CN102796779B - Biological method for preparing gamma-aminobutyric acid - Google Patents
Biological method for preparing gamma-aminobutyric acid Download PDFInfo
- Publication number
- CN102796779B CN102796779B CN2012103045965A CN201210304596A CN102796779B CN 102796779 B CN102796779 B CN 102796779B CN 2012103045965 A CN2012103045965 A CN 2012103045965A CN 201210304596 A CN201210304596 A CN 201210304596A CN 102796779 B CN102796779 B CN 102796779B
- Authority
- CN
- China
- Prior art keywords
- aminobutyric acid
- liquid
- clear liquid
- exchange resin
- gamma
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 183
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 92
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 89
- 238000010170 biological method Methods 0.000 title abstract 3
- 239000007788 liquid Substances 0.000 claims abstract description 66
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000002360 preparation method Methods 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 16
- 239000013078 crystal Substances 0.000 claims abstract description 16
- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
- 239000000843 powder Substances 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 12
- 230000004151 fermentation Effects 0.000 claims abstract description 12
- 238000000909 electrodialysis Methods 0.000 claims abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 238000002425 crystallisation Methods 0.000 claims description 15
- 230000008025 crystallization Effects 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 14
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 13
- 239000004721 Polyphenylene oxide Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 10
- 239000003456 ion exchange resin Substances 0.000 claims description 10
- 229920003303 ion-exchange polymer Polymers 0.000 claims description 10
- 238000005507 spraying Methods 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 239000000463 material Substances 0.000 claims description 9
- 210000001082 somatic cell Anatomy 0.000 claims description 9
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 8
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 235000013922 glutamic acid Nutrition 0.000 claims description 7
- 239000004220 glutamic acid Substances 0.000 claims description 7
- 229910052708 sodium Inorganic materials 0.000 claims description 7
- 239000011734 sodium Substances 0.000 claims description 7
- QWCKQJZIFLGMSD-UHFFFAOYSA-N 2-Aminobutanoic acid Natural products CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 claims description 6
- QWCKQJZIFLGMSD-GSVOUGTGSA-N D-alpha-aminobutyric acid Chemical compound CC[C@@H](N)C(O)=O QWCKQJZIFLGMSD-GSVOUGTGSA-N 0.000 claims description 6
- 230000002378 acidificating effect Effects 0.000 claims description 6
- 230000001580 bacterial effect Effects 0.000 claims description 6
- 239000003729 cation exchange resin Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
- 239000011347 resin Substances 0.000 claims description 6
- 229920005989 resin Polymers 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
- 229920002774 Maltodextrin Polymers 0.000 claims description 5
- 239000005913 Maltodextrin Substances 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 5
- 108010080698 Peptones Proteins 0.000 claims description 5
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 239000002518 antifoaming agent Substances 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 5
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 5
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 5
- 235000019800 disodium phosphate Nutrition 0.000 claims description 5
- 239000008103 glucose Substances 0.000 claims description 5
- 229940035034 maltodextrin Drugs 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 235000019319 peptone Nutrition 0.000 claims description 5
- 229920000570 polyether Polymers 0.000 claims description 5
- 230000001105 regulatory effect Effects 0.000 claims description 5
- 239000001632 sodium acetate Substances 0.000 claims description 5
- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000012138 yeast extract Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 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 4
- 239000012535 impurity Substances 0.000 claims description 4
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 235000013923 monosodium glutamate Nutrition 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 4
- 229940073490 sodium glutamate Drugs 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 239000003957 anion exchange resin Substances 0.000 claims description 3
- 239000002585 base Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 16
- 235000014655 lactic acid Nutrition 0.000 abstract description 8
- 239000004310 lactic acid Substances 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000005516 engineering process Methods 0.000 abstract description 2
