CN114736368A - Method for extracting and purifying gamma-polyglutamic acid from fermentation liquor - Google Patents
Method for extracting and purifying gamma-polyglutamic acid from fermentation liquor Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 43
- 230000004151 fermentation Effects 0.000 title claims abstract description 43
- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 32
- 108010020346 Polyglutamic Acid Proteins 0.000 claims abstract description 28
- 239000003960 organic solvent Substances 0.000 claims abstract description 28
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000001035 drying Methods 0.000 claims abstract description 5
- 229910052799 carbon Inorganic materials 0.000 claims abstract 2
- 238000003756 stirring Methods 0.000 claims description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000002244 precipitate Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000001914 filtration Methods 0.000 claims description 15
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 10
- 238000005119 centrifugation Methods 0.000 claims description 10
- 239000012466 permeate Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 239000008187 granular material Substances 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 abstract description 12
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 230000002776 aggregation Effects 0.000 abstract description 3
- 238000000464 low-speed centrifugation Methods 0.000 abstract description 3
- 238000004220 aggregation Methods 0.000 abstract description 2
- 238000005054 agglomeration Methods 0.000 abstract 1
- 230000001376 precipitating effect Effects 0.000 abstract 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 13
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 12
- 239000008103 glucose Substances 0.000 description 12
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 9
- 239000012153 distilled water Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 229910052760 oxygen Inorganic materials 0.000 description 9
- 239000001301 oxygen Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 239000002245 particle Substances 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- 239000001888 Peptone Substances 0.000 description 6
- 108010080698 Peptones Proteins 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 239000001963 growth medium Substances 0.000 description 6
- 235000019319 peptone Nutrition 0.000 description 6
- WHUUTDBJXJRKMK-GSVOUGTGSA-N D-glutamic acid Chemical compound OC(=O)[C@H](N)CCC(O)=O WHUUTDBJXJRKMK-GSVOUGTGSA-N 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-Glutamic acid Natural products OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 4
- 229960002989 glutamic acid Drugs 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- 241001540526 Leptonotus elevatus Species 0.000 description 3
- 229930003756 Vitamin B7 Natural products 0.000 description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 3
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 235000019796 monopotassium phosphate Nutrition 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012807 shake-flask culturing Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001694 spray drying Methods 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- 235000011912 vitamin B7 Nutrition 0.000 description 3
- 239000011735 vitamin B7 Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical class [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000019800 disodium phosphate Nutrition 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 229910001385 heavy metal Inorganic materials 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000000643 oven drying Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 229930182847 D-glutamic acid Natural products 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000009295 crossflow filtration Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005189 flocculation Methods 0.000 description 1
- 230000016615 flocculation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 235000013557 nattō Nutrition 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 108700022290 poly(gamma-glutamic acid) Proteins 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000001502 supplementing effect Effects 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
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting and purifying gamma-polyglutamic acid from fermentation liquor. The method is an organic solvent precipitation method and mainly comprises the following steps: removing cells and foreign proteins, decoloring by active carbon, precipitating, dehydrating and drying polyglutamic acid. The method can remove cells from the high-viscosity polyglutamic acid fermentation broth in a low-speed centrifugation mode, simultaneously solve the problem of aggregation and agglomeration during organic solvent precipitation, and greatly reduce the dosage of the organic solvent.
Description
The technical field is as follows:
the invention belongs to the technical field of biological fermentation, and particularly relates to a method for extracting and purifying gamma-polyglutamic acid from fermentation liquor.
Background art:
gamma-polyglutamic acid (gamma-PGA for short) is a polypeptide molecule formed by combining L-glutamic acid and D-glutamic acid through a gamma-amide bond, and the molecular weight of the polypeptide molecule is generally between 100 and 2000 KDa. The application fields of the polymer are different according to the difference of molecular weight, such as soil modification, water flocculation, cosmetics, medicines and the like, and generally, the higher the molecular weight is, the higher the viscosity is. The high viscosity of polyglutamic acid makes the separation and purification extremely difficult.
