CN105669560B - A method of the separation and Extraction tetrahydropyrimidine from fermentation liquid - Google Patents
A method of the separation and Extraction tetrahydropyrimidine from fermentation liquid Download PDFInfo
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- C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
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Abstract
The problems such as present invention provides a kind of method of separation and Extraction tetrahydropyrimidine from fermentation liquid, overcomes process route present in existing production technology complicated, isolates and purifies difficulty, and energy consumption water consume is more.Present invention separation and Extraction tetrahydropyrimidine from the fermentation liquid rich in tetrahydropyrimidine, first with thallus, most of albumen and the partial pigment in dual membrane system, that is, microfiltration membranes, Ultra filtration membrane system filtering removal fermentation liquid, then cationic exchange resin adsorption tetrahydropyrimidine is utilized, after again tetrahydropyrimidine being eluted with ammonium hydroxide, obtain tetrahydropyrimidine crystal by operating procedures such as active carbon decoloring, concentration alcohol precipitation, recrystallization, dry finished products.
Description
Technical field
The method of the present invention relates to a kind of from fermentation liquid separation and Extraction tetrahydropyrimidine, belongs to fermentation method production amino acid and spreads out
The technical field of biology.
Background technique
Tetrahydropyrimidine (Ectoine), is called (S)-2-methyl-1,4,5,6-tetra-hydro pyrimidine-4-carboxylic acid, and English scientific name is Isosorbide-5-Nitrae, and 5,
6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid.It is a kind of cyclic amino acid derivativges, phase
To molecular weight 142.16, molecular formula C6H10N2O2, the amino and a carboxyl having on molecular structure makes it have ammonia
The amphotericity of base acid.Tetrahydropyrimidine is energy substance, tonicity-adjusting substances and the cell and macromolecular complex of microbial cell
The Bioprotective of matter, can alleviate hypertonic, high temperature, freeze thawing, drying, radiation and chemical reagent to albumen, nucleic acid, biomembrane and
The toxic action of entire cell.Since tetrahydropyrimidine is in all known compatible solutes to biological cell and macromolecular substances
Protecting effect is best, so it is also increasingly subject to close in cosmetics, enzyme industry and the application in the fields such as biological medicine in recent years
Note.
The production method of tetrahydropyrimidine has extraction method, chemical synthesis, enzyme catalysis method and fermentation method.Extraction method and chemistry close
At method since raw material sources are restricted, isolate and purify difficulty, yield is low, and product purity is low, pollution environment it is serious, mass production at
This height, it is difficult to realize industrialized production.And fermentation method production is low with cost of material, reaction condition is mild, is easily achieved big rule
The advantages that mould produces.
The prior art is to extract tetrahydropyrimidine from Halophiles cell.Patent application 201410754792.1 provides one kind
The extraction process of tetrahydropyrimidine, it is main include pretreatment, tetrahydropyrimidine dissolution, active carbon decoloring, electrodialysis desalination, concentration, from
Son exchange, concentration, freezing and crystallizing.This method pretreatment be related to thalline were collected by centrifugation, it is dry, crush plus water makes tetrahydro
Pyrimidine dissolution and etc., technique very complicated, centrifugation, Grin-ding energ7 are high.Simultaneously because using higher concentration in Halophiles culture
NaCl, therefore two step desalinating process of electrodialysis and cation and anion exchange resin is needed during extracting tetrahydropyrimidine, containing acid
The discharge amount of alkali waste water is big.Patent application 201110097221.1 provides tetrahydropyrimidine and the membrane technology of hydroxy tetrahydro pyrimidine mentions
Method is taken, this method need to use water hypoosmotic shock to discharge tetrahydropyrimidine, and use the desalination of aromatic polyamide composite membrane, UF membrane
At high cost, extract yield is low.A kind of use of novel gene engineering colon bacillus can make tetrahydropyrimidine big in nearest research
Amount runs up in fermentation liquid (application number 201510410080.2), which can be secreted into tetrahydropyrimidine
It is not necessarily to add the NaCl of high concentration in fermentation liquid and in culture medium.Therefore, the present invention provides a kind of from rich in tetrahydropyrimidine
The technique that tetrahydropyrimidine is extracted in fermentation liquid, compared with the technique for extracting tetrahydropyrimidine from Halophiles cell, this simple process
It is easy, separative efficiency is high, separation costs are low, great industrial value.