- 238000003912 environmental pollution Methods 0.000 abstract description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 abstract 2
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 8
- 229960002989 glutamic acid Drugs 0.000 description 6
- 239000013530 defoamer Substances 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 229960000448 lactic acid Drugs 0.000 description 4
- 229960004249 sodium acetate Drugs 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 3
- 244000299461 Theobroma cacao Species 0.000 description 3
- 230000031018 biological processes and functions Effects 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 241000186685 Lactobacillus hilgardii Species 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 235000019219 chocolate Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 241000228212 Aspergillus Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000009470 Theobroma cacao Nutrition 0.000 description 1
- 229940124277 aminobutyric acid Drugs 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000015173 baked goods and baking mixes Nutrition 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 235000019462 natural additive Nutrition 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000001473 noxious effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001256 tonic effect Effects 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The present invention discloses a biological method for preparing gamma-aminobutyric acid, including the steps of: preparing bacteria, producing gamma-aminobutyric acid, decoloring, conducting electrodialysis, preparing gamma-aminobutyric acid powder products or gamma-aminobutyric acid crystal products and the like. The present invention utilizes the free lactic acid bacterium cells for catalyzed synthesis of gamma-aminobutyric acid, and helps to improve the production technology, improve the gamma-aminobutyric acid yield, and reduce the cost, and also establishes the method for preparation of high-purity gamma-aminobutyric acid. The reaction system of the present method does not include carbon sources, nitrogen sources and other culture components, and the reaction liquid components are simple relative to fermentation liquid. The biological method can greatly improve the purity of the product, and has the advantages of simple post-treatment, short production cycle, little environmental pollution, low cost, etc.
Description
Technical field
The present invention relates to a kind of method for preparing γ-aminobutyric acid.
Background technology
γ-aminobutyric acid (Gamma-aminobutyric acid, GABA), claim again aminobutyric acid, is that a kind of naturally occurring nonprotein forms amino acid, is distributed widely in protokaryon and eukaryote.γ-aminobutyric acid is the inhibitory transmitter substance of mammalian central nervous system, have calm neural, promote sleep, reduce blood pressure, brain tonic and intelligence development, delay senility, the important physiological function such as the sharp kidney of strong liver.
Up to now, the preparation method of γ-aminobutyric acid mainly contains two kinds of chemical synthesis and biological processes.Common chemical synthesis process is as using pyrrolidone as raw material, through calcium hydroxide, bicarbonate of ammonia hydrolysis, makes γ-aminobutyric acid.The chemical synthesis cost is higher, and yield is lower, and uses dangerous solvents in production technique, or even deep-etching, noxious solvent.Therefore, the γ-aminobutyric acid of chemical synthesis preparation is difficult in the food factories realization, and goods can not be considered to a kind of natural additive for foodstuff.It is a kind of not only safety, cost but also low method that biological process is compared.According to up-to-date patent report, the bacterial classification that biological process is produced γ-aminobutyric acid has (1) natural original strain, comprises short lactobacillus (patent No. ZL 200510049187.5), aspergillus (publication number CN 101302480 B), plant lactobacillus (publication number CN 101928679 A), milk-acid bacteria (publication number CN 1243101 C) or natural food materials enrichment (publication number CN 1840672 A); (2) recombinant bacterium: intestinal bacteria (publication number CN 1298860 C), Corynebacterium glutamicum (publication number CN 101945997 A) (3) mutagenic strain: short lactobacillus (publication number CN 102174449 A).In existing method, the γ-aminobutyric acid fecund is born in fermented liquid, due to the fermented liquid complicated component, the downstream separation purge process is loaded down with trivial details, the concentration of target product is also lower, these effects limit domestic industry production, and about preparation high purity γ-aminobutyric acid, rarely have especially report.
Summary of the invention
The object of the present invention is to provide a kind of method easy, easy to operate, the method for effective Biological preparation γ-aminobutyric acid.
