GIM1.286 in the step 1.
Preferably, the ingredient of slant medium includes: tryptone 10g/L, yeast extract 5g/L in the step 1,
NaCl 10g/L, agar 15g/L, pH 7.5；Sterilising conditions: 121 DEG C, 20min.
Preferably, seed culture based component includes: sucrose 30g/L, beef extract 10g/L, sodium glutamate in the step 1
30g/L, MgSO4·7H2O 0.25g/L、K2HPO4·3H2O 0.5g/L, pH 7.5；Sterilising conditions: 121 DEG C, 20min.
Preferably, liquid fermentation medium ingredient includes: sucrose 30g/L, beef extract 8g/L, glutamic acid in the step 1
Sodium 30g/L, MgSO4·7H2O 0.25g/L、K2HPO4·3H2O 0.5g/L, pH 7.5；Sterilising conditions: 121 DEG C, 20min.
Preferably, the step 4 and the condition dialysed in step 5 are equal are as follows: dialyse 48h in distilled water, changes one every 4h
Secondary distilled water.
Compared with prior art, the invention has the benefit that
(1) traditional organic solvent precipitation method extracts gamma-polyglutamic acid, and dosage is big, increases cost, present invention determine that
A kind of higher extracting method of recovery rate, organic solvent is recyclable, reduces the pollution to environment.
(2) it is freeze-dried or is dried in vacuo, ensure that product quality.
(3) present invention determine that inverse micellar solution preparation process condition.
(4) concentration can effectively reduce drying cost in industrialized production.
Detailed description of the invention
Fig. 1 is the paper chromatography experimental result picture of gamma-polyglutamic acid prepared by the embodiment of the present invention 1.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
Gamma-polyglutamic acid content in supernatant liquid is detected in various embodiments of the present invention: using CTAB turbidimetry, measurement
Absorbance under 250nm, the specific steps are as follows:
(a) gamma-polyglutamic acid standard items 0.1g accurately is weighed, is placed in 100mL volumetric flask, is dissolved and determined with distilled water
Hold, shake up, obtain the gamma-polyglutamic acid titer of 1g/m L, in the volumetric flask for taking solution 10mL to another 100mL, uses
Distilled water is settled to 100mL, shakes up, and obtains the gamma-polyglutamic acid titer of 100 μ g/m L, prepares two parts in aforementioned manners
The gamma-polyglutamic acid titer of the 100 μ g/mL of 100mL.Precision measures 8,16,24,32,40, the above-mentioned solution of 48mL, sets respectively
In 1~No. 6 volumetric flask, 100mL is settled to distilled water, is shaken up, obtains the gamma-polyglutamic acid titer of 8~48 μ g/mL；
(b) 2% NaOH solution is prepared, and as solvent, CTAB is added, (can suitably heat up and add after CTAB dissolution
Instant solution) it is configured to the CTAB test solution of 25g/L；
(c) titer or sample liquid be in test tube, accurate that 2mL CTAB test solution is added, timing, sufficiently vibration from when being added
It swings, reaction solution is avoided to generate foam as far as possible therebetween, stand to close to 3min, then pour into reaction solution in cuvette, when 3min
Measure absorbance (A of the wavelength at 250nm250), using nonvaccinated culture medium as blank after corresponding position takes 2mL to manage.
The present invention respectively implement in the consummate product of gamma-polyglutamic acid identification method using thin-layer chromatography test, used in
Reagent: (precise 0.0400g ninhydrin, is dissolved in 20mL to hydrochloric acid, 0.5mol/L glutamic acid titer, the color developing agent of 6mol/L
In acetone), developing agent (n-butanol 15mL, formic acid 3mL, distilled water 2mL), glutamic acid sample (120 DEG C of gamma-polyglutamic acid hydrolysis
2h)
Reagent used in various embodiments of the present invention is as follows:
Other reagents are analytical reagents.
Instrument used in various embodiments of the present invention is as follows:
Bacillus subtilis used in the present invention can be bought by commercial sources and be obtained.