Summary of the invention
It is an object of the present invention to provide a kind of separation-extraction technologies of novel tetrahydropyrimidine, overcome in existing production technology and deposit
Process route it is complicated, isolate and purify difficulty, the problems such as energy consumption water consume is more.
The technical solution that the present invention takes to achieve the goals above is summarized as follows:
Present invention separation and Extraction tetrahydropyrimidine from the fermentation liquid rich in tetrahydropyrimidine, first with dual membrane system, that is, micro-filtration
Thallus, most of albumen and partial pigment in film, Ultra filtration membrane system filtering removal fermentation liquid, are then handed over using cation
Resin adsorption tetrahydropyrimidine is changed, then after being eluted tetrahydropyrimidine with ammonium hydroxide, by active carbon decoloring, concentration alcohol precipitation, recrystallization, done
The operating procedures such as dry finished product obtain tetrahydropyrimidine crystal.
Technical solution of the present invention specifically comprises the following steps:
A, the fermentation liquid rich in tetrahydropyrimidine is taken, micro-filtration film separation system is pumped into after being 3.0-6.0 with salt acid for adjusting pH, is received
Collect filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, the tetrahydropyrimidine for the thallus that is removed is micro-
Filtrate；
B, the micro-filtrate that a step obtains is pumped into Ultra filtration membrane system, removing protein and decolorization is carried out to micro-filtrate, received
Collect filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, the tetrahydro of be removed albumen and pigment
Pyrimidine ultrafiltrate；
C, the ultrafiltrate that b step obtains is pumped into ion exchange column, through cationic exchange resin adsorption, sample introduction flow velocity is 1BV/
H, is eluted after adsorption saturation using the ammonium hydroxide of 0.02-0.2mol/L, and elution flow rate 0.05-2BV/h collects the four of pH=7-12
The high flow point eluent of hydrogen pyrimidine；
D, the active carbon decoloring for high flow point eluent addition 0.05%-5% (m/v) for obtaining c step, 20-40 DEG C of temperature,
Stirring decoloration 10-30min, filtering obtain tetrahydropyrimidine destainer；
E, the destainer that d step obtains is concentrated under reduced pressure into the 5%-15% of original volume, it is phonetic that ethanol precipitation acquisition tetrahydro is added
Pyridine crude product；
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, is filtered after being stood still for crystals at 4 DEG C
Crystal is obtained, obtains tetrahydropyrimidine finished product after 60 DEG C of dryings.
Further, the micro-filtration film separation system in above-mentioned steps a uses hollow fiber microfiltration membrane, and micro-filtration membrane aperture is
0.02-0.2 μm, operation temperature is 25-45 DEG C, operating pressure 0.05-0.2MPa.
Further, the Ultra filtration membrane system in above-mentioned steps b uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is
0.001-0.01 μm, operation temperature is 25-45 DEG C, operating pressure 0.1-0.5MPa.
Further, the cation exchange resin in above-mentioned steps c can be gel type resin, be also possible to macroporous type tree
Rouge.
Further, the cation exchange resin in above-mentioned steps c is specifically as follows 001*7, D072, in D061 resin
One kind, preferably D061.
Further, the present invention also provides a kind of method for detecting above-mentioned tetrahydropyrimidine finished product purity, UltiMate is used
3000 (Thermo Scientific) high performance liquid chromatographs measure tetrahydropyrimidine, and chromatographic column is TSK-GEL C18 chromatographic column,
30 DEG C of column temperature, mobile phase is 2% acetonitrile, flow velocity 1mL/min, ultraviolet detection wavelength 210nm.