Technical solution of the present invention is:
A kind of method of Biological preparation γ-aminobutyric acid is characterized in that: comprise the following steps:
(1) thalline preparation:
The Sodium Glutamate that adds 1-30g/L in substratum, as inductor, is induced the product enzyme, and access is produced the bacterial strain of L-Glutamic decarboxylase in born of the same parents and cultivated; Culture condition is: 25～35 ℃ of culture temperature, carry out pH control by adding alkali lye in fermenting process, and fermented liquid pH is maintained in the 4.5-7.0 scope, incubation time 8～24 hours; Obtain bacterium liquid after cultivating end;
(2) ceramic membrane filter is collected thalline: the bacterium liquid that step (1) obtains is collected thalline through ceramic membrane filter, and membrane pore size is 0.05-1 μ m, obtains thalline;
(3) produce γ-aminobutyric acid:
The damping fluid of preparation 0.01-0.5mol/L pH3-7, add the 10-100g/L somatic cells, adds substrate glutamic acid sodium in batches, interpolation concentration is 0-30g/L, be under the condition of 28-40 ℃ in temperature of reaction, by fermentation again, produce γ-aminobutyric acid, fermentation time is 10-50h; After fermentation ends, by centrifugal collection clear liquid;
(4) clear liquid that step (3) is obtained is further removed somatic cells through metal micron membranes strainer, obtains clear liquid;
(5) decolouring: the clear liquid pH that first step (4) is obtained transfers to 7.0-8.0, then by the processing of decolour of granulated active carbon post, and the collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, controls material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-aminobutyric acid clear liquid;
(7) the γ-aminobutyric acid clear liquid that step (6) is obtained, make γ-aminobutyric acid powder product or γ-aminobutyric acid crystal product.
The described method of making the γ-aminobutyric acid powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when feed liquid is concentrated into alpha-aminobutyric acid content for 〉=80g/L, stop concentrating, collect concentrated solution;
(b) spraying drying: add maltodextrin or starch in concentrated solution, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-aminobutyric acid powder product of 20-50%.
The described method of making the γ-aminobutyric acid crystal product is: with the γ-aminobutyric acid clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-aminobutyric acid iso-electric point,, by decrease temperature crystalline, obtain the γ-aminobutyric acid crystal product.
In the described product of step (1) born of the same parents, the bacterial strain of L-Glutamic decarboxylase is milk-acid bacteria or lactobacillus hilgardii.
The composition of the substratum that step (1) is used and take the content of g/L as:
Glucose 10-30, yeast extract paste 5-30, peptone .5-30, sodium acetate 0.5-5, Sodium phosphate dibasic or dipotassium hydrogen phosphate 0.05-10, sal epsom 0.05-10, manganous sulfate 0.05-1, PPE polyether antifoam agent 0.1-3.
Described ion exchange resin is first selected anionite-exchange resin, to remove residual substrate glutamic acid sodium, then passes through acidic cation-exchange resin, to γ-aminobutyric acid adsorb, wash-out processes.
Described anionite-exchange resin is D301, D296 or 201 type highly basic or weak base anion-exchange resins; Described Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin.
The iso-electric point lowering temperature crystallization is adopted in crystallization, the feed liquid that obtains after ion exchange resin treatment is carried out crystallization or add ethanol and carry out crystallization, and adding proportion is ethanol: feed liquid=0～10:1V/V; During crystallization, regulator solution pH value is to the iso-electric point 7.2 of γ-aminobutyric acid, and the greenhouse cooling scope is 90～5 ℃, and the addition of crystal seed is 0. 2-10g/L, and stir speed (S.S.) is 30-100r/min, and rate of temperature fall is 1-10 ℃/h.
The present invention utilizes the free lactic acid mycetocyte to catalyze and synthesize γ-aminobutyric acid, and improvement of production process, and γ-aminobutyric acid output is improved, cost, but also set up the method for preparing the high purity γ-aminobutyric acid.Do not contain carbon source, nitrogenous source etc. in this technique in reaction system and cultivate composition, the reaction solution composition is simple for fermented liquid, can increase substantially product purity, has simultaneously that aftertreatment is simple, with short production cycle and environmental pollution is little, low cost and other advantages.
Description of drawings
The invention will be further described below in conjunction with drawings and Examples.
Fig. 1 is γ-aminobutyric acid formation curve in one embodiment of the invention preparation of industrialization process.
Fig. 2 transforms the amino acid detected result of the conversion fluid that generates γ-aminobutyric acid with lactic-acid bacteria cells.Glu: L-glutamic acid; GABA: γ-aminobutyric acid.