Embodiment 1
Present embodiments provide a kind of extracting method of Gamma-polyglutamic acid from fermentation broth, the specific steps are as follows:
Step 1: the preparation of fermentation liquid:
(a) strain selects: selection bacillus subtilis (Bacillus subtilis GIM1.286) is inoculated in inclined-plane training
It supports in base, the constant temperature incubation 12h at 37 DEG C, as activated spawn；Wherein, slant medium (g/L) ingredient is as follows: tryptone
10, yeast extract 5, NaCl 10, agar 15, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(b) one ring of activated spawn picking is inoculated in seed culture medium, shaking speed 220r/min, 37 DEG C of oscillation trainings
15h is supported as seed liquor；Wherein, seed culture medium (g/L) ingredient is as follows: sucrose 30, beef extract 10, sodium glutamate 30,
MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(c) seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed 220r/min,
37 DEG C obtain the fermentation liquid containing product in shaken cultivation 24 hours；Wherein, liquid fermentation medium (g/L) ingredient is as follows: sucrose
30, beef extract 8, sodium glutamate 30, MgSO4·7H2O 0.25、 K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C,
20min；
Step 2: the fermentation liquid that step 1 obtains being centrifuged 20min in 5000r/min to remove thallus, supernatant is taken, detects
It obtains, gamma-polyglutamic acid content is 12.04g/L in supernatant liquid；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 100mmol/L cetyl trimethylammonium bromide (CTAB)/
80% (v/v) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: the p H of the KBr containing 0.5mol/L
For 4.2 acetate buffer solution；
(b) supernatant pre-processes: 0.1 mol/L is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained
NaCl adjusts pH to 6 with the dilute hydrochloric acid of 6mol/L；
(c) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time is
5min；Then stripping workshop is added, mixing 10min obtains the water phase containing gamma-polyglutamic acid after mutually separating (25 DEG C)；
Step 4: the water phase that step 3 is obtained is placed in 7000Dal bag filter the 48h that dialyses in distilled water, and every 4h changes one
The concentrate of dialysis is dried in vacuo to obtain gamma-polyglutamic acid crude product by secondary distilled water；
Step 5: step 4 is obtained into gamma-polyglutamic acid crude product and is dissolved in distilled water, then acquired solution is put into bag filter,
It is dialysed repeatedly 48h with distilled water, removes small molecule and organic solvent, concentrate carries out vacuum freeze drying, obtains γ-polyglutamic
Sour sterling, yield 9.44g/L, recovery rate 78.40%.With under the conditions of, extracting Product yields with three times ethyl alcohol is 7.73g/
L, recovery rate improve 14.20%.
Paper chromatography experiment (identification gamma-polyglutamic acid):
Rf value (Rf) it is used to indicate that it is sample in two-phase by the moving distance after isolated substance expansion on thin layer
The relative movement speed of solute and solvent caused by middle absorption and parsing degree difference, may be expressed as: Rf=a/b
A-origin to component spot centers distance
B-origin to solvent front distance
As shown in Figure 1, left point is sample in figure, right point is 0.5% standard glutamic acid；
Calculate Rf value: Rf (mark product)=a/b=2/8.3=0.241；
Rf (sample)=a/b=2/8.3=0.241；
The Rf value of two kinds of different aminoacids at least differs 0.05, is calculated according to top as it can be seen that the ratio of sample and mark product moves
It is worth identical, therefore is considered as same amino acid, and as seen from the figure, do not occur variegated, therefore the gamma-polyglutamic acid after dialysis purification is pure
Product.