The utility model has the advantages that
Compared with the prior art, the method have the advantages that:
1) present invention is using thallus, most of albumen and the pigment in dual membrane system removal fermentation liquid, and bacterium is collected by centrifugation
Body is compared, and energy consumption is lower, and the rejection of thallus and protein is higher.
2) present invention utilizes cation exchange resin to adsorb tetrahydropyrimidine according to the characteristics of tetrahydropyrimidine ampholytes,
The impurity such as inorganic salt anionic, acetic acid in fermentation liquid can effectively be removed.Simultaneously because inorganic salt cation and resin cation
Binding ability is stronger, can be selective by four by control ammonia concn and elution flow rate when being eluted using ammonium hydroxide
Hydrogen pyrimidine preferentially elutes, and the ammonium ion in eluent can then volatilize away during reduced pressure, thus effectively
The separation for realizing tetrahydropyrimidine avoids the complex operations such as electrodialysis, cation and anion exchange resin or nanofiltration desalination.
3) present invention passes through the side of ethanol precipitation and recrystallization according to tetrahydropyrimidine the characteristics of being slightly soluble in ethyl alcohol soluble easily in water
Method is refined, and the rate of recovery and purity of product are effectively increased.
Detailed description of the invention
Fig. 1: the extraction process flow chart of tetrahydropyrimidine of the present invention.
Specific embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Range, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
Under the premise of invention spirit and scope, to the various changes or change of material component and dosage progress in these embodiments
It belongs to the scope of protection of the present invention.
Embodiment 1:
A, tetrahydropyrimidine fermentation liquid 5L (tetrahydropyrimidine content 25.0g/L) is taken, is 4.0 with the salt acid for adjusting pH of 4mol/L,
Into micro-filtration film separation system, the tetrahydropyrimidine micro-filtrate of removal thallus is obtained；The micro-filtration film separation system uses hollow fibre
Microfiltration membranes are tieed up, micro-filtration membrane aperture is 0.02 μm, and operation temperature is 30 DEG C, operating pressure 0.2MPa.Filtered fluid is collected, filtering is worked as
Stop collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in liquid, thallus removing rate 99.2%.
B, the micro-filtrate walked through a is pumped into Ultra filtration membrane system, obtains the tetrahydropyrimidine of removing protein and pigment
Ultrafiltrate.The Ultra filtration membrane system uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is 0.003 μm, operation temperature 40
DEG C, operating pressure 0.3MPa collects filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, egg
White removal rate is 80.8%, light transmittance 72.3%.
C, the ultrafiltrate that b step obtains is pumped into D061 cationic exchange resin adsorption with the flow velocity of 1BV/h, after adsorption saturation
It is eluted with the ammonium hydroxide of 0.15mol/L, elution flow rate 0.05BV/h, collects the high flow point eluent of tetrahydropyrimidine that pH is 7-12.
D, c step is obtained into the active carbon decoloring that 3% (m/v) is added in high flow point eluent, 40 DEG C of temperature, stirring is decolourized
15min, filtering obtain tetrahydropyrimidine destainer, light transmittance 99.8%.
E, the destainer that d step obtains is concentrated under reduced pressure into the 10% of substance hydrops, ethanol precipitation is added and obtains tetrahydropyrimidine
Crude product.
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, is filtered after being stood still for crystals at 4 DEG C
Crystal is obtained, obtains tetrahydropyrimidine finished product 101.3g after 60 DEG C of dryings.
G, tetrahydropyrimidine is measured using (Thermo Scientific) high performance liquid chromatograph of UltiMate 3000.Take f
The finished product that step obtains uses micro-sampling pin, 20 μ L of sample volume after being dissolved in water, chromatographic column is TSK-GEL C18 chromatographic column, column
30 DEG C of temperature, mobile phase are 2% acetonitrile, flow velocity 1mL/min, ultraviolet detection wavelength 210nm.Tetrahydropyrimidine is calculated according to standard curve
Purity is 98.2%.
