CN114672016B - Extraction process of degradable material - Google Patents
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- CN114672016B CN114672016B CN202111652288.7A CN202111652288A CN114672016B CN 114672016 B CN114672016 B CN 114672016B CN 202111652288 A CN202111652288 A CN 202111652288A CN 114672016 B CN114672016 B CN 114672016B
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- 238000000605 extraction Methods 0.000 title claims abstract description 37
- 239000000463 material Substances 0.000 title claims abstract description 18
- 239000002244 precipitate Substances 0.000 claims abstract description 64
- 238000000855 fermentation Methods 0.000 claims abstract description 57
- 230000004151 fermentation Effects 0.000 claims abstract description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 43
- 238000003756 stirring Methods 0.000 claims abstract description 28
- 229920002643 polyglutamic acid Polymers 0.000 claims abstract description 27
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 238000009777 vacuum freeze-drying Methods 0.000 claims abstract description 22
- 239000000047 product Substances 0.000 claims abstract description 19
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- 238000005406 washing Methods 0.000 claims abstract description 14
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims abstract description 12
- 229930195725 Mannitol Natural products 0.000 claims abstract description 12
- 238000011033 desalting Methods 0.000 claims abstract description 12
- 235000010355 mannitol Nutrition 0.000 claims abstract description 12
- 239000000594 mannitol Substances 0.000 claims abstract description 12
- 238000009210 therapy by ultrasound Methods 0.000 claims abstract description 9
- 238000007865 diluting Methods 0.000 claims abstract description 8
- 238000001556 precipitation Methods 0.000 claims abstract description 7
- 239000011780 sodium chloride Substances 0.000 claims abstract description 7
- 238000000502 dialysis Methods 0.000 claims description 25
- 238000004108 freeze drying Methods 0.000 claims description 12
- 239000002504 physiological saline solution Substances 0.000 claims description 12
- 235000019441 ethanol Nutrition 0.000 claims description 11
- 238000010790 dilution Methods 0.000 claims description 9
- 239000012895 dilution Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 4
- 238000000034 method Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108010020346 Polyglutamic Acid Proteins 0.000 description 5
- 244000063299 Bacillus subtilis Species 0.000 description 4
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000001291 vacuum drying Methods 0.000 description 4
- 235000014469 Bacillus subtilis Nutrition 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 241000193738 Bacillus anthracis Species 0.000 description 2
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 2
- 229930182847 D-glutamic acid Natural products 0.000 description 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 2
- 229940065181 bacillus anthracis Drugs 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 229960002989 glutamic acid Drugs 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- -1 salt ions Chemical class 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
-
- 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 discloses an extraction process of a degradable material, which comprises the following steps: step 1: pretreatment of fermentation liquor; diluting the fermentation liquor, carrying out ultrasonic treatment on the absorbed fermentation liquor, centrifuging the fermentation liquor, and taking supernatant; step 2: extracting and separating fermentation liquor; respectively extracting the supernatant with absolute ethanol and mannitol for the second time; step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times; step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight; step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and performing vacuum freeze-drying on the precipitate to obtain the pure gamma-polyglutamic acid product.
Description
Technical Field
The invention relates to the technical field of gamma-polyglutamic acid production, in particular to an extraction process of a degradable material.
Background
Gamma-polyglutamic acid (gamma-PGA) was first found in 1937 in Bacillus anthracis (Bacillus anthracis) and in Bacillus subtilis (Bacillus mesentericus), and later in Bacillus subtilis and Bacillus natto. The gamma-polyglutamic acid is a polypeptide molecule formed by condensing D-glutamic acid (D-GLu) and L-glutamic acid (L-GLu) monomers in a peptide bond form by gamma-carboxyl and alpha-amino, has a large number of side chain carboxyl groups with higher activity on a molecular chain, has extremely high moisture retention and water absorption, is easy to combine with some macromolecular substances to generate stable complexes, and is an ideal biodegradable macromolecular material. The gamma-polyglutamic acid has no pollution to the environment, is a green biological product, has a plurality of unique physicochemical and biological characteristics such as excellent biodegradability, film forming property, moisture retention property and the like, is favored by people today focusing on environmental protection and emphasizing sustainable development, is gradually applied to a plurality of fields such as medicine manufacturing, food processing, antifreezing of vegetables, fruits and marine products and the like, and is a multifunctional novel biological product with great development value and wide prospect.
