CN104151385A - Method for extracting cyclic adenosine monophosphate and application of cyclic adenosine monophosphate - Google Patents
Method for extracting cyclic adenosine monophosphate and application of cyclic adenosine monophosphate Download PDFInfo
- Publication number
- CN104151385A CN104151385A CN201410401250.6A CN201410401250A CN104151385A CN 104151385 A CN104151385 A CN 104151385A CN 201410401250 A CN201410401250 A CN 201410401250A CN 104151385 A CN104151385 A CN 104151385A
- Authority
- CN
- China
- Prior art keywords
- precipitation
- gained
- concentrated
- under
- volume
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 51
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 title abstract 5
- 239000006228 supernatant Substances 0.000 claims abstract description 22
- 239000003960 organic solvent Substances 0.000 claims abstract description 18
- 238000000855 fermentation Methods 0.000 claims abstract description 14
- 230000004151 fermentation Effects 0.000 claims abstract description 14
- 239000000047 product Substances 0.000 claims abstract description 11
- 238000001914 filtration Methods 0.000 claims abstract description 10
- 238000001035 drying Methods 0.000 claims abstract description 9
- 239000002244 precipitate Substances 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 7
- 238000001556 precipitation Methods 0.000 claims description 53
- 239000007788 liquid Substances 0.000 claims description 37
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 29
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- -1 cyclic monophosphate Chemical class 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 16
- 239000000706 filtrate Substances 0.000 claims description 16
- 238000001291 vacuum drying Methods 0.000 claims description 13
- 238000005194 fractionation Methods 0.000 claims description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 239000008213 purified water Substances 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000001223 reverse osmosis Methods 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 230000008719 thickening Effects 0.000 claims description 7
- 238000004090 dissolution Methods 0.000 claims description 5
- 241000186063 Arthrobacter Species 0.000 claims description 4
- 239000003513 alkali Substances 0.000 claims description 4
- 230000000813 microbial effect Effects 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 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 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 239000011347 resin Substances 0.000 abstract description 23
- 229920005989 resin Polymers 0.000 abstract description 23
- 238000004587 chromatography analysis Methods 0.000 abstract description 9
- 239000002351 wastewater Substances 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 7
- 238000000605 extraction Methods 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000002845 discoloration Methods 0.000 abstract 2
- 230000001376 precipitating effect Effects 0.000 abstract 2
- 239000013543 active substance Substances 0.000 abstract 1
- 239000003814 drug Substances 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 238000005342 ion exchange Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 238000010828 elution Methods 0.000 description 6
- 239000011230 binding agent Substances 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 230000008025 crystallization Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 230000006837 decompression Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002107 myocardial effect Effects 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000009834 vaporization Methods 0.000 description 2
- 230000008016 vaporization Effects 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 241001524201 Arthrobacter crystallopoietes Species 0.000 description 1
- 241000167367 Arthrobacter sp. A302 Species 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 241000185994 Pseudarthrobacter oxydans Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 229930003756 Vitamin B7 Natural products 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 1
- 229950006790 adenosine phosphate Drugs 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005261 decarburization Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035806 respiratory chain Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011735 vitamin B7 Substances 0.000 description 1
- 235000011912 vitamin B7 Nutrition 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses a method for extracting cyclic adenosine monophosphate and application of cyclic adenosine monophosphate, and belongs to the technical field of extraction of biological active substances. The method provided by the invention comprises the following steps: adjusting the pH of a fermentation liquor by using an alkaline liquor; performing centrifugal separation to obtain a supernatant; concentrating the supernatant, and performing first discoloration; fractionally precipitating a discolored solution by using an organic solvent; dissolving an obtained precipitate, and performing second discoloration; performing micro porous filtration, and concentrating a second discolored solution; adding the organic solvent for precipitating the concentrated second discolored solution; and performing centrifugal separation, and drying the precipitate in vacuum to obtain a finished product. By adopting the method provided by the invention, the conventional ionic exchange and resin chromatography processes can be replaced, and the waste water amount can be lowered by 82.5 percent; meanwhile, the production period is shortened greatly, and the production time is shortened by 36 percent; and the prepared cyclic adenosine monophosphate is stable in quality, can be over 97 percent in purity, and can be widely applied to the fields of medicine, foods, feeds and the like.