Embodiment 2:
A, tetrahydropyrimidine fermentation liquid 5L (tetrahydropyrimidine content 25.0g/L) is taken, is 6.0 with the salt acid for adjusting pH of 4mol/L,
Into micro-filtration film separation system, the tetrahydropyrimidine micro-filtrate of removal thallus is obtained；The micro-filtration film separation system uses hollow fibre
Microfiltration membranes are tieed up, micro-filtration membrane aperture is 0.05 μm, and operation temperature is 35 DEG C, operating pressure 0.15MPa.Filtered fluid is collected, has been served as
Stop collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtrate, thallus removing rate 97.4%.
B, the micro-filtrate walked through a is pumped into Ultra filtration membrane system, obtains the tetrahydropyrimidine of removing protein and pigment
Ultrafiltrate.The Ultra filtration membrane system uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is 0.01 μm, operation temperature 35
DEG C, operating pressure 0.1MPa collects filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, egg
White removal rate is 74.1%, light transmittance 66.5%.
C, the ultrafiltrate that b step obtains is pumped into 001*7 cationic exchange resin adsorption, adsorption saturation with the flow velocity of 1BV/h
It is eluted afterwards with the ammonium hydroxide of 0.2mol/L, elution flow rate 2BV/h, collects the high flow point eluent of tetrahydropyrimidine that pH is 7-12.
D, c step is obtained into the active carbon decoloring that 4% (m/v) is added in high flow point eluent, 20 DEG C of temperature, stirring is decolourized
10min, filtering obtain tetrahydropyrimidine destainer, light transmittance 99.5%.
E, the destainer that d step obtains is concentrated under reduced pressure into the 8% of substance hydrops, it is thick that ethanol precipitation acquisition tetrahydropyrimidine is added
Product.
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, is filtered after being stood still for crystals at 4 DEG C
Crystal is obtained, obtains tetrahydropyrimidine finished product 98.6g after 60 DEG C of dryings.
G, tetrahydropyrimidine is measured using (Thermo Scientific) high performance liquid chromatograph of UltiMate 3000.Take f
The finished product that step obtains uses micro-sampling pin, 20 μ L of sample volume after being dissolved in water, chromatographic column is TSK-GEL C18 chromatographic column, column
30 DEG C of temperature, mobile phase are 2% acetonitrile, flow velocity 1mL/min, ultraviolet detection wavelength 210nm.Tetrahydropyrimidine is calculated according to standard curve
Purity is 98.0%.
Embodiment 3:
A, tetrahydropyrimidine fermentation liquid 5L (tetrahydropyrimidine content 25.0g/L) is taken, is 5.0 with the salt acid for adjusting pH of 4mol/L,
Into micro-filtration film separation system, the tetrahydropyrimidine micro-filtrate of removal thallus is obtained；The micro-filtration film separation system uses hollow fibre
Microfiltration membranes are tieed up, micro-filtration membrane aperture is 0.12 μm, and operation temperature is 45 DEG C, operating pressure 0.1MPa.Filtered fluid is collected, filtering is worked as
Stop collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in liquid, thallus removing rate 94.6%.
B, the micro-filtrate walked through a is pumped into Ultra filtration membrane system, obtains the tetrahydropyrimidine of removing protein and pigment
Ultrafiltrate.The Ultra filtration membrane system uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is 0.006 μm, operation temperature 25
DEG C, operating pressure 0.2MPa collects filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, egg
White removal rate is 77.6%, light transmittance 69.8%.
C, the ultrafiltrate that b step obtains is pumped into D072 cationic exchange resin adsorption with the flow velocity of 1BV/h, after adsorption saturation
It is eluted with the ammonium hydroxide of 0.1mol/L, elution flow rate 1.5BV/h, collects the high flow point eluent of tetrahydropyrimidine that pH is 7-12.