Embodiment 2
Present embodiments provide a kind of extracting method of Gamma-polyglutamic acid from fermentation broth, the specific steps are as follows:
Step 1: the preparation of fermentation liquid:
(a) strain selects: selection bacillus subtilis (Bacillus subtilis GIM1.286) is inoculated in inclined-plane training
It supports in base, the constant temperature incubation 12h at 37 DEG C, as activated spawn；Wherein, slant medium (g/L) ingredient is as follows: tryptone
10, yeast extract 5, NaCl 10, agar 15, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(b) one ring of activated spawn picking is inoculated in seed culture medium, shaking speed 220r/min, 37 DEG C of oscillation trainings
15h is supported as seed liquor；Wherein, seed culture medium (g/L) ingredient is as follows: sucrose 30, beef extract 10, sodium glutamate 30,
MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(c) seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed 220r/min,
37 DEG C of shaken cultivations obtain the fermentation liquid containing product for 24 hours；Wherein, liquid fermentation medium (g/L) ingredient is as follows: sucrose 30,
Beef extract 8, sodium glutamate 30, MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C,
20min；
Step 2: the fermentation liquid that step 1 obtains being centrifuged 20min in 5000r/min to remove thallus, supernatant is taken, detects
It obtains, gamma-polyglutamic acid content is 14.55g/L in supernatant liquid；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 120mmol/L cetyl trimethylammonium bromide (CTAB)/
80% (v/v) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: the p H of the KBr containing 0.5mol/L
For 4.2 acetate buffer solution；
(d) supernatant pre-processes: 0.1 mol/L is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained
NaCl adjusts pH to 8 with the dilute hydrochloric acid of 6mol/L；
(b) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time is
5min；Then stripping workshop is added, mixing 15min obtains the water phase containing gamma-polyglutamic acid after mutually separating (25 DEG C)；
Step 4: the water phase that step 3 is obtained is placed in 7000Dal bag filter the 48h that dialyses in distilled water, and every 4h changes one
Secondary distilled water is dried in vacuo the concentrate of dialysis led to obtain gamma-polyglutamic acid crude product；
Step 5: step 4 is obtained into gamma-polyglutamic acid crude product and is dissolved in distilled water, then acquired solution is put into bag filter,
It is dialysed repeatedly 48h with distilled water, removes small molecule and organic solvent, concentrate carries out vacuum freeze drying, obtains γ-polyglutamic
Sour sterling, yield 12.68g/L, recovery rate 87.15%.With under the conditions of, extracting Product yields with three times ethyl alcohol is
8.92g/L, recovery rate 61.31%, recovery rate improves 25.84%.
Embodiment 3
Present embodiments provide a kind of extracting method of Gamma-polyglutamic acid from fermentation broth, the specific steps are as follows:
Step 1: the preparation of fermentation liquid:
(a) strain selects: selection bacillus subtilis (Bacillus subtilis GIM1.286) is inoculated in inclined-plane training
It supports in base, the constant temperature incubation 12h at 37 DEG C, as activated spawn；Wherein, slant medium (g/L) ingredient is as follows: tryptone
10, yeast extract 5, NaCl 10, agar 15, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(b) one ring of activated spawn picking is inoculated in seed culture medium, shaking speed 220r/min, 37 DEG C of oscillation trainings
15h is supported as seed liquor；Wherein, seed culture medium (g/L) ingredient is as follows: sucrose 30, beef extract 10, sodium glutamate 30,
MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(c) seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed 220r/min,
37 DEG C of shaken cultivations obtain the fermentation liquid containing product for 24 hours；Wherein, liquid fermentation medium (g/L) ingredient is as follows: sucrose 30,
Beef extract 8, sodium glutamate 30, MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C,
20min；
Step 2: the fermentation liquid that step 1 obtains being centrifuged 20min in 5000r/min to remove thallus, supernatant is taken, detects
It obtains, gamma-polyglutamic acid content is 11.03g/L in supernatant liquid；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 130mmol/L cetyl trimethylammonium bromide (CTAB)/
80% (v/v) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: the p H of the KBr containing 0.5mol/L
For 4.2 acetate buffer solution；
(b) supernatant pre-processes: 0.1 mol/L is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained
NaCl adjusts pH to 7 with the dilute hydrochloric acid of 6mol/L；
(c) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time is
5min；Then stripping workshop is added, mixing 10min obtains the water phase containing gamma-polyglutamic acid after mutually separating (25 DEG C)；
Step 4: the water phase that step 3 is obtained is placed in 7000Dal bag filter the 48h that dialyses in distilled water, and every 4h changes one
Secondary distilled water is dried in vacuo the concentrate of dialysis led to obtain gamma-polyglutamic acid crude product；
Step 5: step 4 is obtained into gamma-polyglutamic acid crude product and is dissolved in distilled water, then acquired solution is put into bag filter,
It is dialysed repeatedly 48h with distilled water, removes small molecule and organic solvent, concentrate carries out vacuum freeze drying, obtains γ-polyglutamic
Sour sterling, yield 9.08g/L, recovery rate 82.32%.With under the conditions of, extracting Product yields with three times ethyl alcohol is
8.12g/L, recovery rate 73.62%, recovery rate improves 8.7%.
