CN104805143B - A kind of method for preparing low molecule amount γ polyglutamic acids - Google Patents
A kind of method for preparing low molecule amount γ polyglutamic acids Download PDFInfo
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- CN104805143B CN104805143B CN201510191530.3A CN201510191530A CN104805143B CN 104805143 B CN104805143 B CN 104805143B CN 201510191530 A CN201510191530 A CN 201510191530A CN 104805143 B CN104805143 B CN 104805143B
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- 229920002643 polyglutamic acid Polymers 0.000 title claims abstract description 63
- 238000000034 method Methods 0.000 title claims abstract description 20
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000012530 fluid Substances 0.000 claims abstract description 25
- 238000001556 precipitation Methods 0.000 claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 11
- 239000000843 powder Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 8
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 6
- 239000008213 purified water Substances 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 5
- 238000009413 insulation Methods 0.000 claims abstract description 3
- 244000063299 Bacillus subtilis Species 0.000 claims description 14
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 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
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 6
- 229940073490 sodium glutamate Drugs 0.000 claims description 6
- 230000001954 sterilising effect Effects 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 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 claims description 5
- 238000005352 clarification Methods 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000011218 seed culture Methods 0.000 claims description 5
- 239000002351 wastewater Substances 0.000 claims description 5
- 238000006731 degradation reaction Methods 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 4
- 239000002609 medium Substances 0.000 claims description 4
- 206010001497 Agitation Diseases 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 3
- 235000011187 glycerol Nutrition 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000005720 sucrose Substances 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- 108010020346 Polyglutamic Acid Proteins 0.000 abstract 1
- 108700022290 poly(gamma-glutamic acid) Proteins 0.000 description 24
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 5
- 229920002674 hyaluronan Polymers 0.000 description 5
- 229960003160 hyaluronic acid Drugs 0.000 description 5
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- 241000894006 Bacteria Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
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- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
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- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
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- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 1
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- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of method for preparing low molecule amount γ polyglutamic acids.It adjusts zymotic fluid pH value to 3.5~4.0 first, 75~100 DEG C of temperature, insulation 4 8h of degraded, obtains the low molecule amount γ polyglutamic acids that molecular weight ranges are 10KDa 500KDa；Treat that temperature is down to and be cooled to 0 60 DEG C, add activated carbon decolorizing, plate-frame filtering, except thalline, activated carbon and foreign protein, obtain clear, colorless feed liquid；Feed liquid adjusts pH to 1.5 3.0 with hydrochloric acid, stands 12 24h, separates out precipitation；White powder is added into purified water, and adjusts pH4.5 6.5, stirring and dissolving, obtains the solution that concentration is more than 40%；Then low molecule amount γ polyglutamic acid finished products are obtained by belt drying, crushing.Low molecule amount γ PGA yields prepared by the present invention reach more than 85%, and purity reaches more than 95%, improves product quality.
Description
Technical field
The present invention relates to a kind of method for preparing low molecule weight gamma-polyglutamic acid, belong to industrial microbial technology field.
Background technology
Gamma-polyglutamic acid (γ-PGA) is a kind of macromolecule amino acid polymer by Microbe synthesis.Its to environment without
Pollution, be green bio product, with splendid biodegradability, film forming, into fibroid, plasticity, caking property, moisturizing
The many unique physics and chemistry and biological characteristics such as property.Focusing on today that is environmentally friendly, emphasizing sustainable development, it is this by biosynthesis
Degradable functional material favored by people, be just little by little applied to medical manufacture, food processing, veterinary antibiotics,
Marine product it is antifreeze, fresh-keeping, also can development and application in cosmetics industry, tobacco, leather manufacture industry and vegetable seeds protection etc. are permitted
It is multi-field, it is a kind of to have very big Development volue and the Multifucntional biological products of bright prospects.