D, c step is obtained into the active carbon decoloring that 2% (m/v) is added in high flow point eluent, 35 DEG C of temperature, stirring is decolourized
30min, filtering obtain tetrahydropyrimidine destainer, light transmittance 99.3%.
E, the destainer that d step obtains is concentrated under reduced pressure into the 15% of substance hydrops, ethanol precipitation is added and obtains tetrahydropyrimidine
Crude product.
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, is filtered after being stood still for crystals at 4 DEG C
Crystal is obtained, obtains tetrahydropyrimidine finished product 96.0g after 60 DEG C of dryings.
G, tetrahydropyrimidine is measured using (Thermo Scientific) high performance liquid chromatograph of UltiMate 3000.Take f
The finished product that step obtains uses micro-sampling pin, 20 μ L of sample volume after being dissolved in water, chromatographic column is TSK-GEL C18 chromatographic column, column
30 DEG C of temperature, mobile phase are 2% acetonitrile, flow velocity 1mL/min, ultraviolet detection wavelength 210nm.Tetrahydropyrimidine is calculated according to standard curve
Purity is 96.6%.
Embodiment 4:
A, tetrahydropyrimidine fermentation liquid 5L (tetrahydropyrimidine content 25.0g/L) is taken, is 3.0 with the salt acid for adjusting pH of 4mol/L,
Into micro-filtration film separation system, the tetrahydropyrimidine micro-filtrate of removal thallus is obtained；The micro-filtration film separation system uses hollow fibre
Microfiltration membranes are tieed up, micro-filtration membrane aperture is 0.2 μm, and operation temperature is 25 DEG C, operating pressure 0.05MPa.Filtered fluid is collected, filtering is worked as
Stop collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in liquid, thallus removing rate 92.0%.
B, the micro-filtrate walked through a is pumped into Ultra filtration membrane system, obtains the tetrahydropyrimidine of removing protein and pigment
Ultrafiltrate.The Ultra filtration membrane system uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is 0.001 μm, operation temperature 45
DEG C, operating pressure 0.5MPa collects filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, albumen
Removal rate is 82.4%, light transmittance 75.6%.
C, the ultrafiltrate that b step obtains is pumped into D061 cationic exchange resin adsorption with the flow velocity of 1BV/h, after adsorption saturation
It is eluted with the ammonium hydroxide of 0.02mol/L, elution flow rate 1BV/h, collects the high flow point eluent of tetrahydropyrimidine that pH is 7-12.
D, c step is obtained into the active carbon decoloring that 0.05% (m/v) is added in high flow point eluent, 30 DEG C of temperature, stirring is decolourized
20min, filtering obtain tetrahydropyrimidine destainer, light transmittance 98.8%.
E, the destainer that d step obtains is concentrated under reduced pressure into the 5% of substance hydrops, it is thick that ethanol precipitation acquisition tetrahydropyrimidine is added
Product.
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, is filtered after being stood still for crystals at 4 DEG C
Crystal is obtained, obtains tetrahydropyrimidine finished product 96.8g after 60 DEG C of dryings.
G, tetrahydropyrimidine is measured using (Thermo Scientific) high performance liquid chromatograph of UltiMate 3000.Take f
The finished product that step obtains uses micro-sampling pin, 20 μ L of sample volume after being dissolved in water, chromatographic column is TSK-GEL C18 chromatographic column, column
30 DEG C of temperature, mobile phase are 2% acetonitrile, flow velocity 1mL/min, ultraviolet detection wavelength 210nm.Tetrahydropyrimidine is calculated according to standard curve
Purity is 97.5%.