Embodiment
γ-aminobutyric acid of the present invention refers to 4-Aminobutanoicacid, is the mammalian central nervous system inhibitory transmitter., as new resource food, can add in beverage, cocoa products, chocolate and chocolate, candy, bakery product, puffed food the development of new product to.
Embodiment 1:
One, yeast culture
After lactic acid bacteria culturers is activated, carrying out first order seed cultivates, secondary seed is cultivated, enter finally fermentor cultivation, inoculum size 3%(V/V, seed liquor/fermented liquid), 30 ℃ of standing cultivation 16 h, carry out pH control by adding alkali lye in fermenting process, fermented liquid pH is maintained in the 4.5-7.0 scope, by 0.6 μ m ceramic membrane filter, collect thalline.
The seed culture based raw material is in g/L: glucose 10, yeast extract paste 5, peptone 5, sodium acetate 0.5, Sodium phosphate dibasic (or dipotassium hydrogen phosphate) 1, sal epsom 1, manganous sulfate 1, PPE polyether antifoam agent 0.1(but be not limited only to this defoamer, can select any defoamer that can be used for fermentation industry).
Fermentative medium formula is in g/L: glucose 10, yeast extract paste 5, peptone 5, sodium acetate 0.5, Sodium Glutamate 1, Sodium phosphate dibasic (or dipotassium hydrogen phosphate) 1, sal epsom 1, manganous sulfate 1, PPE polyether antifoam agent 0.1(but be not limited only to this defoamer, can select any defoamer that can be used for fermentation industry).
Two, produce γ-aminobutyric acid
Adopt the lactic-acid bacteria cells conversion method to produce γ-aminobutyric acid, reaction conditions is: acetic acid-sodium-acetate buffer of preparation 0.01mol/L pH7, add the 20g/L somatic cells, add substrate glutamic acid sodium in batches, initial substrate concentration 10g/L, every 4h adds a substrate, interpolation concentration is 5g/L, the total reaction concentration of substrate is 25g/L, and temperature of reaction is 28 ℃, and fermentation time is 17h.After end,, by centrifugal collection clear liquid, obtain content and reach the γ-aminobutyric acid conversion fluid of 12g/L, see Fig. 1.
Three, the detection of alpha-aminobutyric acid content
The trichoroacetic acid(TCA) that adds equal-volume 100g/L after above-mentioned conversion fluid is centrifugal, vibration is even, more centrifugal (10,000 * g, 5 min), 2-10 times of clear liquid dilution, then, through 0.45 μ m membrane filtration, carry out high-efficient liquid phase analysis.Adopt ODS C
18(φ 4.6 * 250nm), and 40
oC analyzes, and γ-aminobutyric acid is quantitative with external standard method.
Four, product preparation:
Filtration sterilization: the γ-aminobutyric acid conversion fluid that will obtain is further removed somatic cells through metal micron membranes strainer, obtains clear liquid;
Decolouring: the clear liquid pH that obtains after filtration sterilization is transferred to 7.0-8.0, then by the processing of decolour of granulated active carbon post, the collection clear liquid;
Electrodialysis: adopt electrodialysis to remove impurity, controls material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-aminobutyric acid clear liquid;
, with the γ-aminobutyric acid clear liquid that step (4) obtains, make γ-aminobutyric acid powder product or γ-aminobutyric acid crystal product.
The described method of making the γ-aminobutyric acid powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when feed liquid is concentrated into alpha-aminobutyric acid content for 〉=50g/L, stop concentrating;
(b) spraying drying: add maltodextrin or starch in concentrated solution, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-aminobutyric acid powder product of 20-50%;
The described method of making the γ-aminobutyric acid powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when feed liquid is concentrated into alpha-aminobutyric acid content for 〉=80g/L, stop concentrating, collect concentrated solution;
(b) spraying drying: add maltodextrin or starch in concentrated solution, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-aminobutyric acid powder product of 20-50%.