γ-PGA production company is fewer at present, and γ-PGA products are in the majority with HMW rank.Low molecule amount γ-
PGA is that the high viscosity gamma-polyglutamic acid zymotic fluid obtained by microbial fermentation obtains by later stage degradation.Low molecule amount
γ-PGA can be used as new pharmaceutical carrier, and experiment shows, and the optimum weight scope that γ-PGA are used as pharmaceutical carrier is 20,000~
60000, its molecular weight is small, small volume, and reactive group ratio is big, is easily combined with medicine, forms relatively stable compound.Low molecule amount
γ-PGA have the function of maintaining and strengthen HA (hyaluronic acid).HA is a kind of composition of human body skin structure, keeps skin
Water moistens and elasticity；It is demonstrated experimentally that low molecule amount γ-PGA can efficiently suppress the activity of hyaluronidase, and HA can be greatly improved
Stability in face of hyaluronidase, so as to slow down the loss of HA in skin.Low molecule amount γ-PGA can improve natural moisture preserving
Factor level.NMF (NFM) is hygroscopic material caused by skin, it is possible to provide positioned at the outermost cuticula of skin
Moisture-keeping functions, research report show, in mankind's TESTSKIN culture medium skins, low molecule amount γ-PGA make as little as 0.1%
Can be improved during dosage in skin several NMF it is horizontal up to 95%, horizontal the continuing to increase of skin NMF can promote skin
Water-retaining property.In addition, low molecule amount γ-PGA also have enhancing epidermal cell structural intergrity, improve moisture of skin and bullet
Property, reduce transepidermal water and lose and slow down the functions such as immune response.
Because low molecule amount γ-PGA preparation technologies are complicated, production cost is high, especially isolates and purifies and process for refining side
Facing large-scale production has obstacle, and numerous domestic research and development institution and enterprise lack experience in this respect, and Innovation Input is not
It is enough, cause product quality not high, production capacity is extremely limited, also limit γ-PGA popularization and application.
The content of the invention
It is in the majority with HMW rank in the market γ-PGA products, the less situation of product type, mesh of the invention
Be to provide a kind of method for preparing low molecule weight gamma-polyglutamic acid, separating-purifying obtains the low molecule amount γ-PGA of high-purity
Product, γ-PGA product types are enriched, expand γ-PGA application market.
The technical scheme is that：A kind of method for preparing low molecule weight gamma-polyglutamic acid, it is characterised in that including
Following steps：
(1) prepared by zymotic fluid：Using bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 as
Bacterial strain is produced, fermenting and producing gamma-polyglutamic acid, obtains zymotic fluid；
(2) high temperature acid degradation obtains harmonic component gamma-polyglutamic acid：Zymotic fluid pH value is adjusted to 3.5~4.0, temperature 75~100
DEG C, the gamma-polyglutamic acid of more than 1000KDa in zymotic fluid is degraded to molecular weight ranges and is by insulation degraded 4-8h, this process
10KDa-500KDa low molecule weight gamma-polyglutamic acid；Treat that temperature is down to and be cooled to 0-60 DEG C, add activated carbon decolorizing 0-2h,
After decolouring terminates, plate-frame filtering, except thalline, activated carbon and foreign protein, clear, colorless feed liquid is obtained；
(3) Precipitation：Feed liquid adjusts pH to 1.5-3.0 with hydrochloric acid, stands 12-24h, separates out precipitation, plate-frame filtering obtains white
Color powder, and untill being washed to waste water achromaticity and clarification with pH1.5-3.0 purifying；
(4) precipitation weight is molten：Obtained white powder will be handled through step (3) and adds purified water, and adjusts pH4.5-6.5, will be stirred
Dissolving is mixed, obtains the solution that concentration is more than 40%；
(5) belt drying：It will be obtained through the high concentration solution that step (4) processing obtains by belt drying, crushing low
Molecule weight gamma-polyglutamic acid finished product.
Further, in the step (1) zymotic fluid preparation method, its step is as follows：
A. seed culture
Bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 are inoculated in and trained equipped with seed
In the triangular flask for supporting base, in 35~37.5 DEG C, 180~220r/min shaking table concussion and cultivate 16-32h, seed liquor is obtained；
B. fermented and cultured
The seed liquor of step (1) is inoculated in fermentation medium, inoculum concentration is 2~3% (mass ratioes), in 35~37.5
DEG C air agitation 45~50h of culture, obtains zymotic fluid.
Each medium component is as follows：Seed culture medium：5~10g/L of glucose, 3~5g/L of yeast extract, ammonium sulfate 2
~10g/L, 5~10g/L of sodium glutamate, 7.2~7.5,115 DEG C of sterilizing 20min of magnesium sulfate 0.2~2g/L, pH；Fermented and cultured
Base：In sucrose, glucose, glycerine, citric acid it is any or at least two 30~150g/L of mixture, 5~30g/ of peptone
L, 5~30g/L of yeast extract, 5~20g/L of ammonium sulfate, 30~100g/L of sodium glutamate, 0.2~2g/L of magnesium sulfate, phosphoric acid hydrogen
7.2~7.5,121 DEG C of sterilizing 20min of dipotassium 2~10g/L, pH.
