CN102796779A - Biological method for preparing gamma-aminobutyric acid - Google Patents
Biological method for preparing gamma-aminobutyric acid Download PDFInfo
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- CN102796779A CN102796779A CN2012103045965A CN201210304596A CN102796779A CN 102796779 A CN102796779 A CN 102796779A CN 2012103045965 A CN2012103045965 A CN 2012103045965A CN 201210304596 A CN201210304596 A CN 201210304596A CN 102796779 A CN102796779 A CN 102796779A
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title abstract description 21
- 229960003692 gamma aminobutyric acid Drugs 0.000 title abstract description 11
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title abstract description 9
- 238000010170 biological method Methods 0.000 title abstract 3
- 239000007788 liquid Substances 0.000 claims abstract description 75
- 238000000034 method Methods 0.000 claims abstract description 45
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 241000894006 Bacteria Species 0.000 claims abstract description 17
- 239000013078 crystal Substances 0.000 claims abstract description 16
- 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
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 238000000909 electrodialysis Methods 0.000 claims abstract description 9
- 235000014655 lactic acid Nutrition 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
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- 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
- 230000031018 biological processes and functions 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
- 238000012545 processing Methods 0.000 claims description 13
- 238000002425 crystallisation Methods 0.000 claims description 12
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- 239000004721 Polyphenylene oxide Substances 0.000 claims description 10
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- 239000003456 ion exchange resin Substances 0.000 claims description 10
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- 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
- 230000001580 bacterial effect Effects 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
- 239000003729 cation exchange resin Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 claims description 6
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- 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
- 229940061607 dibasic sodium phosphate Drugs 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
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019797 dipotassium phosphate Nutrition 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
- 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
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- 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
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- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 241000186685 Lactobacillus hilgardii Species 0.000 claims description 3
- 239000003957 anion exchange resin 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
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- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 229910052757 nitrogen Inorganic materials 0.000 abstract 1
- 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
- 235000013305 food Nutrition 0.000 description 4
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- 229960004249 sodium acetate Drugs 0.000 description 4
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- 244000299461 Theobroma cacao Species 0.000 description 3
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- 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
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- 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
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- 206010020772 Hypertension Diseases 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
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- 235000013361 beverage Nutrition 0.000 description 1
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- 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
- 238000006555 catalytic reaction Methods 0.000 description 1
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- 239000002054 inoculum Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
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- 231100000219 mutagenic Toxicity 0.000 description 1
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- 210000005036 nerve Anatomy 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
- 238000010926 purge Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
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- 239000000126 substance Substances 0.000 description 1
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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-An Jidingsuan.
Background technology
(Gamma-aminobutyric acid GABA), claims aminobutyric acid again to γ-An Jidingsuan, is that a kind of naturally occurring nonprotein is formed amino acid, is distributed widely in protokaryon and the eukaryote.γ-An Jidingsuan is the inhibitory transmitter substance of mammalian central nervous system, have calm nerve, promote sleep, bring high blood pressure down, brain tonic and intelligence development, delay senility, important physical function such as strong liver profit kidney.
Up to now, the preparation method of γ-An Jidingsuan mainly contains two kinds of chemical synthesis and biological processes.Common chemical synthesis process be as with pyrrolidone as raw material, make γ-An Jidingsuan through calcium hydroxide, bicarbonate of ammonia hydrolysis.The chemical synthesis cost is higher, and yield is lower, and in production technique, uses dangerous solvents, or even deep-etching, noxious solvent.Therefore, the γ-An Jidingsuan of chemical synthesis preparation is difficult in food factories and realizes that 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-An Jidingsuan 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) reorganization 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 the existing method, the γ-An Jidingsuan fecund is born in the fermented liquid, because the fermented liquid complicated component; The downstream separation purge process is loaded down with trivial details; The concentration of title product is also lower, these effects limit domestic industry production, and rarely have report especially about preparation high purity γ-An Jidingsuan.