The described method of making the γ-aminobutyric acid crystal product is: with the γ-aminobutyric acid clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-aminobutyric acid iso-electric point,, by decrease temperature crystalline, obtain purity and reach 98% γ-aminobutyric acid crystal product.
Embodiment 2:
A kind of method of Biological preparation γ-aminobutyric acid, comprise the following steps:
(1) thalline preparation:
Add Sodium Glutamate in substratum, addition is 1-30g/L(example 1 g/L, 15 g/L, 30 g/L) as inductor, induce the product enzyme, access is produced the bacterial strain of L-Glutamic decarboxylase in born of the same parents and is cultivated; Culture condition is: 25～35 ℃ of culture temperature (25 ℃, 30 ℃, 35 ℃ of examples), carry out pH control by adding alkali lye in fermenting process, and fermented liquid pH is maintained in the 4.5-7.0 scope, incubation time 8～24 hours (routine 8h, 16h, 24h); Obtain bacterium liquid after cultivating end;
(2) ceramic membrane filter is collected thalline: the bacterium liquid that step (1) obtains is collected thalline through ceramic membrane filter, and membrane pore size is 0.05-1 μ m(example 0.05 μ m, 0.5 μ m, 1 μ m), obtain thalline;
(3) produce γ-aminobutyric acid:
Preparation 0.01-0.5mol/L(example 0.01 mol/L, 0.2 mol/L, 0.5 mol/L) pH3-7(example 3,5,7) damping fluid, add somatic cells, the somatic cells add-on is 10-100g/L(example 10 g/L, 50 g/L, 100 g/L), add substrate glutamic acid sodium in batches, adding concentration is 1-30g/L(example 1g/L, 15 g/L, 30 g/L), be under the condition of 28-40 ℃ (28 ℃, 32 ℃, 40 ℃ of examples) in temperature of reaction, by again fermenting and produce γ-aminobutyric acid, fermentation time is 10-50h(example 10h, 25h, 50h); After fermentation ends, by centrifugal collection clear liquid;
(4) clear liquid that step (3) is obtained is further removed somatic cells through metal micron membranes strainer, obtains clear liquid;
(5) decolouring: the clear liquid pH that first step (4) is obtained transfers to 7.0-8.0, then by the processing of decolour of granulated active carbon post, and the collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, controls material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-aminobutyric acid clear liquid;
(7) the γ-aminobutyric acid clear liquid that step (6) is obtained, make γ-aminobutyric acid powder product or γ-aminobutyric acid crystal product.
The described method of making the γ-aminobutyric acid powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when feed liquid is concentrated into alpha-aminobutyric acid content for 〉=80g/L, stop concentrating, collect concentrated solution;
(b) spraying drying: add maltodextrin or starch in concentrated solution, regulating solid quality content is 5-50%(example 5%, 30%, 50%), carry out spraying drying, making mass content is the γ-aminobutyric acid powder product of 20-50%.
The described method of making the γ-aminobutyric acid crystal product is: with the γ-aminobutyric acid clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-aminobutyric acid iso-electric point,, by decrease temperature crystalline, obtain the γ-aminobutyric acid crystal product.
In the described product of step (1) born of the same parents, the bacterial strain of L-Glutamic decarboxylase is milk-acid bacteria or lactobacillus hilgardii.
The composition of the substratum that step (1) is used and take the content of g/L as:
Glucose 10-30(example 10,20,30), yeast extract paste 5-30(example 5,20,30), peptone .5-30(example 5,20,30), sodium acetate 0.5-5(example 0.5,3,5), Sodium phosphate dibasic or dipotassium hydrogen phosphate 0.05-10(example 0.05,5,10), sal epsom 0.05-10(example 0.05,5,10), manganous sulfate 0.05-1(example 0.05,0.5,1), PPE polyether antifoam agent 0.1-3(example 0.1,1,3).
Described ion exchange resin is first selected anionite-exchange resin, to remove residual substrate glutamic acid sodium, then passes through acidic cation-exchange resin, to γ-aminobutyric acid adsorb, wash-out processes.
Described anionite-exchange resin is D301, D296 or 201 type highly basic or weak base anion-exchange resins; Described Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin.