Description
Technical field
The present invention relates to a kind of method and application of extracting cyclic monophosphate, belong to microbial fermentation product extractive technique field.
Background technology
CAMP is for participating in regulating second messenger's material of cell function, and its effect is very extensive, can make myocardial contraction strengthen, and causes elevation of the blood pressure, and cardiac output increases.And can diastole unstriated muscle, coronary artery dilator blood vessel, improve liver function, promote neurotization, suppress the division of skin outer layer epithelial cell and transform paracytic function, promote the oxidasic activity of respiratory chain and improve myocardial anoxia etc.
At present the main production method of cyclic monophosphate is chemical synthesis, exists that yield is low, cost is high, the shortcoming that toxic side effect is large; In order to overcome these shortcomings, develop a kind of biological method and prepare cyclic monophosphate and become research tendency, mainly contain at present two kinds of research directions of red date extraction method and biological fermentation process.And these two kinds of methods are compared, biological fermentation process has and not limited by starting material, the cheap and easily buying of starting material, scale is easily amplified, constant product quality, and on cost, have very big advantage, so this method is a kind of method of the tool potentiality of instead of chemical synthesis method.
To the technique of fermentative Production cyclic monophosphate product extraction aspect, be mainly ion-exchange and adsorption chromatography at present, the shortcoming of this method maximum is that wastewater flow rate is large, this is also the common problem that all resin isolation exist, raising along with environmental requirement, cost for wastewater treatment also starts to rise, therefore from economic and social benefit angle, a kind of develop instead of resins separation method just seems quite necessary.
Summary of the invention
For addressing the above problem, the invention provides a kind of method of extracting cyclic monophosphate from fermented liquid, the technical scheme of taking is as follows:
The object of the present invention is to provide a kind of method of extracting cyclic monophosphate from fermented liquid, the method is that after regulating the pH of fermented liquid, supernatant liquor is obtained in centrifugation, after concentrated supernatant, decolour for the first time, utilize organic solvent fractionation precipitation destainer, after dissolving again gained precipitation, decolour for the second time, concentrated the second destainer after millipore filtration, adds the second destainer after organic solvent deposit concentrates, and after centrifugation vacuum-drying precipitation, obtains finished product.
The step of described method is as follows:
1) utilize alkali lye to regulate the pH to 8.0-8.5 of fermented liquid, obtain supernatant liquor after centrifugal;
2) enrichment step 1) gained supernatant liquor, and the processing of decolouring, obtain the first destainer;
3) utilizing organic solvent to step 2) the first destainer of gained carries out fractionation precipitation, the fractional precipitation of centrifugation collection unit;
4) utilize purified water dissolving step 3) partly precipitated of gained, the processing of decolouring for the second time after dissolving, obtains filtrate after millipore filtration;
5) enrichment step 4) gained filtrate, then add organic solvent to carry out precipitation process, within centrifugal minute, obtain precipitation;
6) vacuum drying step 5) gained precipitation acquisition finished product.
Step 1) described fermented liquid is Arthrobacter fermented liquid; Described alkali lye is oxyhydroxide or the carbonate compound solution of sodium or potassium; Described centrifugal, be centrifugal 5-6min under 4500-5000rpm.
Step 2) described concentrated, be concentrating under reduced pressure, vacuum tightness is-0.065---0.075MPa that thickening temperature is 45-50 ℃, concentrated final concentration 20-25g/L; Described decolouring is processed, and uses craboraffin, under 55-60 ℃ of condition, processes 15-20min, and gac addition is 1% of concentrated solution volume.
Step 3) described fractionation precipitation, is to use dehydrated alcohol or acetone, and the mode adding by stream is carried out; Described collection unit fractional precipitation is the precipitation of collecting organic solvent concentration 40%-50%.
Step 4) described purified water, is two-pass reverse osmosis water, specific conductivity≤2 μ S/cm, and amount of water is 5 times of precipitation quality; Described decolouring is for the second time processed, and is the 15min that decolours under normal temperature condition with injection active carbon, and the addition of injection active carbon is 6% of precipitation quality.
Step 5) described concentrated, be at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, the volume of filtrate is concentrated into 1/3 of original volume; Described precipitation process is that to add dehydrated alcohol or acetone to solvent volume concentration be 70%.