Summary of the invention
The object of the present invention is to provide a kind of method easy, easy to operate, effective biological process prepares the method for γ-An Jidingsuan.
Technical solution of the present invention is:
A kind of biological process prepares the method for γ-An Jidingsuan, it is characterized in that: comprise the following steps:
(1) thalline preparation:
The Sodium Glutamate that in substratum, adds 1-30g/L is induced the product enzyme as inductor, inserts the bacterial strain that produces L-Glutamic decarboxylase in the born of the same parents and cultivates; Culture condition is: 25～35 ℃ of culture temperature, carry out pH control through adding alkali lye in the 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-An Jidingsuan:
The damping fluid of preparation 0.01-0.5mol/L pH3-7 adds the 10-100g/L somatic cells, adds substrate glutamic acid sodium in batches; Interpolation concentration is 0-30g/L; Be under 28-40 ℃ the condition in temperature of reaction, produce γ-An Jidingsuan through fermentation once more, fermentation time is 10-50h; After the fermentation ends, through 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 earlier step (4) is obtained transfers to 7.0-8.0, then through processings of decolouring of granulated active carbon post, collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, control material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-An Jidingsuan clear liquid;
(7) the γ-An Jidingsuan clear liquid that step (6) is obtained is processed γ-An Jidingsuan powder product or γ-An Jidingsuan crystal product.
The said method of processing the γ-An Jidingsuan powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when treating that feed liquid is concentrated into alpha-aminobutyric acid content for >=80g/L, stop to concentrate, collect liquid concentrator;
(b) spraying drying: in liquid concentrator, add maltodextrin or starch, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-An Jidingsuan powder product of 20-50%.
The said method of processing the γ-An Jidingsuan crystal product is: with the γ-An Jidingsuan clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-An Jidingsuan iso-electric point,, obtain the γ-An Jidingsuan crystal product through decrease temperature crystalline.
The bacterial strain of L-Glutamic decarboxylase is milk-acid bacteria or lactobacillus hilgardii in the said product of step (1) born of the same parents.
The content that the composition of the employed substratum of step (1) reaches in g/L is:
Glucose 10-30, yeast extract paste 5-30, peptone .5-30, sodium acetate 0.5-5, Sodium phosphate, dibasic or potassium hydrogenphosphate 0.05-10, sal epsom 0.05-10, manganous sulfate 0.05-1, PPE polyether antifoam agent 0.1-3.
Said ion exchange resin is selected anionite-exchange resin earlier for use, to remove residual substrate glutamic acid sodium, passes through acidic cation-exchange resin again, to γ-An Jidingsuan adsorb, wash-out handles.
Said anionite-exchange resin is D301, D296 or 201 type highly basic or weakly basic anion exchange resins; Said Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin.
Iso-electric point decrease temperature crystalline method is adopted in crystallization, and the feed liquid that after ion exchange resin treatment, obtains is carried out crystallization or added ethanol and carry out crystallization, and adding proportion is an ethanol: feed liquid=0～10:1V/V; The iso-electric point 7.2 of regulator solution pH value to γ-An Jidingsuan during crystallization, 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 catalysis of free lactic acid mycetocyte to synthesize γ-An Jidingsuan, and improves production technique, and γ-An Jidingsuan output is improved, and cost reduces, but also has set up the method for preparing the high purity γ-An Jidingsuan.Do not contain carbon source, nitrogenous source etc. in this technology in the reaction system and cultivate composition, the reaction solution composition is simple for fermented liquid, can increase substantially product purity, and it is simple, with short production cycle and environmental pollution is little, low cost and other advantages to have aftertreatment simultaneously.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is described further.
Fig. 1 is a γ-An Jidingsuan formation curve in the one embodiment of the invention preparation of industrialization process.
Fig. 2 is the amino acid detected result that transforms the conversion fluid that generates γ-An Jidingsuan with lactic-acid bacteria cells.Glu: L-glutamic acid; GABA: γ-An Jidingsuan.
