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.