Above-mentioned fermentation process can also use the method in the patent of invention of the applicant oneself；The application number of the patent：
201210555304.5；The applying date：2012.11.02；Denomination of invention：One plant of production gamma-polyglutamic acid genetic engineering bacterium and its high yield
Gamma-polyglutamic acid method (see claim 6 and embodiment 5-6).
Further, in the step (2), activated carbon dosage is the 1%~2% of zymotic fluid weight, it is stirred at room temperature 60~
90min。
Further, step (5) the band-type baking condition is：Heating Zone Temperature is 75~95 DEG C, sample introduction speed
For 20~30L/h, crawler track speeds are 20~30cm/min.
The beneficial effects of the invention are as follows：
(1) more than 1000KDa HMW γ-PGA are degraded to 10KDa- by the high temperature in the present invention, acid degradation process
500KDa low molecule amount γ-PGA, enrich γ-PGA product types, expand γ-PGA application market；
(2) present invention has been reached point using the characteristic for there was only gamma-polyglutamic acid Precipitation in the clarified solution of low ph value
From the purpose of purification of high-purity low molecule weight gamma-polyglutamic acid.Acid out goes out precipitation and replaces alcohol precipitation process, not only increases product and returns
Yield, industrial cost and danger are reduced, also save the energy；
(3) low molecule amount γ-PGA yields prepared by the present invention reach more than 85%, and purity reaches more than 95%, improves
Product quality.
Embodiment
Embodiment 1：
(1) prepared by zymotic fluid：Bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 are entered
Row fermented and cultured, obtains zymotic fluid, and specific steps include：
A. the strain 1 of glycerol stocks under the conditions of -20 DEG C in refrigerator is taken, oese connects ring species in equipped with 100ml kinds
In the 500ml triangular flasks of sub- culture medium, in 35~37.5 DEG C, 200r/min shaking table concussion and cultivate 24h, seed liquor, strain are obtained
Concentration reaches OD6001.5~2.0；
B. the seed liquor got ready is inoculated in fermentation medium, inoculum concentration 2.5%, in 35~37.5 DEG C of air agitations
48h is cultivated, obtains zymotic fluid, fermentation yield is up to 67g/l.
Seed culture medium：Glucose 10g/L, yeast extract 5g/L, ammonium sulfate 5g/L, sodium glutamate 10g/L, magnesium sulfate
7.2~7.5,115 DEG C of sterilizing 20min of 1g/L, pH；
Fermentation medium：Sucrose 40g/L, peptone 10g/L, yeast extract 5g/L, ammonium sulfate 5g/L, sodium glutamate
50g/L, magnesium sulfate 1g/L, 7.2~7.5,121 DEG C of sterilizing 20min of dipotassium hydrogen phosphate 10g/L, pH.
(2) harmonic component gamma-polyglutamic acid obtains：
Zymotic fluid pH to 3.5 is adjusted, is warming up to 85 DEG C, 4h is incubated, treats that temperature is down to 60 DEG C, 1% activated carbon is added and takes off
Color 90min, after decolouring terminates, plate-frame filtering, except thalline, activated carbon and foreign protein, obtain clear, colorless feed liquid；This process will ferment
More than 1000KDa gamma-polyglutamic acid is degraded to low molecule amount γ-polyglutamic that molecular weight ranges are 10KDa-500KDa in liquid
Acid；
(3) Precipitation：
Above-mentioned feed liquid is adjusted into pH to 1.5 with hydrochloric acid, 12h is stood, separates out precipitation, plate-frame filtering obtains white powder, is used in combination
Untill pH1.5 purifying is washed to waste water achromaticity and clarification；
(4) precipitation weight is molten：
The white powder that above-mentioned processing is obtained adds purified water, and adjusts pH4.5, stirring and dissolving, obtains concentration as 40%
Solution above；
(5) belt drying：
Above-mentioned highly concentrated solution is subjected to belt drying, Heating Zone Temperature is 95 DEG C, and sample introduction speed is 30L/h, and crawler belt is fast
Spend for 30cm/min, obtain mobility and the higher high purity and low molecular weight gamma-polyglutamic acid (10KDa- of dissolubility after crushed
500KDa)。
Low molecule amount γ-PGA yields reach 85.5%, purity 95.8%.