Step 6) described vacuum-drying, drying temperature is 45 ℃, time of drying 10h.
The concrete steps of described method are as follows:
1) utilize the pH to 8.0-8.5 of sodium hydroxide solution adjusting joint bacillus fermentation liquid, then by fermented liquid centrifugal 5min under 5000rpm, obtain supernatant liquor;
2) at vacuum tightness-0.065---0.075MPa, under the condition of thickening temperature 45-50 ℃ by step 1) gained supernatant concentration to concentration is 25g/L, to the craboraffin that adds concentrated solution volume 1% in concentrated solution, at 60 ℃, 15min is processed in decolouring, remove after gac, obtain the first destainer;
3) mode adding with stream is to step 2) add dehydrated alcohol or acetone to carry out fractionation precipitation processing in the first destainer of gained, collect the precipitation of organic solvent concentration 40%-50%;
4) to step 3) add the two-pass reverse osmosis water dissolution precipitation of the specific conductivity≤2 μ S/cm of 5 times of quality in the partly precipitated of gained, then add the injection active carbon that precipitate quality 6%, at room temperature decolouring is processed 15min and is had millipore filtration acquisition filtrate;
5) at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, by step 4) volume of gained filtrate is concentrated into 1/3 of original volume, then to add dehydrated alcohol or acetone to solvent volume concentration be 70%, centrifugal 20min under 8000rpm, obtains precipitation;
6) under 45 ℃ of conditions, vacuum drying step 5) gained precipitation 10h.
Described method is for extracting cyclic monophosphate from microbial fermentation solution.
The present invention preferably uses tubular-bowl centrifuge to the separation of fermented liquid; The preferred single-action of concentrating under reduced pressure equipment or multiple-effect decompression evaporator; The separated preferably explosion-proof type cloth bag loading and unloading material whizzer of organic solvent deposit, the preferred tubular type decarburizing machine of decarburization.
Beneficial effect of the present invention:
1, take the method for twice crystalline deposit, substitution ion exchange and resin chromatography technique, reduce wastewater flow rate more than 70%.
2, adopt tubular-bowl centrifuge to carry out separation to intermediates, than traditional pressure filter and membrane filter plant, good separating effect, continuous production is strong, reduces labour intensity, water saving.
3, the production cycle shortens greatly, and by original 72 hours foreshorten to 48 hours.
4, product with stable quality, purity is not less than 95%, and heavy metal content meets enterprise-quality standard.One time yield is greater than 72%.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described, but the present invention is not subject to the restriction of embodiment.
Method therefor of the present invention, reagent and instrument, without special instruction, be ordinary method, reagent and instrument in the art.
The preparation of embodiment 1 fermented liquid
The present invention's fermented liquid used is Arthrobacter fermented liquid, wherein, and preferred strain Arthrobacter crystallopoietes, Arthrobacter oxidans, Arthrobacter CCTCC NO:M2013431, the fermented liquid of Arthrobacter sp.A302.The present embodiment provides a kind of preparation method of above-mentioned bacterial strains fermented liquid.
The present embodiment used medium is specifically composed as follows:
Slant medium (g/L): glucose 10, peptone 10, yeast extract paste 10, extractum carnis 10, NaCl3, agar 20.pH?6.8。
Seed culture medium (g/L): glucose 10, peptone 10, yeast extract paste 10, extractum carnis 10, NaCl3.pH?6.8。
Fermention medium (g/L): glucose 30, K
2hPO
415, KH
2pO
45, MgSO
40.1, urea 10, vitamin H 0.005, CoCl
20.005, NaCl0.4, xanthoglobulin 5, pH 7.0.
Fermentation condition is as follows:
-80 ℃ of Refrigerator store bacterial classifications are inoculated in to slant medium activation, cultivate 48h for 30 ℃.Lawn on slant medium is inoculated in seed culture medium, 30 ℃, 280r/min shaking table cultivation 24h; By cultured seed culture medium, with 8% inoculum size access fermention medium, 30 ℃, 280r/min shaking table are cultivated 72h.Above seed culture medium and fermention medium are all used 500mL shaking flask, and liquid amount is 30mL.