The extraction and purification of the polyglutamic acid are mainly divided into two parts. First, solid-liquid separation removes cells from a high viscosity fermentation broth; secondly, the purity of the polyglutamic acid is improved by various purification methods, and the polyglutamic acid is dried to obtain an ideal solid form of the product, is not easy to absorb water and regain moisture during subpackaging and storage, and is convenient to transport.
First, the cell removal method reported in the prior art is either an acid-regulated centrifugation method, a dilution centrifugation method, or a filtration method. The direct acid adjustment centrifugation requires higher centrifugation rotating speed, and is difficult to realize in an industrial scale; dilution centrifugation needs to dilute the fermentation broth by more than 6 times to obviously reduce the viscosity, but due to the large increase of the volume, the subsequent ultrafiltration procedure has to be increased, and the high viscosity of polyglutamic acid causes poor liquid fluidity during ultrafiltration, thereby affecting the ultrafiltration concentration capability; the vertical filtration for removing cells leads to the great increase of the dosage of the diatomite due to the great cell content in the fermentation liquor, while the cross-flow filtration limits the application of the polyglutamic acid due to the great molecular weight of the polyglutamic acid, and is difficult to effectively separate the cells from the polyglutamic acid through a microfiltration membrane.
Secondly, the currently reported purification methods mainly include an organic solvent precipitation method, a heavy metal precipitation method, an alcohol-free purification method, and the like. The heavy metal precipitation method can adopt saturated copper sulfate for precipitation, and the usage amount of the copper sulfate is too large, and the environmental pollution is serious. The alcohol-free purification method is proposed in recent years, and only membrane filtration, diatomite filtration and other methods are adopted for purification, but the method can only remove macroscopic suspended matters and pigments, and the product purity is low; the sample has high viscosity and small particles, so that the filtering speed is low, and the filtering process is easy to block; the filter aid is frequently replaced, so that solid waste is seriously generated; finally, freeze drying or spray drying is needed to obtain solid powder, the energy consumption is high, and the powder is easy to absorb water and regain moisture. The organic solvent precipitation method mainly adopts methanol, ethanol or isopropanol for precipitation to remove impurities, but in the currently reported organic solvent precipitation method, the use amount of an organic solvent is huge and is often 3-4 times of the volume of fermentation liquor; the precipitate is aggregated into a mass, and is difficult to dry, while the spray drying has larger energy consumption, and the powder is easy to absorb water and regain moisture.
The invention content is as follows:
the invention aims to provide a method for extracting and purifying gamma-polyglutamic acid from fermentation liquor, which is simple to operate and easy to industrialize, aiming at the defects of the prior art.
The invention provides a method for extracting and purifying gamma-polyglutamic acid from fermentation liquor, which comprises the following steps:
1. removal of cells and foreign proteins
And (3) adjusting the pH value of the fermentation solution to 2.0-3.0 by using acid, adding a certain amount of organic solvent, uniformly stirring, and centrifuging at a low speed to collect supernatant.
2. Decolorizing with activated carbon
And taking the centrifugal supernatant, adding a certain amount of activated carbon, stirring for 0.5-1 h, filtering and collecting the permeate.
3. Precipitation of polyglutamic acid
And (3) adding a certain amount of sulfate into the filtered permeate, dissolving and uniformly stirring, reducing the temperature to 0-10 ℃, adjusting the pH to 5.0-7.5, supplementing a certain amount of organic solvent, continuously stirring for 10-30 min, centrifuging and collecting the precipitate.
4. Dewatering and drying
And (4) taking the precipitate collected by centrifugation, adding a certain amount of organic solvent, and stirring until the precipitate is white and small particles. Filtering and collecting white particles, and drying at the temperature of 35-60 ℃ to constant weight to obtain the polyglutamic acid.
Further, the acid in step 1 is selected from sulfuric acid, hydrochloric acid, preferably sulfuric acid.
Further, the organic solvent in step 1 is selected from methanol, ethanol or isopropanol, preferably ethanol.
Furthermore, the volume of the organic solvent in the step 1 is 0.5-1 time of the volume of the fermentation liquor.