Embodiment
γ-An Jidingsuan of the present invention is meant the 4-propalanine, is the mammalian central nervous system inhibitory transmitter.As new resource food, can add in beverage, cocoa products, chocolate and chocolate, candy, bakery product, the puffed food development of new product to.
Embodiment 1:
One, yeast culture
After lactic acid bacteria culturers is activated, carries out first order seed and cultivate, secondary seed is cultivated; Get into fermentor cultivation at last, inoculum size 3% (V/V, seed liquor/fermented liquid); 30 ℃ leave standstill cultivation 16 h; Carry out pH control through adding alkali lye in the fermenting process, fermented liquid pH is maintained in the 4.5-7.0 scope, collect thalline through 0.6 μ m ceramic membrane filter.
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 potassium hydrogenphosphate) 1, sal epsom 1; Manganous sulfate 1, and PPE polyether antifoam agent 0.1 (but be not limited only to this skimmer, can select any skimmer that can be used for fermentation industry for use).
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 potassium hydrogenphosphate) 1; Sal epsom 1; Manganous sulfate 1, and PPE polyether antifoam agent 0.1 (but be not limited only to this skimmer, can select any skimmer that can be used for fermentation industry for use).
Two, produce γ-An Jidingsuan
Adopt the lactic-acid bacteria cells conversion method to produce γ-An Jidingsuan, reaction conditions is: acetic acid-sodium-acetate buffer of preparation 0.01mol/L pH7 adds 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 the end,, obtain the γ-An Jidingsuan conversion fluid that content reaches 12g/L, see Fig. 1 through centrifugal collection clear liquid.
Three, the detection of alpha-aminobutyric acid content
With the trichoroacetic acid(TCA) of the centrifugal back adding of above-mentioned conversion fluid equal-volume 100g/L, vibration is even, and centrifugal again (10,000 * g, 5 min), clear liquid dilute 2-10 doubly, and warp 0.45 μ m membrane filtration carries out high-efficient liquid phase analysis again.Adopt ODS C
18(φ 4.6 * 250nm), and 40
oC analyzes, and γ-An Jidingsuan is quantitative with external standard method.
Four, product prepn:
Filtration sterilization: the γ-An Jidingsuan conversion fluid that obtains is further removed somatic cells through metal micron membranes strainer, obtain clear liquid;
Decolouring: the clear liquid pH that obtains after the filtration sterilization is transferred to 7.0-8.0, then through processings of decolouring of granulated active carbon post, the collection clear liquid;
Electrodialysis: adopt electrodialysis to remove impurity, control material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-An Jidingsuan clear liquid;
With the γ-An Jidingsuan clear liquid that step (4) obtains, process γ-An Jidingsuan powder product or γ-An Jidingsuan crystal product.
The said method of processing the γ-An Jidingsuan powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when treating that feed liquid is concentrated into alpha-aminobutyric acid content for >=50g/L, stop to concentrate;
(b) spraying drying: add maltodextrin or starch in the liquid concentrator, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-An Jidingsuan powder product of 20-50%;
The said method of processing the γ-An Jidingsuan powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when treating that feed liquid is concentrated into alpha-aminobutyric acid content for >=80g/L, stop to concentrate, collect liquid concentrator;
(b) spraying drying: in liquid concentrator, add maltodextrin or starch, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-An Jidingsuan powder product of 20-50%.
The said method of processing the γ-An Jidingsuan crystal product is: with the γ-An Jidingsuan clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-An Jidingsuan iso-electric point,, obtain purity and reach 98% γ-An Jidingsuan crystal product through decrease temperature crystalline.