Embodiment 2
The present embodiment provides a kind of method that adopts ion-exchange and resin chromatography method to extract adenosine phosphate, and step is as follows:
1) get the prepared fermented liquid 1L of embodiment 1, fermentation unit 3g/L, centrifugal 5min under 5000rpm rotating speed, collects supernatant liquor, with concentrated hydrochloric acid, adjusts pH to 2.0.
2) the separated resin used of ion-exchange is 001 * 7 type Zeo-karb, and resin dress column volume 200mL, is processed into Hydrogen with 4% hydrochloric acid.Upper column flow rate 0.3 column volume per hour is per hour, uses the purified water binder of 2 times of bed volumes after upper prop finishes again, and binder finishes rear with 0.1N sodium hydroxide wash-out, and elution flow rate is the same, collects elutriant 450mL.
3) resin chromatography chromatographic resin is macroporous adsorbent resin HP-20, resin dress column volume 300mL, will be from handing over the rear elutriant of collecting to add in resin, loading flow velocity 0.3 column volume is per hour, after end of the sample, adopt 20% methanol solution stepwise elution of 3 times of column volume purified water and 5 times of column volumes, collect methanol solution wash-out part, elution flow rate is the same.
4) concentrated that elutriant is concentrated in decompression evaporator, vacuum tightness-0.065--0.075MPa, vaporization temperature 40-45 ℃, is concentrated into 1/5 of fermentating liquid volume.
5) crystallization adds dehydrated alcohol to whole dense degree 70%, stirred crystallization 12 hours toward concentrated solution
6) separated drying crystalline utilizes whizzer separation to obtain wet crystal after completing, and is then placed in vacuum drying oven dry, drying temperature 40-45 ℃.
Embodiment 3
1) get the prepared fermented liquid 1L of embodiment 1, fermentation unit 4g/L, centrifugal 5min under 5000rpm rotating speed, collects supernatant liquor, with concentrated hydrochloric acid, adjusts pH to 3.0.
2) the separated resin used of ion-exchange is 001 * 7 type Zeo-karb, and resin dress column volume 200mL, is processed into Hydrogen with 4% hydrochloric acid.Upper column flow rate 0.3 column volume per hour is per hour, uses the purified water binder of 2 times of bed volumes after upper prop finishes again, and binder finishes rear with 0.1N sodium hydroxide wash-out, and elution flow rate is the same, collects elutriant 450mL.
3) resin chromatography chromatographic resin is macroporous adsorbent resin HP-20, resin dress column volume 300mL, will be from handing over the rear elutriant of collecting to add in resin, loading flow velocity 0.3 column volume is per hour, after end of the sample, adopt 20% methanol solution stepwise elution of 3 times of column volume purified water and 5 times of column volumes, collect methanol solution wash-out part, elution flow rate is the same.
4) concentrated that elutriant is concentrated in decompression evaporator, vacuum tightness-0.065--0.075MPa, vaporization temperature 40-45 ℃, is concentrated into 1/5 of fermentating liquid volume.
5) crystallization adds dehydrated alcohol to whole dense degree 70%, stirred crystallization 12 hours toward concentrated solution
6) separated drying crystalline utilizes whizzer separation to obtain wet crystal after completing, and is then placed in vacuum drying oven dry, drying temperature 40-45 ℃.
Embodiment 4
The present embodiment provides a kind of method of extracting cyclic monophosphate from fermented liquid, and step is as follows:
1) get the prepared fermented liquid 1L of embodiment 1, fermentation unit 4g/L utilizes sodium hydroxide solution to regulate the pH to 8.0-8.5 of fermented liquid, then by fermented liquid centrifugal 5min under 5000rpm, obtains supernatant liquor;
2) at vacuum tightness-0.065---0.075MPa, under the condition of thickening temperature 45-50 ℃ by step 1) gained supernatant concentration to concentration is 25g/L, to the craboraffin that adds concentrated solution volume 1% in concentrated solution, at 60 ℃, 15min is processed in decolouring, remove after gac, obtain the first destainer;
3) mode adding with stream is to step 2) add dehydrated alcohol or acetone to carry out fractionation precipitation processing in the first destainer of gained, collect the precipitation of organic solvent concentration 40%-50%;
4) to step 3) add the two-pass reverse osmosis water dissolution precipitation of the specific conductivity≤2 μ S/cm of 5 times of quality in the partly precipitated of gained, then add the injection active carbon that precipitate quality 6%, at room temperature decolouring is processed 15min and is had millipore filtration acquisition filtrate;
5) at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, by step 4) volume of gained filtrate is concentrated into 1/3 of original volume, then to add dehydrated alcohol or acetone to solvent volume concentration be 70%, centrifugal 10min under 8000rpm, obtains precipitation;
6) under 45 ℃ of conditions, vacuum drying step 5) gained precipitation 10h.