Embodiment 4
Present embodiments provide a kind of extracting method of Gamma-polyglutamic acid from fermentation broth, the specific steps are as follows:
Step 1: the preparation of fermentation liquid:
(a) strain selects: selection bacillus subtilis (Bacillus subtilis GIM1.286) is inoculated in inclined-plane training
It supports in base, the constant temperature incubation 12h at 37 DEG C, as activated spawn；Wherein, slant medium (g/L) ingredient is as follows: tryptone
10, yeast extract 5, NaCl 10, agar 15, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(b) one ring of activated spawn picking is inoculated in seed culture medium, shaking speed 220r/min, 37 DEG C of oscillation trainings
15h is supported as seed liquor；Wherein, seed culture medium (g/L) ingredient is as follows: sucrose 30, beef extract 10, sodium glutamate 30,
MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C, 20min；
(c) seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed 220r/min,
37 DEG C of shaken cultivations obtain the fermentation liquid containing product for 24 hours；Wherein, liquid fermentation medium (g/L) ingredient is as follows: sucrose 30,
Beef extract 8, sodium glutamate 30, MgSO4·7H2O 0.25、K2HPO4·3H2O 0.5, pH 7.5；Sterilising conditions: 121 DEG C,
20min；
Step 2: the fermentation liquid that step 1 obtains being centrifuged 20min in 5000r/min to remove thallus, supernatant is taken, detects
It obtains, gamma-polyglutamic acid content is 13.27g/L in supernatant liquid；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 140mmol/L cetyl trimethylammonium bromide (CTAB)/
80% (v/v) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: the p H of the KBr containing 0.5mol/L
For 4.2 acetate buffer solution；
(b) supernatant pre-processes: 0.1 mol/L is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained
NaCl adjusts pH to 6 with the dilute hydrochloric acid of 6mol/L；
(b) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time is
5min；Then stripping workshop is added, mixing 10min obtains the water phase containing gamma-polyglutamic acid after mutually separating (25 DEG C)；
Step 4: the water phase that step 3 is obtained is placed in 7000Dal bag filter the 48h that dialyses in distilled water, and every 4h changes one
Secondary distilled water is dried in vacuo the concentrate of dialysis led to obtain gamma-polyglutamic acid crude product；
Step 5: step 4 is obtained into gamma-polyglutamic acid crude product and is dissolved in distilled water, then acquired solution is put into bag filter,
It is dialysed repeatedly 48h with distilled water, removes small molecule and organic solvent, concentrate carries out vacuum freeze drying, obtains γ-polyglutamic
Sour sterling, yield 9.88g/L, recovery rate 74.45%.With under the conditions of, extracting Product yields with three times ethyl alcohol is
7.91g/L, recovery rate 59.61%, recovery rate improves 14.84%.
Claims (6)
1. a kind of extracting method of Gamma-polyglutamic acid from fermentation broth, which comprises the following steps:
Step 1: the preparation of fermentation liquid:
(b) bacillus subtilis is inoculated in slant medium, 37 DEG C of culture 12h in constant incubator, as activation bacterium
Kind；
(b) one ring of strain picking after (a) activation is inoculated in seed culture medium, shaking speed 220r/min, 37 DEG C of vibrations
Culture 15h is swung as seed liquor；
(c) (b) resulting seed liquor is inoculated into liquid fermentation medium by the inoculum concentration of 5wt%, shaking speed 220r/
Min, 37 DEG C of shaken cultivations obtain the fermentation liquid containing product for 24 hours；
Step 2: fermentation liquor pretreatment:
The fermentation liquid that step 1 obtains is centrifuged 20min in 5000r/min to remove thallus, takes supernatant；
Step 3: inverse micelle abstraction:
(a) preparation of inverse micellar solution: forward extraction solution are as follows: 100~150mmol/L cetyl trimethylammonium bromide/80% (v/
V) isooctane/5% (v/v) n-hexyl alcohol/15% (v/v) n-butanol；Stripping workshop are as follows: the vinegar that the pH of 0.5mol/L KBr is 4.2
Acid buffer；
(b) supernatant pre-processes: 0.1mol/L NaCl is added in the supernatant containing gamma-polyglutamic acid that step 2 is obtained, and uses
Dilute HCl of 6mol/L adjusts pH to 6~8；
(c) inverse micelle abstraction: isometric forward extraction solution is mixed with pretreated supernatant, incorporation time be 5~
15min；Then stripping workshop is added, 5~15min of mixing obtains the water phase containing gamma-polyglutamic acid after mutually separating；
Step 4: dialysing to the water phase that step 3 obtains, the concentrate that dialysis is obtained carries out vacuum freeze drying, obtains
Gamma-polyglutamic acid crude product；
Step 5: the gamma-polyglutamic acid crude product that step 4 obtains being dissolved in distilled water, is dialysed again, concentrate carries out true
Vacuum freecing-dry obtains gamma-polyglutamic acid sterling.