The iso-electric point lowering temperature crystallization is adopted in crystallization, the feed liquid that obtains after ion exchange resin treatment is carried out crystallization or add ethanol and carry out crystallization, and adding proportion is ethanol: feed liquid=0～10:1V/V(example 1:1,5:1,10:1); During crystallization, regulator solution pH value is to the iso-electric point 7.2 of γ-aminobutyric acid, the greenhouse cooling scope is 90～5 ℃ (90 ℃, 40 ℃, 5 ℃ of examples), the addition of crystal seed is 0. 2-10g/L(example 0.2 g/L, 5 g/L, 10 g/L), stir speed (S.S.) is 30-100r/min, and rate of temperature fall is 1 ℃/h of 1-10 ℃/h(example, 5 ℃/h, 10 ℃/h).
Claims (1)
1. the method for a Biological preparation γ-aminobutyric acid, is characterized in that: comprise the following steps:
(1) thalline preparation:
The Sodium Glutamate that adds 1-30g/L in substratum, as inductor, is induced the product enzyme, and access is produced the bacterial strain of L-Glutamic decarboxylase in born of the same parents and cultivated; Culture condition is: 25～35 ℃ of culture temperature, carry out pH control by adding alkali lye in fermenting process, and fermented liquid pH is maintained in the 4.5-7.0 scope, incubation time 8～24 hours; Obtain bacterium liquid after cultivating end; The composition of the substratum that uses and take the content of g/L as:
Glucose 10-30, yeast extract paste 5-30, peptone 5-30, sodium acetate 0.5-5, Sodium phosphate dibasic or dipotassium hydrogen phosphate 0.05-10, sal epsom 0.05-10, manganous sulfate 0.05-1, PPE polyether antifoam agent 0.1-3;
(2) ceramic membrane filter is collected thalline: the bacterium liquid that step (1) obtains is collected thalline through ceramic membrane filter, and membrane pore size is 0.05-1 μ m, obtains thalline;
(3) produce γ-aminobutyric acid:
The damping fluid of preparation 0.01-0.5mol/L pH3-7, add the 10-100g/L somatic cells, adds substrate glutamic acid sodium in batches, interpolation concentration is 25g/L, be under the condition of 28-40 ℃ in temperature of reaction, by fermentation again, produce γ-aminobutyric acid, fermentation time is 10-50h; After fermentation ends, by centrifugal collection clear liquid;
(4) clear liquid that step (3) is obtained is further removed somatic cells through metal micron membranes strainer, obtains clear liquid;
(5) decolouring: the clear liquid pH that first step (4) is obtained transfers to 7.0-8.0, then by the processing of decolour of granulated active carbon post, and the collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, controls material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-aminobutyric acid clear liquid;
(7) the γ-aminobutyric acid clear liquid that step (6) is obtained, make γ-aminobutyric acid powder product or γ-aminobutyric acid crystal product;
The described method of making the γ-aminobutyric acid powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when feed liquid is concentrated into alpha-aminobutyric acid content for 〉=80g/L, stop concentrating, collect concentrated solution;
(b) spraying drying: add maltodextrin or starch in concentrated solution, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-aminobutyric acid powder product of 20-50%;
The described method of making the γ-aminobutyric acid crystal product is: with the γ-aminobutyric acid clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-aminobutyric acid iso-electric point,, by decrease temperature crystalline, obtain the γ-aminobutyric acid crystal product; Described ion exchange resin is first selected anionite-exchange resin, to remove residual substrate glutamic acid sodium, then passes through acidic cation-exchange resin, to γ-aminobutyric acid adsorb, wash-out processes; Described anionite-exchange resin is D301, D296 or 201 type highly basic or weak base anion-exchange resins; Described Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin; The iso-electric point lowering temperature crystallization is adopted in crystallization, the feed liquid that obtains after ion exchange resin treatment is carried out crystallization or add ethanol and carry out crystallization, and adding proportion is ethanol: feed liquid=1～10:1(V/V); During crystallization, regulator solution pH value is to the iso-electric point 7.2 of γ-aminobutyric acid, and the greenhouse cooling scope is 90～5 ℃, and the addition of crystal seed is 0. 