Further, the centrifugation can adopt a table centrifuge, a tubular centrifuge or a disk centrifuge, the centrifugal force is more than 2500 Xg, and the centrifugation time is more than 3 min.
Further, the mass of the activated carbon added in the step 2 is 0.25-2% of the volume of the centrifugal supernatant, and preferably 0.5-0.75%.
Furthermore, the sulfate added in step 3 is selected from sodium sulfate, ammonium sulfate or magnesium sulfate, and the addition amount is 0.5-4% of the permeate, preferably 2-3%.
Furthermore, the organic solvent supplemented in the step 3 is consistent with the organic solvent used in the step 1, and the addition amount of the organic solvent is 0.2-0.5 times of the volume of the original fermentation liquor.
Furthermore, the temperature of the feed liquid is controlled to be 0-10 ℃ in the centrifugal process in the step 3.
Further, the organic solvent added in the step 4 is consistent with the organic solvent used in the step 1, and the mass ratio of the added volume of the solvent to the collected precipitate is 1: 1-3: 1.
furthermore, the organic solvent added in the step 4 needs to be pre-cooled to 0-10 ℃, and then added into the polyglutamic acid precipitate.
The method has the advantages that a proper amount of organic solvent is added under an acidic condition, the precipitation and aggregation of cells and a large amount of foreign proteins can be promoted under the condition that polyglutamic acid is not precipitated, the viscosity of feed liquid is reduced, solid-liquid separation can be realized under the low-speed centrifugation condition, and a clarified polyglutamic acid solution is obtained, so that the cells are easily removed, and a large amount of impurities such as proteins and nucleic acids are removed.
The polyglutamic acid solution is subjected to organic solvent precipitation at low temperature on the basis of adding a certain amount of sulfate. The precipitate is not easy to be bonded into a mass, the precipitate can be collected only by a low-speed centrifugation mode, and granular polyglutamic acid can be obtained by further dehydrating through a small amount of organic solvent. Multiple organic solvent dehydration is avoided, the organic solvent consumption is reduced, and the high-energy-consumption working procedures such as freeze drying or spray drying are also avoided.
Detailed Description
The technical content of the present invention is further described below with reference to specific examples for better understanding of the content of the present invention, but the scope of the present invention is not limited thereto.
Example 1
50 mu L of glycerol pipefish strain liquid (Bacillus subtilis, separated from natto food) is absorbed into 100mL of LB liquid culture medium, and shake culture is carried out on the shake flask culture medium for 24h at the temperature of 37 ℃ and the rpm of 160 in a constant temperature incubator as seed liquid.
The seed solution was inoculated into 5L fermentation medium (10L fermenter) under flame protection, and fermentation was carried out at 37 ℃. During the fermentation process, the dissolved oxygen is controlled to be more than 10% by adjusting the stirring speed, the ventilation quantity and the tank pressure, and the pH is controlled to be 7.00 +/-0.02 by using 25% ammonia water. After the dissolved oxygen level rises to more than 80% within 5min, the glucose solution is supplemented, so that the dissolved oxygen level is maintained at 10-20%. And (5) fermenting for 24 hours, stopping fermentation, and collecting fermentation liquor.
Taking 1L fermentation liquor, and using 6mol/L H2SO4Adjusting pH to 2.0, adding 0.8L anhydrous methanol under stirring, centrifuging at 2500 × g centrifugal force for 5min, and collecting supernatant. 4.5g of activated carbon was added thereto, the mixture was stirred for 30min, 10g of diatomaceous earth was added thereto, and 1.7L of the permeate was collected by filtration. Adding 68g of sodium sulfate, dissolving and stirring uniformly, cooling to 10 ℃, adjusting the pH to 5.0, adding 0.2L of anhydrous methanol while stirring, continuously stirring for 30min, and centrifuging at 4 ℃ to collect 105g of precipitate. And (3) adding 315mL of anhydrous methanol precooled to 0 ℃ into the precipitate, and stirring to enable the polyglutamic acid product to be white granules. Filtering with 200 mesh screen, collecting polyglutamic acid particles, and oven drying at 35 deg.C to constant weight to obtain white granular polyglutamic acid product 41.2 g.