Claims (7)
1. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid, includes the following steps:
A, the fermentation liquid rich in tetrahydropyrimidine is taken, micro-filtration film separation system is pumped into after being 3.0-6.0 with salt acid for adjusting pH, collected
Filtrate stops collecting, the tetrahydropyrimidine micro-filtrate for the thallus that is removed when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid；
B, the micro-filtrate that a step obtains is pumped into Ultra filtration membrane system, removing protein and decolorization is carried out to micro-filtrate, collected
Filtrate stops collecting, the tetrahydropyrimidine of be removed albumen and pigment when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid
Ultrafiltrate；
C, the ultrafiltrate that b step obtains is pumped into ion exchange column, through cationic exchange resin adsorption, sample introduction flow velocity is 1BV/h, is inhaled
It is eluted after attached saturation using the ammonium hydroxide of 0.02-0.2mol/L, elution flow rate 0.05-2BV/h, the tetrahydro for collecting pH=7-12 is phonetic
The high flow point eluent of pyridine；
D, the high flow point eluent that c step obtains is added to the active carbon decoloring of 0.05%-5% m/v, 20-40 DEG C of temperature, stirring is de-
Color 10-30 min, filtering obtain tetrahydropyrimidine destainer；
E, the destainer that d step obtains is concentrated under reduced pressure into the 5%-15% of original volume, ethanol precipitation is added and obtains tetrahydropyrimidine crude product；
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, filters and obtains after being stood still for crystals at 4 DEG C
Crystal obtains tetrahydropyrimidine finished product after 60 DEG C of dryings.
2. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1, which is characterized in that step a
Described in micro-filtration film separation system use hollow fiber microfiltration membrane, micro-filtration membrane aperture be 0.02-0.2 μm, operation temperature 25-
45 DEG C, operating pressure 0.05-0.2MPa.
3. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1, which is characterized in that step b
Described in Ultra filtration membrane system use hollow fiber ultrafiltration membrane, ultrafiltration membrane aperture is 0.001-0.01 μm, and operation temperature is
25-45 DEG C, operating pressure 0.1-0.5MPa.
4. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1, which is characterized in that step c
Described in cation exchange resin be gel type resin or macroporous ion-exchange resin.
5. a kind of method of separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1 or 4, which is characterized in that step
Cation exchange resin described in rapid c is 001*7, D072, one of D061 resin.
6. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1, which is characterized in that further include
Detection to the obtained tetrahydropyrimidine finished product purity of step f, it is efficient using 3000 Thermo Scientific of UltiMate
Hplc determination tetrahydropyrimidine, chromatographic column are TSK-GEL C18 chromatographic column, and 30 DEG C of column temperature, mobile phase is 2% acetonitrile,
Flow velocity 1mL/min, ultraviolet detection wavelength 210nm.
7. a kind of method of the separation and Extraction tetrahydropyrimidine from fermentation liquid as described in claim 1, includes the following steps:
A, tetrahydropyrimidine fermentation liquid 5L, tetrahydropyrimidine content 25.0g/L are taken, is 4.0 with the salt acid for adjusting pH of 4mol/L, entrance is micro-
Membrane separation system obtains the tetrahydropyrimidine micro-filtrate of removal thallus；The micro-filtration film separation system uses doughnut micro-filtration
Film, micro-filtration membrane aperture are 0.02 μm, and operation temperature is 30 DEG C, operating pressure 0.2MPa, filtered fluid are collected, when in filtered fluid four
Hydrogen pyrimidine concentration stops collecting when being lower than 1.0g/L；Thallus removing rate is 99.2%；