Embodiment 2：
(1) prepared by zymotic fluid：Bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 are carried out
Fermented and cultured, zymotic fluid is obtained, specific steps are the same as embodiment 1；
(2) harmonic component gamma-polyglutamic acid obtains：
Zymotic fluid pH to 3.5 is adjusted, is warming up to 80 DEG C, 6h is incubated, treats that temperature is down to about 60 DEG C, add 1.5% activity
Carbon decoloring 75min, after decolouring terminates, plate-frame filtering, except thalline, activated carbon and foreign protein, obtain clear, colorless feed liquid；This process will
More than 1000KDa gamma-polyglutamic acid is degraded to low molecule amount γ that molecular weight ranges are 10KDa-500KDa-poly- in zymotic fluid
Glutamic acid；
(3) Precipitation：
Above-mentioned feed liquid is adjusted into pH to 2.5 with hydrochloric acid, 24h is stood, separates out precipitation, plate-frame filtering obtains white powder, is used in combination
Untill pH2.5 purifying is washed to waste water achromaticity and clarification；
(4) precipitation weight is molten：
The white powder that above-mentioned processing is obtained adds purified water, and adjusts pH to 5.5, and stirring and dissolving, obtaining concentration is
More than 40% solution；
(5) belt drying：
Above-mentioned highly concentrated solution is subjected to belt drying, Heating Zone Temperature is 85 DEG C, and sample introduction speed is 25L/h, and crawler belt is fast
Spend for 25cm/min, obtain mobility and the higher high purity and low molecular weight gamma-polyglutamic acid (10KDa- of dissolubility after crushed
500KDa)。
Low molecule amount γ-PGA yields reach 86.1%, purity 95.8%.
Embodiment 3：
(1) prepared by zymotic fluid：Bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 are carried out
Fermented and cultured, zymotic fluid is obtained, specific steps are the same as embodiment 1；
(2) harmonic component gamma-polyglutamic acid obtains：
Zymotic fluid pH to 4.0 is adjusted, is warming up to 85 DEG C, 5h is incubated, treats that temperature is down to 60 DEG C, 2% activated carbon is added and takes off
Color 60min, after decolouring terminates, plate-frame filtering, except thalline, activated carbon and foreign protein, obtain clear, colorless feed liquid；This process will ferment
More than 1000KDa gamma-polyglutamic acid is degraded to low molecule amount γ-polyglutamic that molecular weight ranges are 10KDa-500KDa in liquid
Acid；
(3) Precipitation：
Above-mentioned feed liquid is adjusted into pH to 3.0 with hydrochloric acid, 24h is stood, separates out precipitation, plate-frame filtering obtains white powder, is used in combination
Untill pH3.0 purifying is washed to waste water achromaticity and clarification；
(4) precipitation weight is molten：
The white powder that above-mentioned processing is obtained adds purified water, and adjusts pH to 6.5, and stirring and dissolving, obtaining concentration is
More than 40% solution；
(5) belt drying：
Above-mentioned highly concentrated solution is subjected to belt drying, Heating Zone Temperature is 75 DEG C, and sample introduction speed is 20L/h, and crawler belt is fast
Spend for 20cm/min, obtain mobility and the higher high purity and low molecular weight gamma-polyglutamic acid (10KDa- of dissolubility after crushed
500KDa)。
Low molecule amount γ-PGA yields reach 85.5%, purity 95.8%.