Wherein the culture medium comprises the following components:
LB liquid medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1L of distilled water and pH 7.2.
Fermentation medium: 125g of glucose, 40g of yeast powder, 25g of peptone, 150g of L-glutamic acid, 50g of ammonium sulfate, 50g of citric acid, 2.5g of monopotassium phosphate, 5g of disodium hydrogen phosphate dodecahydrate, 2.5g of magnesium sulfate heptahydrate, 1g of calcium chloride, 2.5mg of biotin (vitamin H), 5mL of natural killer, 5L of distilled water and pH 7.2.
Additional glucose solution: 1400g of glucose, 100g of citric acid and 10g of magnesium sulfate are dissolved by adding distilled water and the volume is adjusted to 2L.
Example 2
100 mu L of glycerol pipefish strain liquid (the strain is the same as the above) is sucked into 400mL of LB liquid culture medium, and the shake flask culture medium is shake-cultured for 24h at 120rpm in a constant temperature incubator at 39 ℃ to be used as seed liquid.
The seed liquid was put into 15L fermentation medium (30L fermenter) under flame protection, and fermentation cultured at 39 ℃. During the fermentation process, the dissolved oxygen is controlled to be more than 10 percent by adjusting the stirring speed, the ventilation quantity and the tank pressure, and the pH is controlled to be 6.80 +/-0.02 by using 25 percent ammonia water. And after the dissolved oxygen level rises to more than 80% within 5min, beginning to supplement glucose, so that the dissolved oxygen level is maintained at 20-30%. Fermenting for 20h, stopping fermentation, and collecting fermentation liquor.
Taking 2L of fermentation liquor, and adding 1mol/L H2SO4Adjusting pH to 3.0, adding 2L anhydrous ethanol under stirring, centrifuging at 2500 × g centrifugal force for 3min, and collecting supernatant. 20g (w/v) of activated carbon was added thereto, the mixture was stirred for 30min, and 20g of celite was added to collect 3.9L of a permeate by filtration. Adding 19.5g magnesium sulfate, dissolving and stirring uniformly, cooling to 4 deg.C, adjusting pH to 7.5, adding 0.6L anhydrous ethanol while stirring, stirring for 30min, and centrifuging at 4 deg.C to collect 209g precipitate. And (3) taking the precipitate, adding 209mL of anhydrous ethanol precooled to 4 ℃, and stirring to enable the polyglutamic acid product to be white granules. Filtering with 200 mesh screen, collecting polyglutamic acid particles, and oven drying at 50 deg.C to constant weight to obtain white granular polyglutamic acid product 71.2 g.
Wherein the culture medium comprises the following components:
LB liquid medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 1L of distilled water and pH 7.1.
Fermentation medium: 225g of glucose, 60g of yeast powder, 120g of peptone, 300g of L-glutamic acid, 80g of ammonium sulfate, 300g of citric acid, 15g of monopotassium phosphate, 45g of disodium hydrogen phosphate dodecahydrate, 7.5g of magnesium sulfate heptahydrate, 3g of calcium chloride, 7.5mg of biotin (vitamin H), 15mL of sodium hydrogen phosphate, 15L of distilled water and pH 7.0.
Additional glucose solution: 4200g of glucose, 300g of citric acid and 30g of magnesium sulfate are dissolved by adding distilled water and the volume is 6L.
Example 3
Sucking 200 mu L of glycerol pipefish strain liquid (the strain is the same as the above) into 400mL of LB liquid culture medium, inoculating 3 bottles in total, and shake-culturing the shake-flask culture medium in a constant-temperature incubator at 35 ℃ and 160rpm for 24h to serve as seed liquid.
The seed liquid was put into 15L fermentation medium (30L fermenter) under flame protection, and fermentation cultured at 35 ℃. During the fermentation process, the dissolved oxygen is controlled to be more than 10% by adjusting the stirring speed, the ventilation quantity and the tank pressure, and the pH is controlled to be 7.20 +/-0.02 by using 25% ammonia water. And after the dissolved oxygen level rises to more than 80% within 5min, beginning to supplement glucose, so that the dissolved oxygen level is maintained at 20-30%. Fermenting for 26h, stopping fermentation, and collecting fermentation liquor.