In the production process of gamma-polyglutamic acid, the fermentation liquor obtained by the liquid fermentation method has great viscosity, so that great difficulty is brought to downstream separation and purification, and the ideal effect cannot be obtained by the common separation and extraction method, so that the improvement of the separation efficiency of gamma-polyglutamic acid is another technical key of the fermentation production of the material.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides an extraction process of a degradable material so as to improve the separation efficiency of gamma-polyglutamic acid.
The invention is realized by the following technical scheme:
an extraction process of degradable materials comprises the following steps:
step 1: pretreatment of fermentation liquor; diluting the fermentation liquor, carrying out ultrasonic treatment on the absorbed fermentation liquor, centrifuging the fermentation liquor, and taking supernatant;
step 2: extracting and separating fermentation liquor; respectively extracting the supernatant with absolute ethanol and mannitol for the second time;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times;
step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and performing vacuum freeze-drying on the precipitate to obtain the pure gamma-polyglutamic acid product.
Preferably, the step 1 specifically includes the following operations: firstly diluting 15ml of fermentation liquor with physiological saline, wherein the volume ratio of the fermentation liquor to the physiological saline is 1:1, then carrying out ultrasonic treatment on the fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃, the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant.
Preferably, the step 2 specifically includes the following operations:
(1) Primary extraction: adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
(2) And (3) secondary extraction: adding 45ml mannitol into the precipitate obtained by one extraction, stirring for 30 min at 400r/min, centrifuging, and collecting precipitate.
Preferably, the dialysis desalting in the step 5 comprises the following operations of adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then performing vacuum freeze-drying on a sample of the dialysis bag to obtain the gamma-polyglutamic acid pure product.
Preferably, the sample of the dialysis bag is placed in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for precooling 12h, the pre-frozen freeze-dried bottle is placed in a material tray, an organic glass cover is covered, a vacuum pump is started for vacuum freeze drying, the air pressure is kept at 1pa until the freeze drying is finished, and the freeze drying time is 12 hours.
The beneficial effects of the invention are as follows:
(1) The invention firstly reduces the viscosity of the fermentation liquor by adopting an ultrasonic and normal saline dilution method, and introduces a large amount of Na by utilizing the normal saline for dilution + The high concentration of salt ions suppresses repulsive force between carboxyl groups, so that the viscosity of the gamma-PGA fermentation broth is greatly reduced, and the extraction rate of polyglutamic acid can be greatly improved.
(2) The method for extracting the mannitol and the ethanol for the first time adopts the method for extracting the mannitol for the second time, and the ethanol can be used for extracting a part of impurity molecules to a large extent, and the mannitol is purified for the second time, so that the purity of the polyglutamic acid product is ensured.
(3) In the drying mode, firstly, the dialysis bag sample is precooled, so that the sample is completely frozen and small ice crystals are formed, the time required by freeze drying is reduced, and compared with vacuum drying, the gamma-PGA yield is higher, and the properties and quality of the finally obtained finished product are better and better.
Drawings
FIG. 1 is a graph showing comparison of results of the extraction yield of gamma-PGA according to various embodiments.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following description will further describe the technical scheme of the present invention in connection with the preferred embodiments of the present invention, but the present invention is not limited to the embodiments.
Example 1
An extraction process of degradable materials comprises the following steps:
step 1: pretreatment of fermentation liquor; firstly diluting 15ml of fermentation liquor with physiological saline, wherein the volume ratio of the fermentation liquor to the physiological saline is 1:1, then carrying out ultrasonic treatment on the fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃, the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant.
Step 2: extracting and separating fermentation liquor;
(1) Primary extraction: adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
(2) And (3) secondary extraction: adding 45ml of mannitol into the precipitate obtained by the primary extraction, stirring for 30 minutes at a stirring speed of 400r/min, and centrifugally separating to obtain the precipitate;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times.
Step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and performing vacuum freeze-drying on the precipitate to obtain the pure gamma-polyglutamic acid product.
The dialysis desalting in the step 5 comprises the following operations of adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then carrying out vacuum freeze-drying on a sample of the dialysis bag to obtain the gamma-polyglutamic acid pure product.
The vacuum freeze-drying operation is as follows: and (3) pre-cooling the sample of the dialysis bag in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for 12h, putting the pre-frozen freeze-dried bottle in a material tray, covering an organic glass cover, starting a vacuum pump for vacuum freeze-drying, and keeping the air pressure of 1pa until the freeze-drying is finished, wherein the freeze-drying time is 12h.