B, the micro-filtrate walked through a is pumped into Ultra filtration membrane system, obtains the tetrahydropyrimidine ultrafiltration of removing protein and pigment
Liquid；The Ultra filtration membrane system uses hollow fiber ultrafiltration membrane, and ultrafiltration membrane aperture is 0.003 μm, and operation temperature is 40 DEG C, behaviour
Making pressure is 0.3MPa, collects filtered fluid, stops collecting when tetrahydropyrimidine concentration is lower than 1.0g/L in filtered fluid, albumen removal
Rate is 80.8%, light transmittance 72.3%；
C, the ultrafiltrate that b step obtains is pumped into D061 cationic exchange resin adsorption with the flow velocity of 1BV/h, is used after adsorption saturation
The ammonium hydroxide of 0.15mol/L elutes, elution flow rate 0.05BV/h, collects the high flow point eluent of tetrahydropyrimidine that pH is 7-12；
D, c step is obtained into the active carbon decoloring that 3% m/v is added in high flow point eluent, 40 DEG C of temperature, stirs decoloration 15min, filtering
Obtain tetrahydropyrimidine destainer, light transmittance 99.8%；
E, the destainer that d step obtains is concentrated under reduced pressure into the 10% of substance hydrops, ethanol precipitation is added and obtains tetrahydropyrimidine crude product；
F, the tetrahydropyrimidine crude product that e step obtains is redissolved in deionized water to being saturated, filters and obtains after being stood still for crystals at 4 DEG C
Crystal obtains tetrahydropyrimidine finished product 101.3g after 60 DEG C of dryings；
G, tetrahydropyrimidine is measured using 3000 Thermo Scientific high performance liquid chromatograph of UltiMate, f step is taken to obtain
The finished product obtained uses micro-sampling pin, 20 μ L of sample volume after being dissolved in water, chromatographic column is TSK-GEL C18 chromatographic column, column temperature
30 DEG C, mobile phase is 2% acetonitrile, and flow velocity 1mL/min, ultraviolet detection wavelength 210nm calculate tetrahydropyrimidine according to standard curve
Purity is 98.2%.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1080172A (en) * | 1992-01-30 | 1994-01-05 | 耶达研究及发展有限公司 | The tetrahydropyrimidinederivatives derivatives of purification and comprise their Pharmaceutical composition |
| CA2271519A1 (en) * | 1998-05-13 | 1999-11-13 | Degussa-Huls Aktiengesellschaft | Process for the separation of tetrahydropyrimidine derivatives |
| CN102226210A (en) * | 2011-04-19 | 2011-10-26 | 朱道辰 | Tetrahydropyrimidine and method for extracting tetrahydropyrimidine by film technology |
| CN104450819A (en) * | 2014-01-03 | 2015-03-25 | 威海韩孚生化药业有限公司 | Preparation method and application of ectoine compounds |
| CN104557729A (en) * | 2014-12-11 | 2015-04-29 | 山东福田药业有限公司 | Tetrahydropyrimidine extraction process |
| CN105018403A (en) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof |
-
2016
- 2016-01-12 CN CN201610017537.8A patent/CN105669560B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1080172A (en) * | 1992-01-30 | 1994-01-05 | 耶达研究及发展有限公司 | The tetrahydropyrimidinederivatives derivatives of purification and comprise their Pharmaceutical composition |
| CA2271519A1 (en) * | 1998-05-13 | 1999-11-13 | Degussa-Huls Aktiengesellschaft | Process for the separation of tetrahydropyrimidine derivatives |
| CN102226210A (en) * | 2011-04-19 | 2011-10-26 | 朱道辰 | Tetrahydropyrimidine and method for extracting tetrahydropyrimidine by film technology |
| CN104450819A (en) * | 2014-01-03 | 2015-03-25 | 威海韩孚生化药业有限公司 | Preparation method and application of ectoine compounds |
| CN104557729A (en) * | 2014-12-11 | 2015-04-29 | 山东福田药业有限公司 | Tetrahydropyrimidine extraction process |
| CN105018403A (en) * | 2015-07-14 | 2015-11-04 | 天津科技大学 | Genetically engineered bacterium producing tetrahydropyrimidine and structuring method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| Process Optimization of the Integrated Synthesis and Secretion of Ectoine and Hydroxyectoine Under Hyper/Hypo-Osmotic Stress;C. Fallet et al.;《Biotechnology Bioengineering》;20100408;第107卷(第1期);第124-133页 |
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