Taking 10L of fermentation liquor, and using 6mol/L H2SO4The pH was adjusted to 2.5, 7L of isopropanol was added with stirring and stirred uniformly, and the mixture was centrifuged continuously by a tube centrifuge 16000 Xg centrifugal force to collect the supernatant. 85g (w/v) of activated carbon was added thereto, and the mixture was stirred for 30min, and then 100g of celite was added to collect 16.5L of a permeate by filtration. 247.5g ammonium sulfate was added, dissolved and stirred uniformly, the temperature was then lowered to 10 ℃ and the pH was adjusted to 6.5, 5L isopropanol was added with stirring, and after stirring for 30min, 1026g of precipitate was collected by centrifugation at 4 ℃. The precipitate was taken and 1026mL of anhydrous ethanol pre-cooled to 4 ℃ was added and stirred to make the polyglutamic acid product as small white particles. Filtering and collecting polyglutamic acid particles by using a 200-mesh screen, and drying at 60 ℃ to constant weight to obtain 368g of white granular polyglutamic acid product.
Wherein the culture medium comprises the following components:
LB liquid medium: peptone 20g, yeast powder 10g, sodium chloride 20g, distilled water 2L, pH 7.1.
Fermentation medium: 750g of glucose, 400g of yeast powder, 500g of peptone, 1000g of L-glutamic acid, 150g of ammonium sulfate, 750g of citric acid, 50g of monopotassium phosphate, 75g of disodium hydrogen phosphate dodecahydrate, 25g of magnesium sulfate heptahydrate, 10g of calcium chloride, 25mg of biotin (vitamin H), 50mL of sodium hydrogen phosphate, 50L of distilled water and pH 6.8.
Additional glucose solution: 14000g of glucose, 1000g of citric acid and 100g of magnesium sulfate are dissolved by adding distilled water and the volume is set to 20L.
Claims (6)
1. A method for extracting and purifying gamma-polyglutamic acid from fermentation liquor is characterized by comprising the following steps:
(1) and (3) adjusting the pH value of the fermentation solution to 2.0-3.0 by using acid, adding a certain amount of organic solvent, uniformly stirring, and centrifuging at a low speed to collect supernatant.
(2) Taking the above centrifugated supernatant, adding a certain amount of active carbon, stirring, filtering, and collecting the permeate.
(3) And (3) adding a certain amount of sulfate into the filtered permeate, dissolving and uniformly stirring, reducing the temperature to 0-10 ℃, adjusting the pH to 5.0-7.5, adding a certain amount of organic solvent, continuously stirring, centrifuging and collecting the precipitate.
(4) And (3) adding a certain amount of organic solvent into the centrifugally collected precipitate, stirring until the precipitate is white granules, filtering, collecting, and drying until the weight is constant to obtain the purified polyglutamic acid.
2. The method for extracting and purifying gamma-polyglutamic acid from fermentation broth of claim 1, wherein the acid in said step 1 is selected from sulfuric acid, hydrochloric acid.
3. The method for extracting and purifying gamma-polyglutamic acid from fermentation broth of claim 1, wherein the organic solvent in step 1 is selected from methanol, ethanol or isopropanol.
4. The method for extracting and purifying gamma-polyglutamic acid from fermentation broth according to claim 1, wherein the mass of activated carbon added in the step 2 is 0.25-2% of the volume of the supernatant liquid after centrifugation.
5. The method for extracting and purifying gamma-polyglutamic acid from a fermentation broth according to claim 1, wherein the sulfate added in the step 3 is selected from sodium sulfate, ammonium sulfate or magnesium sulfate, and the addition amount is 0.5-4% of the permeate.
6. The method for extracting and purifying gamma-polyglutamic acid from fermentation broth according to claim 1, wherein the organic solvent added in the step 4 is pre-cooled to 0-10 ℃ and then added into polyglutamic acid precipitate.
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