Claims (6)
1. a kind of method for preparing low molecule weight gamma-polyglutamic acid, it is characterized in that, comprise the following steps：
(1) prepared by zymotic fluid：Using bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 as production
Bacterial strain, fermenting and producing gamma-polyglutamic acid, obtains zymotic fluid；
(2) high temperature acid degradation obtains harmonic component gamma-polyglutamic acid：Zymotic fluid pH value is adjusted to 3.5~4.0,75~100 DEG C of temperature,
Insulation degraded 4-8h, obtains the low molecule weight gamma-polyglutamic acid that molecular weight ranges are 10KDa-500KDa；Treat that temperature is down to 0-60
DEG C, activated carbon decolorizing 0-2h is added, after decolouring terminates, plate-frame filtering, except thalline, activated carbon and foreign protein, obtains clear, colorless material
Liquid；
(3) Precipitation：Feed liquid adjusts pH to 1.5-3.0 with hydrochloric acid, stands 12-24h, separates out precipitation, and plate-frame filtering obtains white powder
End, and untill being washed to waste water achromaticity and clarification with pH1.5-3.0 purifying；
(4) precipitation weight is molten：Obtained white powder will be handled through step (3) and adds purified water, and adjusts pH4.5-6.5, stirring is molten
Solution, obtain the solution that concentration is more than 40%；
(5) belt drying：By obtained high concentration solution is handled through step (4) low molecule is obtained by belt drying, crushing
Weight gamma-polyglutamic acid finished product.
2. a kind of method for preparing low molecule weight gamma-polyglutamic acid as claimed in claim 1, it is characterized in that,
A. seed culture
Bacillus subtilis (Bacillus subtilis) FRD518CGMCC NO.6772 are inoculated in equipped with seed culture medium
Triangular flask in, in 35~37.5 DEG C, 180~220r/min shaking table concussion and cultivate 16-32h, obtain seed liquor；
B. fermented and cultured
The seed liquor of step (1) is inoculated in fermentation medium, inoculum concentration is 2~3%, is trained in 35~37.5 DEG C of air agitations
45~50h is supported, obtains zymotic fluid.
3. a kind of method for preparing low molecule weight gamma-polyglutamic acid as claimed in claim 2, it is characterized in that, the seed training
Support base component and content be：5~10g/L of glucose, 3~5g/L of yeast extract, 2~10g/L of ammonium sulfate, sodium glutamate 5
7.2~7.5,115 DEG C of sterilizing 20min of~10g/L, magnesium sulfate 0.2~2g/L, pH.
4. a kind of method for preparing low molecule weight gamma-polyglutamic acid as claimed in claim 2, it is characterized in that, the fermentation training
Support base component and content be：In sucrose, glucose, glycerine, citric acid it is any or at least two 30~150g/ of mixture
L, 5~30g/L of peptone, 5~30g/L of yeast extract, 5~20g/L of ammonium sulfate, 30~100g/L of sodium glutamate, magnesium sulfate
7.2~7.5,121 DEG C of sterilizing 20min of 0.2~2g/L, dipotassium hydrogen phosphate 2~10g/L, pH.
5. a kind of method for preparing low molecule weight gamma-polyglutamic acid as described in any one in claim 1-4, its feature
It is that in the step (2), activated carbon dosage is the 1%~2% of zymotic fluid weight, and 60~90min is stirred at room temperature.
6. a kind of method for preparing low molecule weight gamma-polyglutamic acid as described in any one in claim 1-4, its feature
It is that step (5) the band-type baking condition is：Heating Zone Temperature is 75~95 DEG C, and sample introduction speed is 20~30L/h, is carried out
Tape speed is 20~30cm/min.
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| CN105968383B (en) * | 2016-04-05 | 2019-03-22 | 山东省药学科学院 | A kind of low molecular weight polyglutamic acid and the preparation method and application thereof |
| CN110498919B (en) * | 2019-07-26 | 2022-03-01 | 河南师范大学 | A kind of synthetic method of γ-PGA-Bn with controllable esterification degree |
| CN110372858A (en) * | 2019-08-30 | 2019-10-25 | 尚科生物医药（上海）有限公司 | A kind of method of polyglutamic acid extraction and purification |
| CN114774488A (en) * | 2022-05-13 | 2022-07-22 | 山东福瑞达生物科技有限公司 | Production method of low-endotoxin gamma-polyglutamic acid |
| CN116970162A (en) * | 2023-06-09 | 2023-10-31 | 天津北洋百川生物技术有限公司 | Low endotoxin gamma-polyglutamic acid and separation and extraction method and application thereof |
| CN117186395B (en) * | 2023-09-13 | 2024-07-23 | 南京轩凯生物科技股份有限公司 | Preparation method and application of calcium polyglutamate |
| CN117720722A (en) * | 2023-12-19 | 2024-03-19 | 山东福瑞达生物科技有限公司 | Preparation method and application of oligoglutamic acid |
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