2. the extracting method of Gamma-polyglutamic acid from fermentation broth as described in claim 1, which is characterized in that withered in the step 1
Careless bacillus is Bacillus subtilis GIM1.286.
3. the extracting method of Gamma-polyglutamic acid from fermentation broth as described in claim 1, which is characterized in that in the step 1 tiltedly
The ingredient of face culture medium includes: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, agar 15g/L, pH 7.5；
Sterilising conditions: 121 DEG C, 20min.
4. the extracting method of Gamma-polyglutamic acid from fermentation broth as described in claim 1, which is characterized in that planted in the step 1
Sub- medium component includes: sucrose 30g/L, beef extract 10g/L, sodium glutamate 30g/L, MgSO4·7H2O 0.25g/L、
K2HPO4·3H2O 0.5g/L, pH 7.5；Sterilising conditions: 121 DEG C, 20min.
5. the extracting method of Gamma-polyglutamic acid from fermentation broth as described in claim 1, which is characterized in that liquid in the step 1
Body fermentation medium components include: sucrose 30g/L, beef extract 8g/L, sodium glutamate 30g/L, MgSO4·7H2O 0.25g/L、
K2HPO4·3H2O 0.5g/L, pH 7.5；Sterilising conditions: 121 DEG C, 20min.
6. the extracting method of Gamma-polyglutamic acid from fermentation broth as described in claim 1, which is characterized in that the step 4 and step
The condition dialysed in rapid 5 is equal are as follows: dialyse 48h in distilled water, changes primary distilled water every 4h.
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| CN111592670A (en) * | 2020-06-24 | 2020-08-28 | 上海应用技术大学 | Preparation method of gamma-polyglutamic acid molecularly imprinted polymer membrane |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100196482A1 (en) * | 2007-04-04 | 2010-08-05 | Massachusetts Institute Of Technology | Polymer-encapsulated reverse micelles |
| CN101864073A (en) * | 2010-05-25 | 2010-10-20 | 上海应用技术学院 | Method for extracting Gamma-polyglutamic acid from fermentation broth |
| CN103172542A (en) * | 2013-03-26 | 2013-06-26 | 山东轻工业学院 | Method for separating and purifying L-citrulline |
| CN107760657A (en) * | 2017-09-07 | 2018-03-06 | 贵州大学 | The selective extracellular lipase culture mediums of sn 2 and its application method |
-
2019
- 2019-09-17 CN CN201910875651.8A patent/CN110511374A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100196482A1 (en) * | 2007-04-04 | 2010-08-05 | Massachusetts Institute Of Technology | Polymer-encapsulated reverse micelles |
| CN101864073A (en) * | 2010-05-25 | 2010-10-20 | 上海应用技术学院 | Method for extracting Gamma-polyglutamic acid from fermentation broth |
| CN103172542A (en) * | 2013-03-26 | 2013-06-26 | 山东轻工业学院 | Method for separating and purifying L-citrulline |
| CN107760657A (en) * | 2017-09-07 | 2018-03-06 | 贵州大学 | The selective extracellular lipase culture mediums of sn 2 and its application method |
Non-Patent Citations (3)
| Title |
|---|
| W.WANG ET AL: "Effect of the concentration of DODMAC and 1-decanol on the behavior of reverse micelles in the extraction of amino acids", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 杨继生: "《表面活性剂原理与应用》", 31 December 2012, 东南大学出版社 * |
| 鞠蕾 等: "γ-聚谷氨酸提取条件的优化", 《食品工业科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111592670A (en) * | 2020-06-24 | 2020-08-28 | 上海应用技术大学 | Preparation method of gamma-polyglutamic acid molecularly imprinted polymer membrane |
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