Embodiment 5
The present embodiment provides a kind of method of extracting cyclic monophosphate from fermented liquid, and step is as follows:
1) get the prepared fermented liquid 1L of embodiment 1, fermentation unit 4g/L utilizes sodium hydroxide solution to regulate the pH to 8.0-8.5 of fermented liquid, then by fermented liquid centrifugal 5min under 5000rpm, obtains supernatant liquor;
2) at vacuum tightness-0.065---0.075MPa, under the condition of thickening temperature 45-50 ℃ by step 1) gained supernatant concentration to concentration is 25g/L, to the craboraffin that adds concentrated solution volume 1% in concentrated solution, at 60 ℃, 10min is processed in decolouring, remove after gac, obtain the first destainer;
3) mode adding with stream is to step 2) add dehydrated alcohol or acetone to carry out fractionation precipitation processing in the first destainer of gained, collect the precipitation of organic solvent concentration 40%-50%;
4) to step 3) add the two-pass reverse osmosis water dissolution precipitation of the specific conductivity≤2 μ S/cm of 5 times of quality in the partly precipitated of gained, then add the injection active carbon that precipitate quality 6%, at room temperature decolouring is processed 15min and is had millipore filtration acquisition filtrate;
5) at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, by step 4) volume of gained filtrate is concentrated into 1/3 of original volume, then to add dehydrated alcohol or acetone to solvent volume concentration be 70%, with centrifugal 15min under 8000rpm, obtain precipitation;
6) under 45 ℃ of conditions, vacuum drying step 5) gained precipitation 10h.
Embodiment 6
The present embodiment provides a kind of method of extracting cyclic monophosphate from fermented liquid, and step is as follows:
1) get the prepared fermented liquid 1L of embodiment 1, fermentation unit 4g/L utilizes sodium hydroxide solution to regulate the pH to 8.0-8.5 of fermented liquid, then by fermented liquid centrifugal 5min under 4000rpm, obtains supernatant liquor;
2) at vacuum tightness-0.065---0.075MPa, under the condition of thickening temperature 45-50 ℃ by step 1) gained supernatant concentration to concentration is 25g/L, to the craboraffin that adds concentrated solution volume 1% in concentrated solution, at 50 ℃, 15min is processed in decolouring, remove after gac, obtain the first destainer;
3) mode adding with stream is to step 2) add dehydrated alcohol or acetone to carry out fractionation precipitation processing in the first destainer of gained, collect the precipitation of organic solvent concentration 40%-50%;
4) to step 3) add the two-pass reverse osmosis water dissolution precipitation of the specific conductivity≤2 μ S/cm of 5 times of quality in the partly precipitated of gained, then add the injection active carbon that precipitate quality 6%, at room temperature decolouring is processed 15min and is had millipore filtration acquisition filtrate;
5) at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, by step 4) volume of gained filtrate is concentrated into 1/3 of original volume, then to add dehydrated alcohol or acetone to solvent volume concentration be 70%, with centrifugal 15min under 8000rpm, obtain precipitation;
6) under 45 ℃ of conditions, vacuum drying step 5) gained precipitation 10h.
Embodiment 7
The purity of embodiment 2-6 gained sample that the present embodiment has utilized high effective liquid chromatography for measuring, result is as shown in table 1;
Table 1 embodiment 2-6 prepares purity and the extraction yield of cyclic monophosphate
| ? | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 |
| Purity | 99 | 97 | 97 | 99 | 98 |
| Extraction yield | 65％ | 62 | 72％ | 72.5 | 71％ |
As can be seen from Table 1, the purity of the method products therefrom of embodiment 4-6 and embodiment 2 and 3 ion-exchange and resin chromatography methods used do not have marked difference, but the extraction yield of product is but apparently higher than embodiment 2 and 3 ion-exchange and resin chromatography methods used.Illustrate that method provided by the present invention is more efficient than ion-exchange and resin chromatography method.