Example 2
An extraction process of degradable materials comprises the following steps:
step 1: pretreatment of fermentation liquor; firstly, diluting 15ml of fermentation liquor with distilled water, wherein the volume ratio of the fermentation liquor to the distilled water is 1:1, then performing ultrasonic treatment on the fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃, the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant.
Step 2: extracting and separating fermentation liquor;
(1) Primary extraction: adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
(2) And (3) secondary extraction: adding 45ml of mannitol into the precipitate obtained by the primary extraction, stirring for 30 minutes at a stirring speed of 400r/min, and centrifugally separating to obtain the precipitate;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times.
Step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and performing vacuum freeze-drying on the precipitate to obtain the pure gamma-polyglutamic acid product.
The dialysis desalting in the step 5 comprises the following operations of adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then carrying out vacuum freeze-drying on a sample of the dialysis bag to obtain the gamma-polyglutamic acid pure product.
The vacuum freeze-drying operation is as follows: and (3) pre-cooling the sample of the dialysis bag in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for 12h, putting the pre-frozen freeze-dried bottle in a material tray, covering an organic glass cover, starting a vacuum pump for vacuum freeze-drying, and keeping the air pressure of 1pa until the freeze-drying is finished, wherein the freeze-drying time is 12h.
Example 1 differs from example 2 in that distilled water was used for dilution.
Example 3
An extraction process of degradable materials comprises the following steps:
step 1: pretreatment of fermentation liquor; firstly diluting 15ml of fermentation liquor with physiological saline, wherein the volume ratio of the fermentation liquor to the physiological saline is 1:1, then carrying out ultrasonic treatment on the fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃, the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant.
Step 2: extracting and separating fermentation liquor;
adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 with 80% ethanol for 2-3 times.
Step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and performing vacuum freeze-drying on the precipitate to obtain the pure gamma-polyglutamic acid product.
The dialysis desalting in the step 5 comprises the following operations of adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then carrying out vacuum freeze-drying on a sample of the dialysis bag to obtain the gamma-polyglutamic acid pure product.
The vacuum freeze-drying operation is as follows: and (3) pre-cooling the sample of the dialysis bag in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for 12h, putting the pre-frozen freeze-dried bottle in a material tray, covering an organic glass cover, starting a vacuum pump for vacuum freeze-drying, and keeping the air pressure of 1pa until the freeze-drying is finished, wherein the freeze-drying time is 12h.
Example 1 and example 3 differ in that only one extraction was performed using ethanol.
Example 4
An extraction process of degradable materials comprises the following steps:
step 1: pretreatment of fermentation liquor; firstly diluting 15ml of fermentation liquor with physiological saline, wherein the volume ratio of the fermentation liquor to the physiological saline is 1:1, then carrying out ultrasonic treatment on the fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃, the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant.
Step 2: extracting and separating fermentation liquor;
(1) Primary extraction: adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
(2) And (3) secondary extraction: adding 45ml of mannitol into the precipitate obtained by the primary extraction, stirring for 30 minutes at a stirring speed of 400r/min, and centrifugally separating to obtain the precipitate;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times.
Step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: and (3) centrifugally separating the precipitate in the step (4), collecting the precipitate, dialyzing for desalting, and vacuum drying the precipitate to obtain the gamma-polyglutamic acid pure product.
The dialysis desalting in the step 5 comprises the following operations of adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then carrying out vacuum freeze-drying on a sample of the dialysis bag to obtain the gamma-polyglutamic acid pure product.
Example 4 differs from example 1 in that example 4 was dried by vacuum drying.
The extraction rate of the gamma-PGA obtained by calculation after the experiment of each embodiment is finished is calculated as follows: because of the turbidity generated by the reaction of cetyltrimethylammonium bromide (CTAB) solution and gamma-PGA, the turbidity is reflected by the absorbance of the reaction system, and the extraction rate of gamma-PGA is calculated by the linear relation between the turbidity and the concentration of gamma-PGA, and the calculation result is shown in the following figure 1:
the results of examples 1 and 2 show that the extraction rate of polyglutamic acid can be improved by the ultrasonic and physiological saline dilution method, the mechanism is that the viscosity of the fermentation broth can be reduced by ultrasonic treatment, a large amount of Na+ is introduced by dilution with physiological saline, and the repulsive force between carboxyl groups is inhibited by salt ions with high concentration, so that the viscosity of the gamma-PGA fermentation broth is greatly reduced, and the extraction rate of polyglutamic acid is increased.