Embodiment 2:
A kind of biological process prepares the method for γ-An Jidingsuan, comprises the following steps:
(1) thalline preparation:
In substratum, add Sodium Glutamate, addition be 1-30g/L (routine 1 g/L, 15 g/L, 30 g/L) as inductor, induce the product enzyme, insert to produce the bacterial strain of L-Glutamic decarboxylase in the born of the same parents and cultivate; Culture condition is: 25～35 ℃ of culture temperature (25 ℃, 30 ℃, 35 ℃ of examples), carry out pH control through adding alkali lye in the 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 (routine 0.05 μ m, 0.5 μ m, 1 μ m), obtains thalline;
(3) produce γ-An Jidingsuan:
The damping fluid of preparation 0.01-0.5mol/L (routine 0.01 mol/L, 0.2 mol/L, 0.5 mol/L) pH3-7 (example 3,5,7); Add somatic cells; The somatic cells add-on is 10-100g/L (routine 10 g/L, 50 g/L, 100 g/L); Add substrate glutamic acid sodium, interpolation concentration is 1-30g/L (routine 1g/L, 15 g/L, 30 g/L) in batches, is under the condition of 28-40 ℃ (28 ℃, 32 ℃, 40 ℃ of examples) in temperature of reaction; Produce γ-An Jidingsuan through fermenting once more, fermentation time is 10-50h (routine 10h, 25h, 50h); After the fermentation ends, through 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 earlier step (4) is obtained transfers to 7.0-8.0, then through processings of decolouring of granulated active carbon post, collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, control material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-An Jidingsuan clear liquid;
(7) the γ-An Jidingsuan clear liquid that step (6) is obtained is processed γ-An Jidingsuan powder product or γ-An Jidingsuan crystal product.
The said method of processing the γ-An Jidingsuan powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when treating that feed liquid is concentrated into alpha-aminobutyric acid content for >=80g/L, stop to concentrate, collect liquid concentrator;
(b) spraying drying: in liquid concentrator, add maltodextrin or starch, regulating solid quality content is 5-50% (example 5%, 30%, 50%), carries out spraying drying, and making mass content is the γ-An Jidingsuan powder product of 20-50%.
The said method of processing the γ-An Jidingsuan crystal product is: with the γ-An Jidingsuan clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-An Jidingsuan iso-electric point,, obtain the γ-An Jidingsuan crystal product through decrease temperature crystalline.
The bacterial strain of L-Glutamic decarboxylase is milk-acid bacteria or lactobacillus hilgardii in the said product of step (1) born of the same parents.
The content that the composition of the employed substratum of step (1) reaches in g/L is:
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 potassium hydrogenphosphate 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).
Said ion exchange resin is selected anionite-exchange resin earlier for use, to remove residual substrate glutamic acid sodium, passes through acidic cation-exchange resin again, to γ-An Jidingsuan adsorb, wash-out handles.
Said anionite-exchange resin is D301, D296 or 201 type highly basic or weakly basic anion exchange resins; Said Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin.
Iso-electric point decrease temperature crystalline method is adopted in crystallization, and the feed liquid that after ion exchange resin treatment, obtains is carried out crystallization or added ethanol and carry out crystallization, and adding proportion is an ethanol: feed liquid=0～10:1V/V (routine 1:1,5:1,10:1); The iso-electric point 7.2 of regulator solution pH value to γ-An Jidingsuan during crystallization; The greenhouse cooling scope is 90～5 ℃ (90 ℃, 40 ℃, 5 ℃ of examples); The addition of crystal seed is 0. 2-10g/L (routine 0.2 g/L, 5 g/L, 10 g/L); Stir speed (S.S.) is 30-100r/min, rate of temperature fall be 1-10 ℃/h (example 1 ℃/h, 5 ℃/h, 10 ℃/h).