Table 2 is that embodiment 2-6 prepares cyclic monophosphate wastewater flow rate used and time.
Table 2 embodiment 2-6 prepares waste water consumption and the time used of cyclic monophosphate
| ? | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Embodiment 6 |
| Wastewater flow rate | 10L | 12L | 2.5L | 2.1L | 2.3L |
| Preparation total time | 73 | 72 | 49 | 47 | 49 |
As can be seen from Table 2, embodiment 4-6 wastewater flow rate used is obviously less than embodiment 2-3 wastewater flow rate used, meanwhile, prepares also obviously shortening of time used.Compare with resin chromatography method with ion-exchange, method therefor of the present invention can be saved the water of 75%-82.5%, shortens the time of 31%-36%.
Although the present invention with preferred embodiment openly as above; but it is not in order to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; can do various changes and modification, so protection scope of the present invention should be with being as the criterion that claims were defined.
Claims (10)
1. a method of extracting cyclic monophosphate, it is characterized in that, after the pH of adjusting fermented liquid, supernatant liquor is obtained in centrifugation, after concentrated supernatant, decolour for the first time, utilize organic solvent fractionation precipitation destainer, then decolour for the second time after dissolving gained precipitation, concentrated the second destainer after millipore filtration, the second destainer after adding organic solvent deposit concentrated, obtains finished product after centrifugation vacuum-drying precipitation.
2. method described in claim 1, is characterized in that, step is as follows:
1) utilize alkali lye to regulate the pH to 8.0-8.5 of fermented liquid, after centrifugation, collect supernatant liquor;
2) enrichment step 1) gained supernatant liquor, and the processing of decolouring, obtain the first destainer;
3) utilizing organic solvent to step 2) the first destainer of gained carries out fractionation precipitation, the fractional precipitation of centrifugation collection unit;
4) utilize purified water dissolving step 3) partly precipitated of gained, the processing of decolouring for the second time after dissolving, obtains filtrate after millipore filtration;
5) enrichment step 4) gained filtrate, then add organic solvent to carry out precipitation process, within centrifugal minute, obtain precipitation;
6) vacuum drying step 5) gained precipitation acquisition finished product.
3. method described in claim 2, is characterized in that step 1) described fermented liquid is Arthrobacter fermented liquid; Described alkali lye is oxyhydroxide or the carbonate compound solution of sodium or potassium; Described centrifugal, be centrifugal 5-6min under 4500-5000rpm.
4. method described in claim 2, is characterized in that step 2) described concentrated, be concentrating under reduced pressure, vacuum tightness is-0.065---0.075MPa that thickening temperature is 45-50 ℃, concentrated final concentration 20-25g/L; Described decolouring is processed, and uses craboraffin, under 55-60 ℃ of condition, processes 15-20min, and gac addition is 1% of concentrated solution volume.
5. method described in claim 2, is characterized in that step 3) described fractionation precipitation, be to use dehydrated alcohol or acetone, the mode adding by stream is carried out; Described collection unit fractional precipitation is the precipitation of collecting organic solvent concentration 40%-50%.
6. method described in claim 2, is characterized in that step 4) described purified water, be two-pass reverse osmosis water, specific conductivity≤2 μ S/cm, amount of water is 5 times of precipitation quality; Described decolouring is for the second time processed, and is the 15min that decolours under normal temperature condition with injection active carbon, and the addition of injection active carbon is 6% of precipitation quality.
7. method described in claim 2, is characterized in that step 5) described concentrated, be at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, the volume of filtrate is concentrated into 1/3 of original volume; Described precipitation process is that to add dehydrated alcohol or acetone to solvent volume concentration be 70%.
8. method described in claim 2, is characterized in that step 6) described vacuum-drying, drying temperature is 45 ℃, time of drying 10h.