Compared with the embodiment 1 and the embodiment 3, the ethanol and mannitol secondary extraction method is adopted, and part of impurity molecules can be greatly extracted by adopting the ethanol, and the mannitol secondary purification ensures the purity of the polyglutamic acid product.
In example 1 and example 4, the dialysis bag sample is first pre-cooled in the drying mode of vacuum freeze drying, so that the sample is completely frozen and small ice crystals are formed, the time required for freeze drying is reduced, and compared with vacuum drying, the gamma-PGA extraction rate is higher, and the properties and quality of the finally obtained finished product are better and better.
Claims (1)
1. An extraction process of a degradable material is characterized in that: the method comprises the following steps:
step 1: pretreatment of fermentation liquor: firstly diluting 15ml of gamma-polyglutamic acid fermentation liquor with physiological saline, wherein the volume ratio of the fermentation liquor to the physiological saline is 1:1, then carrying out ultrasonic treatment on the gamma-polyglutamic acid fermentation liquor under 40-70W power, wherein the ultrasonic temperature is 50-70 ℃ and the ultrasonic time is 50-120 minutes, centrifuging the fermentation liquor after ultrasonic dilution, and taking supernatant;
step 2: extraction and separation of fermentation broth:
(1) Primary extraction: adding 80% absolute ethyl alcohol with the volume being 2 times that of the fermentation broth obtained in the step 1, stirring for 30 minutes at the stirring speed of 400r/min, and centrifugally separating to obtain a precipitate;
(2) And (3) secondary extraction: adding 45ml of mannitol into the precipitate obtained by the primary extraction, stirring for 30 minutes at a stirring speed of 400r/min, and centrifugally separating to obtain the precipitate;
step 3: washing the precipitate: washing the precipitate obtained in the step 2 by using 80% ethanol for 2-3 times;
step 4: adding 4% sodium chloride solution into the precipitate obtained in the step 3 according to the volume ratio of 1:1, controlling the pH value to be 8-9.5, stirring for 30 minutes at the rotating speed of 400r/min, and standing for precipitation overnight;
step 5: centrifuging the precipitate in the step 4, collecting the precipitate, dialyzing for desalting, and vacuum freeze-drying the precipitate to obtain pure gamma-polyglutamic acid;
adding 3 times of volume of ultrapure water into the precipitate for dissolution, completely dissolving by ultrasonic, centrifuging to obtain supernatant, adding the supernatant into a dialysis bag, dialyzing for 48 hours, and then carrying out vacuum freeze-drying on a sample of the dialysis bag to obtain a gamma-polyglutamic acid pure product;
the sample of the dialysis bag is subjected to vacuum freeze drying in the following operation mode: and (3) pre-cooling the sample of the dialysis bag in an ultralow temperature refrigerator at the temperature of minus 80 ℃ for 12h, putting the pre-frozen freeze-dried bottle in a material tray, covering an organic glass cover, starting a vacuum pump for vacuum freeze-drying, and keeping the air pressure at 1pa until the freeze-drying is finished, wherein the freeze-drying time is 12h.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908178A (en) * | 2005-08-06 | 2007-02-07 | 加拿大时代科技投资管理有限公司 | Method of low cost preparing gamma-polyglutamic acid utilizing farm and side line products solid fermentation |
| JP2007297559A (en) * | 2006-05-02 | 2007-11-15 | Rohto Pharmaceut Co Ltd | AMINO ACID-MODIFIED-(gamma-POLYGLUTAMIC ACID) OR ITS SALT AND THEIR USES |
| 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 |
-
2021
- 2021-12-31 CN CN202111652288.7A patent/CN114672016B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908178A (en) * | 2005-08-06 | 2007-02-07 | 加拿大时代科技投资管理有限公司 | Method of low cost preparing gamma-polyglutamic acid utilizing farm and side line products solid fermentation |
| JP2007297559A (en) * | 2006-05-02 | 2007-11-15 | Rohto Pharmaceut Co Ltd | AMINO ACID-MODIFIED-(gamma-POLYGLUTAMIC ACID) OR ITS SALT AND THEIR USES |
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 聚谷氨酸的生物合成及应用前景;刘晓鸥 等;《食品工程》(第1期);第23-26页 * |
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