Claims (8)
1. a biological process prepares the method for γ-An Jidingsuan, it is characterized in that: comprise the following steps:
(1) thalline preparation:
The Sodium Glutamate that in substratum, adds 1-30g/L is induced the product enzyme as inductor, inserts the bacterial strain that produces L-Glutamic decarboxylase in the born of the same parents and cultivates; Culture condition is: 25～35 ℃ of culture temperature, carry out pH control through adding alkali lye in the 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-An Jidingsuan:
The damping fluid of preparation 0.01-0.5mol/L pH3-7 adds the 10-100g/L somatic cells, adds substrate glutamic acid sodium in batches; Interpolation concentration is 0-30g/L; Be under 28-40 ℃ the condition in temperature of reaction, produce γ-An Jidingsuan through fermentation once more, fermentation time is 10-50h; After the fermentation ends, through 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 earlier step (4) is obtained transfers to 7.0-8.0, then through processings of decolouring of granulated active carbon post, collection clear liquid;
(6) electrodialysis: adopt electrodialysis to remove impurity, control material liquid pH is 7.0-8.0, drops in specific conductivity≤during 1000 μ s/cm, stop circulation, collection γ-An Jidingsuan clear liquid;
(7) the γ-An Jidingsuan clear liquid that step (6) is obtained is processed γ-An Jidingsuan powder product or γ-An Jidingsuan crystal product.
2. biological process according to claim 1 prepares the method for γ-An Jidingsuan, it is characterized in that: the said method of processing the γ-An Jidingsuan powder product comprises the following steps: successively
(a) vacuum concentration: adopt the triple effect plate-type evaporator to concentrate, when treating that feed liquid is concentrated into alpha-aminobutyric acid content for >=80g/L, stop to concentrate, collect liquid concentrator;
(b) spraying drying: in liquid concentrator, add maltodextrin or starch, regulating solid quality content is 5-50%, carries out spraying drying, and making mass content is the γ-An Jidingsuan powder product of 20-50%.
3. biological process according to claim 1 prepares the method for γ-An Jidingsuan, it is characterized in that: the said method of processing the γ-An Jidingsuan crystal product is: with the γ-An Jidingsuan clear liquid that step (6) obtains, and the process ion exchange resin treatment; Then material liquid pH is transferred to the γ-An Jidingsuan iso-electric point,, obtain the γ-An Jidingsuan crystal product through decrease temperature crystalline.
4. prepare the method for γ-An Jidingsuan according to claim 1,2 or 3 described biological processes, it is characterized in that: the bacterial strain of L-Glutamic decarboxylase is milk-acid bacteria or lactobacillus hilgardii in the said product of step (1) born of the same parents.
5. prepare the method for γ-An Jidingsuan according to claim 1,2 or 3 described biological processes, it is characterized in that: the content that the composition of the employed substratum of step (1) reaches in g/L is:
Glucose 10-30, yeast extract paste 5-30, peptone .5-30, sodium acetate 0.5-5, Sodium phosphate, dibasic or potassium hydrogenphosphate 0.05-10, sal epsom 0.05-10, manganous sulfate 0.05-1, PPE polyether antifoam agent 0.1-3.
6. biological process according to claim 3 prepares the method for γ-An Jidingsuan; It is characterized in that: said ion exchange resin is selected anionite-exchange resin earlier for use; To remove residual substrate glutamic acid sodium; Pass through acidic cation-exchange resin again, to γ-An Jidingsuan adsorb, wash-out handles.
7. biological process according to claim 6 prepares the method for γ-An Jidingsuan, it is characterized in that: said anionite-exchange resin is D301, D296 or 201 type highly basic or weakly basic anion exchange resins; Said Zeo-karb is 001 or D113 type strong acid or weakly acidic cation-exchange resin.
8. biological process according to claim 3 prepares the method for γ-An Jidingsuan; It is characterized in that: iso-electric point decrease temperature crystalline method is adopted in crystallization; The feed liquid that after ion exchange resin treatment, obtains is carried out crystallization or added ethanol and carry out crystallization, and adding proportion is an ethanol: feed liquid=0～10:1V/V; The iso-electric point 7.2 of regulator solution pH value to γ-An Jidingsuan during crystallization, 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.
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