9. method described in claim 2, is characterized in that, concrete steps are as follows:
1) utilize the pH to 8.0-8.5 of sodium hydroxide solution adjusting joint bacillus fermentation liquid, then by fermented liquid centrifugal 5min under 5000rpm, obtain supernatant liquor;
2) at vacuum tightness-0.065---0.075MPa, under the condition of thickening temperature 45-50 ℃ by step 1) gained supernatant concentration to concentration is 20-25g/L, to the craboraffin that adds concentrated solution volume 1% in concentrated solution, at 60 ℃, 15min is processed in decolouring, remove after gac, obtain the first destainer;
3) mode adding with stream is to step 2) add dehydrated alcohol or acetone to carry out fractionation precipitation processing in the first destainer of gained, collect the precipitation of organic solvent concentration 40%-50%;
4) to step 3) add the two-pass reverse osmosis water dissolution precipitation of the specific conductivity≤2 μ S/cm of 5 times of quality in the partly precipitated of gained, then add the injection active carbon that precipitate quality 6%, at room temperature decolouring is processed 15min and is had millipore filtration acquisition filtrate;
5) at vacuum tightness-0.065---0.075MPa, under the condition of temperature 45-50 ℃, by step 4) volume of gained filtrate is concentrated into 1/3 of original volume, then to add dehydrated alcohol or acetone to solvent volume concentration be 70%, centrifugal 20min under 8000rpm, obtains precipitation;
6) under 45 ℃ of conditions, vacuum drying step 5) gained precipitation 10h.
10. method described in claim 1, for extracting cyclic monophosphate from microbial fermentation solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410401250.6A CN104151385B (en) | 2014-08-15 | 2014-08-15 | A kind of method extracting cyclic adenosine monophosphate and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410401250.6A CN104151385B (en) | 2014-08-15 | 2014-08-15 | A kind of method extracting cyclic adenosine monophosphate and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104151385A true CN104151385A (en) | 2014-11-19 |
| CN104151385B CN104151385B (en) | 2016-12-07 |
Family
ID=51877038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410401250.6A Expired - Fee Related CN104151385B (en) | 2014-08-15 | 2014-08-15 | A kind of method extracting cyclic adenosine monophosphate and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104151385B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104788522A (en) * | 2015-03-26 | 2015-07-22 | 安徽省皖北药业股份有限公司 | Method for extracting cyclic adenosine monophosphate from fermentation liquid |
| CN105541946A (en) * | 2016-03-11 | 2016-05-04 | 阎虎林 | Adenosine cyclophosphate crystalline compound |
| CN107417750A (en) * | 2017-05-16 | 2017-12-01 | 河南科技学院 | A kind of method that CAMP is extracted from microbial fermentation solution |
| CN109851651A (en) * | 2019-02-18 | 2019-06-07 | 江苏集萃工业生物技术研究所有限公司 | A kind of method that macroporous absorbent resin separates cyclic adenosine monophosphate from catalytic liquid |
| CN112300968A (en) * | 2020-11-19 | 2021-02-02 | 河南巨龙生物工程股份有限公司 | Arthrobacter producing adenosine cyclophosphate and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899080A (en) * | 2010-01-28 | 2010-12-01 | 山东鼎力枣业食品集团有限公司 | Production method of golden-silk jujube cyclic adenosine monophosphate |
| CN102268469A (en) * | 2010-06-04 | 2011-12-07 | 南京工业大学 | Preparation method of cyclic adenosine monophosphate |
-
2014
- 2014-08-15 CN CN201410401250.6A patent/CN104151385B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101899080A (en) * | 2010-01-28 | 2010-12-01 | 山东鼎力枣业食品集团有限公司 | Production method of golden-silk jujube cyclic adenosine monophosphate |
| CN102268469A (en) * | 2010-06-04 | 2011-12-07 | 南京工业大学 | Preparation method of cyclic adenosine monophosphate |
Non-Patent Citations (2)
| Title |
|---|
| 王春霞等，: "纳膜过滤在提取红枣环磷酸腺苷( cAMP) 中的应用.", 《食品与发酵工业》, vol. 37, no. 2, 31 December 2011 (2011-12-31), pages 186 - 190 * |
| 王荔等，: "大枣环磷酸腺苷提取纯化工艺的初步研究.", 《食品科技》, vol. 37, no. 4, 31 December 2012 (2012-12-31), pages 190 - 195 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104788522A (en) * | 2015-03-26 | 2015-07-22 | 安徽省皖北药业股份有限公司 | Method for extracting cyclic adenosine monophosphate from fermentation liquid |