2-10g/L, and stir speed (S.S.) is 30-100r/min, and rate of temperature fall is 1-10 ℃/h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103045965A CN102796779B (en) | 2012-08-24 | 2012-08-24 | Biological method for preparing gamma-aminobutyric acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012103045965A CN102796779B (en) | 2012-08-24 | 2012-08-24 | Biological method for preparing gamma-aminobutyric acid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102796779A CN102796779A (en) | 2012-11-28 |
| CN102796779B true CN102796779B (en) | 2013-11-20 |
Family
ID=47196076
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012103045965A Active CN102796779B (en) | 2012-08-24 | 2012-08-24 | Biological method for preparing gamma-aminobutyric acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102796779B (en) |
Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102978250B (en) * | 2012-12-20 | 2014-07-30 | 江苏久吾高科技股份有限公司 | Method for producing Gamma-aminobutyric acid through centrifugal mother liquid of glutamic acid |
| CN104561160A (en) * | 2014-12-22 | 2015-04-29 | 南通励成生物工程有限公司 | Method for preparing theanine by using biological method |
| CN104531795A (en) * | 2015-01-13 | 2015-04-22 | 北京格力森生物工程技术有限公司 | Method for producing high-purity gamma-aminobutyric acid |
| CN105671098A (en) * | 2016-04-06 | 2016-06-15 | 河南巨龙生物工程股份有限公司 | Method for producing L-2-aminobutyric acid by fermentation process |
| CN106544372A (en) * | 2016-11-04 | 2017-03-29 | 广西多得乐生物科技有限公司 | A kind of method that gamma aminobutyric acid is purified from zymotic fluid |
| CN106417480B (en) * | 2016-11-21 | 2019-11-08 | 昆明理工大学 | A kind of production technology of the bread rich in γ-aminobutyric acid |
| CN106417533A (en) * | 2016-11-21 | 2017-02-22 | 昆明理工大学 | Making method of gamma-aminobutyric acid containing biscuits |
| CN107827765A (en) * | 2016-12-05 | 2018-03-23 | 南通励成生物工程有限公司 | A kind of gamma aminobutyric acid hemi-hydrate crystalline and preparation method thereof |
| CN107326052B (en) * | 2017-06-28 | 2020-12-22 | 岭南师范学院 | Method for improving glutamate decarboxylase activity by using D101 macroporous adsorption resin |
| CN107252021A (en) * | 2017-07-10 | 2017-10-17 | 秦皇岛长胜营养健康科技有限公司 | Reduce asparagus drink of Intima-medial thickness effect and its preparation method and application |
| CN108300742A (en) * | 2017-07-26 | 2018-07-20 | 南通励成生物工程有限公司 | A kind of method that current adding substrate enzyme prepares γ-aminobutyric acid |
| CN109369431B (en) * | 2018-11-30 | 2021-06-18 | 沧州信联化工有限公司 | Crystallization method of gamma-aminobutyric acid |
| CN109761832A (en) * | 2019-03-05 | 2019-05-17 | 内蒙古精晶生物科技有限公司 | A kind of purification process of 4-Aminobutanoicacid |
| CN115322450B (en) * | 2022-08-25 | 2023-12-08 | 华熙生物科技股份有限公司 | Composition and preparation method and application thereof |
| CN116655485B (en) * | 2023-06-14 | 2024-01-23 | 山东福瑞达生物科技有限公司 | Process for converting and extracting gamma-aminobutyric acid |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1276087C (en) * | 2005-03-07 | 2006-09-20 | 浙江大学 | Process for biologically synthesizing gamma-amino butyric acid |
| CN101333548A (en) * | 2008-06-26 | 2008-12-31 | 南昌大学 | Method for preparing gamma-aminobutyric acid by lactobacillus brevis |
| CN101838672B (en) * | 2010-05-10 | 2014-12-17 | 江南大学 | Method for producing gamma-amino butyric acid by using immobilized lactobacillus plantarum |
| CN102108370B (en) * | 2010-12-02 | 2013-03-06 | 中国农业大学 | Semicontinuous fermentation method for producing gamma-aminobutyric acid by lactic acid bacteria |
| CN102174449B (en) * | 2011-03-04 | 2012-08-22 | 天津科技大学 | Method for producing high-yield gamma-propalanine and application thereof |
| CN102199636A (en) * | 2011-04-13 | 2011-09-28 | 王健 | Efficient preparation process of gamma-amino-n-butyric acid |
-
2012
- 2012-08-24 CN CN2012103045965A patent/CN102796779B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102796779A (en) | 2012-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102796779B (en) | Biological method for preparing gamma-aminobutyric acid | |
| CN101838672B (en) | Method for producing gamma-amino butyric acid by using immobilized lactobacillus plantarum | |
| CN100562581C (en) | A kind of method and special-purpose reaction column thereof of producing γ-An Jidingsuan | |
| CN103320362B (en) | One strain is produced the bacterial strain of L-Glutamic decarboxylase and is produced the method for γ-aminobutyric acid with it | |
| CN109504720B (en) | Green production process of glutamic acid | |
| CN104694612A (en) | Method for highly producing L-tryptophan employing industrial fermentation | |
| CN104531795A (en) | Method for producing high-purity gamma-aminobutyric acid | |
| CN103642853B (en) | A kind of L MALIC ACID novel technology for extracting | |
| CN103966274A (en) | Method for biotransformation production of gamma-aminobutyric acid with aquatic products and processing leftovers thereof as raw materials | |
| CN106544372A (en) | A kind of method that gamma aminobutyric acid is purified from zymotic fluid | |
| CN105219661B (en) | The special strain therefore of synthesis of oligonucleotides galactolipin and method with its synthesis of oligonucleotides galactolipin | |
| CN103130664A (en) | Process method of extracting gamma-aminobutyric acid through membrane separation technique | |
| CN113321580B (en) | Method for producing malic acid | |
| CN110904163A (en) | Method for improving lactic acid content of corn steep liquor | |
| CN109136299B (en) | Method for preparing, extracting and purifying threonine | |
| CN101974151B (en) | Method for preparing bio-based nylon polybutyrolactam | |
| JP6008505B2 (en) | Method for producing GABA-rich yeast | |
| CN109609565B (en) | A kind of method of fermenting and producing l-Isoleucine | |
| CN104694614B (en) | A kind of extraction process of L-Trp | |
| CN107083407A (en) | A kind of preparation, separation and the edulcoration purification process of L malic acid | |
| Hoeks et al. | Process integration aspects for the production of fine chemicals illustrated with the biotransformation of γ-butyrobetaine into l-carnitine | |
| CN107326052B (en) | Method for improving glutamate decarboxylase activity by using D101 macroporous adsorption resin | |
| CN100526470C (en) | Preparation method of functional sweetener D-tatai sugar | |
| CN110004192A (en) | A kind of method of preparing granular type threonine | |
| CN104561160A (en) | Method for preparing theanine by using biological method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220325 Address after: 226010 No. 333, Xinxing East Road, development zone, Nantong City, Jiangsu Province Patentee after: Nantong Licheng Biotechnology Co.,Ltd. Address before: No. 333, Xinxing East Road, development zone, Nantong City, Jiangsu Province, 226000 Patentee before: NANTONG LICHENG BIOLOGICAL ENGINEERING Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Method for preparing g-aminobutyric acid by biological method Effective date of registration: 20220831 Granted publication date: 20131120 Pledgee: Industrial Bank Co.,Ltd. Shanghai Putuo sub branch Pledgor: Nantong Licheng Biotechnology Co.,Ltd. Registration number: Y2022310000220 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20231023 Granted publication date: 20131120 Pledgee: Industrial Bank Co.,Ltd. Shanghai Putuo sub branch Pledgor: NANTONG LICHENG BIOLOGICAL ENGINEERING Co.,Ltd.|Nantong Licheng Biotechnology Co.,Ltd. Registration number: Y2022310000220 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right |