| CN105541946A (en) * | 2016-03-11 | 2016-05-04 | 阎虎林 | Adenosine cyclophosphate crystalline compound |
| CN107417750A (en) * | 2017-05-16 | 2017-12-01 | 河南科技学院 | A kind of method that CAMP is extracted from microbial fermentation solution |
| CN107417750B (en) * | 2017-05-16 | 2020-04-21 | 河南科技学院 | Method for extracting cyclic adenosine monophosphate from microbial fermentation liquid |
| CN109851651A (en) * | 2019-02-18 | 2019-06-07 | 江苏集萃工业生物技术研究所有限公司 | A kind of method that macroporous absorbent resin separates cyclic adenosine monophosphate from catalytic liquid |
| CN112300968A (en) * | 2020-11-19 | 2021-02-02 | 河南巨龙生物工程股份有限公司 | Arthrobacter producing adenosine cyclophosphate and application thereof |
| CN112300968B (en) * | 2020-11-19 | 2023-10-27 | 河南巨龙生物工程股份有限公司 | Arthrobacter for producing adenosine cyclophosphate and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104151385B (en) | 2016-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104151385A (en) | Method for extracting cyclic adenosine monophosphate and application of cyclic adenosine monophosphate | |
| CN101702984B (en) | Phellinus igniarius mycelium liquid fermentation method by utilizing fungal elicitor for improving yield of flavone of phellinus igniarius | |
| CN108823261A (en) | A kind of Ultra-low molecular weight Dendrobium officinale polysaccharide and its preparation and application | |
| CN102746348A (en) | Method for separation of lincomycin | |
| CN104788522A (en) | Method for extracting cyclic adenosine monophosphate from fermentation liquid | |
| CN107151685B (en) | Method for producing chondroitin sulfate by fermentation method | |
| CN103992953B (en) | Dongxiang wild rice endophytic fungus for converting glycyrrhizic acid to generate glycyrrhetinic acid glycoside | |
| CN113480587A (en) | Method for efficiently extracting N6- (2-hydroxyethyl) adenosine from cordyceps sobolifera sporocarp | |
| CN106544372A (en) | A kind of method that gamma aminobutyric acid is purified from zymotic fluid | |
| CN109576324A (en) | A kind of astragalus polyose and its biological extraction method | |
| CN104357332B (en) | Aspergillus niger JH-2 and application to biotransformation and synthesis of asiatic acid | |
| CN103342668A (en) | Simple method for extracting natural taurine from abalone viscera | |
| CN106167753A (en) | A kind of synchronize to adsorb common bacterium fermentation method and remove inferior Fructus Mali pumilae completely and brewage the method for Penicillium patulum toxin in applejack | |
| CN103193854A (en) | Method for separation and purification of betulinol and biological and chemical conversion of betulinic acid | |
| CN100393738C (en) | Natamycin extracting and purifying method | |
| CN103504286A (en) | Method for extracting grifola frondosa mycelium based on membrane technology | |
| CN104031109A (en) | Method for purifying tea saponin by microbial fermentation | |
| CN106636252A (en) | Thelephora ganbajun Zang exopolysaccharide, preparation method thereof and application of exopolysaccharide | |
| CN108117558B (en) | Method for separating teddenox A and teddenox B from fermented tea | |
| CN108276467A (en) | A kind of Tea Saponin and its extraction process and application | |
| CN104357527A (en) | Method for extracting tea saponin from tea seed meal with microbial fermentation method | |
| CN106491661B (en) | Method for extracting fresh cordyceps sinensis raw pulp | |
| CN104774794A (en) | Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same | |
| CN108384814A (en) | A kind of preparation method of phloretin | |
| CN104045723A (en) | Method for extracting tea polysaccharide by biotechnology |
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 | ||
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20161201 Address after: 150028, Heilongjiang, Harbin Province, Songbei Innovation Road 600, No. three Patentee after: Wu Li Patentee after: Zhang Liang Patentee after: Feng Dienan Patentee after: Liu Weizhuo Address before: 150030 Xiangfang City, Harbin Province Wood Street, No. 59, No. Patentee before: Liu Wanyi |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20161207 Termination date: 20190815 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |