CN112358478A - Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method - Google Patents
Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method Download PDFInfo
- Publication number
- CN112358478A CN112358478A CN202011308354.4A CN202011308354A CN112358478A CN 112358478 A CN112358478 A CN 112358478A CN 202011308354 A CN202011308354 A CN 202011308354A CN 112358478 A CN112358478 A CN 112358478A
- Authority
- CN
- China
- Prior art keywords
- pyrroloquinoline quinone
- supramolecular solvent
- supramolecular
- solution
- phase containing
- 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
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Nitrogen Condensed Heterocyclic Rings (AREA)
Abstract
The invention relates to a method for separating and purifying pyrroloquinoline quinone by using a supramolecular solvent extraction method, which comprises the following steps of (1) preparing fermentation liquor containing pyrroloquinoline quinone by using pyrroloquinoline quinone producing bacteria; (2) dissolving quaternary ammonium salt surfactant in alkane, slowly adding alkanol into the solution while stirring, and stirring until the solution is clear and transparent, thus obtaining the supramolecular solvent; (3) extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent to obtain a supramolecular solvent phase containing pyrroloquinoline quinone; (4) performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution to obtain a water phase containing pyrroloquinoline quinone; (5) standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone. The method has the advantages of simple operation, low cost, less pollution, high separation efficiency and convenient rapid and continuous operation, and can be applied to the industrial production of PQQ.
Description
Technical Field
The invention relates to a method for separating and purifying pyrroloquinoline quinone by using a supramolecular solvent extraction method, belonging to the technical field of biochemical engineering.
Background
Pyrroloquinoline quinone (PQQ) is a water-soluble quinone compound capable of reversibly transferring electrons, and is a third coenzyme found in life systems after riboflavin and nicotinamide, as a redox enzyme cofactor. As a prosthetic group of various enzymes, it has important physiological activities for living bodies. Studies have found that PQQ-deficient mice grow slowly, have fragile skin, osteoporosis, abnormal bone, poor immune response, poor reproductive performance, and are prone to joint inflammation. Notably, PQQ cannot be synthesized in mammals and must be taken in from the outside to meet the needs of life activities. Therefore, PQQ is considered to be a vitamin and a nutrient essential to the human body, and is useful as a novel functional food material and has a great potential for development in the fields of medicine, food, health, and the like.
However, the method for separating and purifying PQQ is a bottleneck in the large-scale development and application thereof. Currently, methods for preparing PQQ mainly include microbial fermentation and chemical synthesis. The reported chemical synthesis method of PQQ can be obtained only by more than ten steps of reaction, has low yield, needs a large amount of toxic reagents and heavy metal catalysts, and has great damage to the environment. The production and preparation of bioactive substances by a fermentation method is a method for obtaining biological medicine raw materials in a green and environment-friendly manner which is generally accepted at home and abroad at present. However, pyrroloquinoline quinone belongs to a product generated by modifying polypeptide after gene transcription and translation, is influenced by factors such as gene transcription control, modification reaction speed limit and the like, and compared with other fermented fine chemical products, the concentration of a PQQ fermentation product is still lower. In addition, due to the characteristics of strong polarity, water solubility and complex fermentation liquid components of PQQ, the conventional natural product separation and purification means is only suitable for separating medium-polarity fat-soluble compounds. Therefore, the difficulty of separating and purifying PQQ from fermentation broth is increased, and the technical problem of separation and purification must be solved for the industrial development of PQQ.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for separating and purifying PQQ by using a supramolecular solvent extraction method.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for separating and purifying pyrroloquinoline quinone by using a supramolecular solvent extraction method comprises the following steps:
(1) preparing fermentation liquor: preparing fermentation liquor containing pyrroloquinoline quinone by using pyrroloquinoline quinone producing bacteria;
(2) preparation of supramolecular solvent: dissolving a quaternary ammonium salt surfactant in alkane, stirring at 60 ℃ and 200rpm, slowly adding alkanol into the solution, and stirring until the solution is clear and transparent to obtain a supramolecular solvent;
(3) extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, stirring for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing and layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase;
(4) back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, stirring for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing for layering, wherein the upper layer is a recovered supramolecular solvent phase, and the lower layer is a water phase containing pyrroloquinoline quinone;
(5) and (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
The pyrroloquinoline quinone producing strain comprises: demethylating bacillus, klebsiella pneumoniae, gluconobacter oxydans or pseudomonas aeruginosa.
The quaternary ammonium salt is dimethyl benzyl ammonium chloride, trioctyl methyl ammonium chloride or dodecyl dimethyl benzyl ammonium chloride; the alkanol is n-pentanol, n-hexanol or n-heptanol; the alkane is n-pentane, n-hexane or n-heptane.
The dosage of the quaternary ammonium salt is 0.1-2.0g, the volume of the alkane is 8-10mL, and the volume of the alkanol is 1-2 mL.
The volume ratio of the fermentation liquid containing pyrroloquinoline quinone to the supramolecular solvent is 1: 1.
the volume ratio of the sodium chloride solution to the supramolecular solvent phase containing pyrroloquinoline quinone is 1: 1; the mass concentration of the sodium chloride solution is 10-15%.
The supermolecule solvent phase recovered in the back extraction separation can be reused after being adsorbed by active carbon for 4-8 h.
The volume ratio of the using amount of the activated carbon to the supermolecule solvent phase is 0.5-1.5 g: 1L of the compound.
The active carbon is wood active carbon.
The invention has the beneficial effects that:
the supermolecule solvent extraction method effectively overcomes the defect of low extraction rate of water-soluble PQQ component by the traditional organic solvent physical extraction method, and the high-purity PQQ is prepared by extracting and enriching PQQ from fermentation liquor by using the supermolecule solvent. The supermolecule extractant used in the invention is formed by the self-assembly of quaternary ammonium cationic surfactant, alkane and alkanol. Based on the ion pair complexation between the PQQ carboxylic acid group and the cationic surfactant and the pi-pi complexation between the benzyl group on the surfactant and the PQQ pyrrole ring, the supramolecular extractant efficiently and selectively extracts PQQ from fermentation liquor. The supermolecule solvent extraction method overcomes the defects of chemical extraction and absorbs the advantages of physical extraction.
The present invention is based on the recognition of the specificity of the PQQ structure, and a specific supramolecular solvent for the PQQ structure is constructed. The supramolecular solvent is a liquid with a nano structure formed by an amphiphilic compound through intermolecular non-covalent bond interaction and a self-assembly process in the solvent, and the forming process is shown in figure 2. Firstly, compounds with amphiphilic properties (such as amphiphilic molecular monomers) are self-assembled to form micelle droplets (or colloidal solution) of amphiphilic aggregates under the induction of amphiphilic structures/concentrations; these single micellar droplets then aggregate under hydrophobic interaction to form larger aggregates; finally, under the induction of external environmental conditions (such as pH, temperature, electrolyte and solvent), water-insoluble nano-scale aggregates are formed and separated from the water phase, and a supramolecular solvent, namely water-insoluble forward micelles, reverse micelles and vesicles, is formed. Depending on the type of amphiphilic molecule and dispersant or organic solvent, some supramolecular solvents are in the upper aqueous phase and some in the lower aqueous phase. Because the supermolecule solvent is in a nano state, the contact area of the supermolecule solvent and the extraction liquid is large, and the mass transfer efficiency and the extraction efficiency are high.
The invention utilizes the swing effect of the solvent and carries out back extraction by using the sodium chloride salt solution, thereby realizing the regeneration process of the extractant, reducing the solvent loss, having relatively simple operation and high separation efficiency, wherein the yield of PQQ is more than 90 percent, the purity can reach 97 percent, the yield is higher than that of the conventional separation and purification method (the yield is lower than 70 percent, the purity is lower than 80 percent), the cost is low, the pollution is less, the rapid and continuous operation is convenient, and the method can be applied to the industrial production of PQQ.
Drawings
FIG. 1 is a schematic diagram of the multi-directional complexation of a supramolecular solvent to PQQ;
FIG. 2 is a schematic diagram of the process of self-assembly of supramolecular solvents;
FIG. 3 is a UV spectrum of PQQ;
wherein, A is a PQQ standard substance, and B is a product of the example 1 of the invention;
FIG. 4 is a mass spectrum of the product of example 1 of the present invention;
FIG. 5 is a mass spectrum of a PQQ standard;
FIG. 6 shows the result of HPLC analysis of PQQ standard;
FIG. 7 shows the results of HPLC analysis of the product of example 1 of the present invention.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
Example 1
The method for separating and purifying pyrroloquinoline quinone by using the supramolecular solvent extraction method comprises the following steps:
(1) preparing fermentation liquor: the method for preparing the fermentation liquor containing pyrroloquinoline quinone by utilizing gluconobacter oxydans comprises the following steps:
inoculating gluconobacter oxydans into a culture medium according to the inoculation amount of 5%, and performing shake culture for 3d at the temperature of 28 ℃ to obtain a seed solution; then inoculating the seed solution into a culture medium according to the inoculation amount of 10%, performing shake culture at 28 ℃ for 5d, centrifuging the culture solution at 5 ℃ and 9000r/min for 15min, and collecting the supernatant to obtain the fermentation liquid rich in pyrroloquinoline quinone with the concentration of 100-200 mg/L.
Wherein each liter of the culture medium contains 40g sorbitol, 20g yeast extract, 10g (NH)4)2SO4、4g KH2PO4、10g MgSO4·H2O。
(2) Preparation of supramolecular solvent: weighing 1g of dimethylbenzyl dodecyl ammonium chloride, dissolving in 9mL of n-hexane, magnetically stirring at 60 ℃ and 200rpm, slowly adding 1mL of n-amyl alcohol into the solution, and stirring until the solution is clear and transparent, namely the supramolecular solvent.
(3) Extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, wherein the volume ratio of the fermentation liquor containing pyrroloquinoline quinone to the supramolecular solvent is 1: 1, placing the mixture into a round-bottom flask, magnetically stirring the mixture for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing the mixture for layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase.
(4) Back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, wherein the volume ratio of the 15% sodium chloride solution to the supramolecular solvent phase containing pyrroloquinoline quinone is 1: 1, pouring the mixed reagent into a separating funnel for standing and layering after magnetically stirring for 10min at normal temperature, wherein the upper layer is a recycled supramolecular solvent phase and can be recycled after being adsorbed by activated carbon for 8h, and the volume ratio of the using amount of the activated carbon to the supramolecular solvent phase is 0.5 g: 1L of the compound. The lower layer is a water phase containing pyrroloquinoline quinone.
(5) And (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
Example 2
The method for separating and purifying pyrroloquinoline quinone by using the supramolecular solvent extraction method comprises the following steps:
(1) preparing fermentation liquor: the method for preparing the fermentation liquor containing pyrroloquinoline quinone by utilizing gluconobacter oxydans comprises the following steps:
inoculating gluconobacter oxydans into a culture medium according to the inoculation amount of 5%, and performing shake culture for 3d at the temperature of 28 ℃ to obtain a seed solution; then inoculating the seed solution into a culture medium according to the inoculation amount of 10%, carrying out shake culture at 28 ℃ for 5d, centrifuging the culture solution at 5 ℃ and 9000r/min for 15min, and collecting the supernatant to obtain the fermentation liquor rich in pyrroloquinoline quinone with the concentration of 100-200 mg/L.
Wherein each liter of the culture medium contains 40g sorbitol, 20g yeast extract, 10g (NH)4)2SO4、4g KH2PO4、10g MgSO4·H2O。
(2) Preparation of supramolecular solvent: weighing 1g of dimethylbenzyl dodecyl ammonium chloride, dissolving in 9mL of n-hexane, magnetically stirring at 60 ℃ and 200rpm, slowly adding 1mL of n-hexanol into the solution, and stirring until the solution is clear and transparent, thus obtaining the supramolecular solvent.
(3) Extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, wherein the volume ratio of the fermentation liquor containing pyrroloquinoline quinone to the supramolecular solvent is 1: 1, placing the mixture into a round-bottom flask, magnetically stirring the mixture for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing the mixture for layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase.
(4) Back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, wherein the volume ratio of the 15% sodium chloride solution to the supramolecular solvent phase containing pyrroloquinoline quinone is 1: 1, pouring the mixed reagent into a separating funnel for standing and layering after magnetic stirring for 10min at normal temperature, wherein the upper layer is a recycled supramolecular solvent phase and can be recycled after being adsorbed by wood activated carbon for 6h, and the volume ratio of the using amount of the activated carbon to the supramolecular solvent phase is 0.8 g: 1L of the compound. The lower layer is a water phase containing pyrroloquinoline quinone.
(5) And (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
Example 3
The method for separating and purifying pyrroloquinoline quinone by using the supramolecular solvent extraction method comprises the following steps:
(1) preparing fermentation liquor: the method for preparing fermentation liquor containing pyrroloquinoline quinone by utilizing the demethylating bacillus comprises the following steps:
inoculating the demethylating bacillus into a culture medium according to the inoculation amount of 5%, and performing shake culture for 4d at the temperature of 30 ℃ to obtain a seed solution; then inoculating the seed solution into a culture medium according to the inoculation amount of 10%, carrying out shake culture at 30 ℃ for 4d, centrifuging the culture solution at 5 ℃ and 9000r/min for 15min, and collecting the supernatant to obtain the fermentation liquor rich in pyrroloquinoline quinone with the concentration of 100-200 mg/L.
Wherein each liter of the culture medium contains 10mL of methanol and 3g (NH)4)2SO4、1.5g KH2PO4、0.2g MgSO4·7H2O、0.03gCaCl2·7H2O、0.005gZnSO4·7H2O、0.005gCuSO4·5H2O, 10g methionine.
(2) Preparation of supramolecular solvent: weighing 1g of dimethylbenzyl dodecyl ammonium chloride, dissolving in 9mL of n-hexane, magnetically stirring at 60 ℃ and 200rpm, slowly adding 1mL of n-amyl alcohol into the solution, and stirring until the solution is clear and transparent, namely the supramolecular solvent.
(3) Extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, wherein the volume ratio of the fermentation liquor containing pyrroloquinoline quinone to the supramolecular solvent is 1: 1, placing the mixture into a round-bottom flask, magnetically stirring the mixture for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing the mixture for layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase.
(4) Back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, wherein the volume ratio of the 15% sodium chloride solution to the supramolecular solvent phase containing pyrroloquinoline quinone is 1: 1, pouring the mixed reagent into a separating funnel for standing and layering after magnetic stirring for 10min at normal temperature, wherein the upper layer is a recycled supramolecular solvent phase, and the mixed reagent can be repeatedly utilized after being adsorbed by activated carbon for 4h, wherein the volume ratio of the using amount of the activated carbon to the supramolecular solvent phase is 1 g: 1L of the compound. The lower layer is a water phase containing pyrroloquinoline quinone.
(5) And (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
Example 4
The method for separating and purifying pyrroloquinoline quinone by using the supramolecular solvent extraction method comprises the following steps:
(1) preparing fermentation liquor: the method for preparing fermentation liquor containing pyrroloquinoline quinone by utilizing the demethylating bacillus comprises the following steps:
inoculating the demethylating bacillus into a culture medium according to the inoculation amount of 5%, and performing shake culture for 4d at the temperature of 30 ℃ to obtain a seed solution; then inoculating the seed solution into a culture medium according to the inoculation amount of 10%, carrying out shake culture at 30 ℃ for 4d, centrifuging the culture solution at 5 ℃ and 9000r/min for 15min, and collecting the supernatant to obtain the fermentation liquor rich in pyrroloquinoline quinone with the concentration of 100-200 mg/L.
Wherein each liter of the culture medium contains 10mL of methanol and 3g (NH)4)2SO4、1.5g KH2PO4、0.2g MgSO4·7H2O、0.03gCaCl2·7H2O、0.005gZnSO4·7H2O、0.005gCuSO4·5H2O, 10g methionine.
(2) Preparation of supramolecular solvent: weighing 1g of dodecyl benzyl ammonium chloride, dissolving in 9mL of n-hexane, magnetically stirring at 60 ℃ and 200rpm, slowly adding 1mL of n-hexanol into the solution, and stirring until the solution is clear and transparent, thus obtaining the supramolecular solvent.
(3) Extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, wherein the volume ratio of the fermentation liquor containing pyrroloquinoline quinone to the supramolecular solvent is 1: 1, placing the mixture into a round-bottom flask, magnetically stirring the mixture for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing the mixture for layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase.
(4) Back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, wherein the volume ratio of the 15% sodium chloride solution to the supramolecular solvent phase containing pyrroloquinoline quinone is 1: 1, pouring the mixed reagent into a separating funnel for standing and layering after magnetic stirring for 10min at normal temperature, wherein the upper layer is a recycled supramolecular solvent phase, and the supramolecular solvent phase can be recycled after being adsorbed by wood activated carbon for 4h, wherein the volume ratio of the consumption of the activated carbon to the supramolecular solvent phase is 1.5 g: 1L of the compound. The lower layer is a water phase containing pyrroloquinoline quinone.
(5) And (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
Examples of the experiments
1. Analysis of the product
1.1 ultraviolet Spectroscopy
The results of simultaneous detection of the PQQ standard and the crystalline product of example 1 of the present invention using an ultraviolet spectrometer are shown in fig. 3A and 3B, respectively. The result shows that the ultraviolet spectrum result of the invention is consistent with that of the PQQ standard product, and the final product of the invention is PQQ.
1.2 Mass Spectrometry
The crystalline product of example 1 of the present invention was examined using a mass spectrometer and the results are shown in fig. 4. Comparing the mass spectrum data of the crystalline product of the present invention with the mass spectrum data of the PQQ standard shown in fig. 5, the results show that the mass spectrum data of the crystalline product of the present invention is consistent with the mass spectrum data of the PQQ standard, indicating that the final product of the present invention is PQQ.
1.3 detection by HPLC method
The detection method comprises the following steps: HPLC detection of the product: a Waters Symmetry 300C18 liquid chromatography column was used, with methanol: water (methanol and water both contain 0.1% (volume concentration) trifluoroacetic acid) is used as a mobile phase, the flow rate is 1mL/min, gradient elution (the volume concentration of methanol aqueous solution is 30-90%, the elution time is 30min), and the detection wavelength is 230 nm.
Detecting an object: pyrroloquinoline quinone standards, crystalline products of examples 1-4 of the present invention.
And (4) analyzing results: the HPLC analysis results are shown in FIG. 6 (pyrroloquinoline quinone standard) and FIG. 7 (inventive example 1). The result shows that the final product of the invention has the same chromatographic peak with the PQQ standard substance, and can obtain better chromatographic analysis result, and the product purity is shown in the following table 1.
1.4 yield and purity of pyrroloquinoline quinone
Taking the fermentation broth of pyrroloquinoline quinone in examples 1 to 4 of the present invention as a sample solution, studying the separation effect of the molecularly imprinted solid-phase extraction on pyrroloquinoline quinone, detecting the content of pyrroloquinoline quinone in the aqueous phase after the back extraction of sodium chloride solution by using a high performance liquid chromatography technique, and obtaining the yield as shown in table 1 below. The yield of the pyrroloquinoline quinone is the ratio of the mass of the pyrroloquinoline quinone in the water phase after the back extraction of the sodium chloride solution to the mass of the pyrroloquinoline quinone in the original fermentation liquor.
Wherein: PQQ yield ═ C1V1/C0V0)×100％，
C1: the concentration of pyrroloquinoline quinone in the water phase after the back extraction of the sodium chloride solution; v1: volume of aqueous phase after stripping of sodium chloride solution;
C0: the concentration of pyrroloquinoline quinone in the fermentation broth; v0: volume of fermentation broth.
TABLE 1 yield and purity of PQQ of the present invention
| Item | Example 1 | Example 2 | Example 3 | Example 4 |
| Yield (%) | 92.1 | 93.5 | 95.6 | 96.3 |
| Purity (%) | 97.2 | 97.8 | 97.0 | 97.6 |
Claims (9)
1. A method for separating and purifying pyrroloquinoline quinone by using a supramolecular solvent extraction method is characterized by comprising the following steps:
(1) preparing fermentation liquor: preparing fermentation liquor containing pyrroloquinoline quinone by using pyrroloquinoline quinone producing bacteria;
(2) preparation of supramolecular solvent: dissolving a quaternary ammonium salt surfactant in alkane, stirring at 60 ℃ and 200rpm, slowly adding alkanol into the solution, and stirring until the solution is clear and transparent to obtain a supramolecular solvent;
(3) extraction and separation: extracting fermentation liquor containing pyrroloquinoline quinone by using a supramolecular solvent, stirring for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing and layering, wherein the upper layer is a supramolecular solvent phase containing pyrroloquinoline quinone, and the lower layer is a water phase;
(4) back extraction and separation: performing back extraction on the supramolecular solvent phase containing pyrroloquinoline quinone by using a sodium chloride solution, stirring for 10min at normal temperature, pouring the mixed reagent into a separating funnel, standing for layering, wherein the upper layer is a recovered supramolecular solvent phase, and the lower layer is a water phase containing pyrroloquinoline quinone;
(5) and (3) crystallization and purification: standing the water phase containing pyrroloquinoline quinone for 24h at 4 ℃, and filtering when red crystals appear to obtain the pyrroloquinoline quinone.
2. The method according to claim 1, wherein the pyrroloquinoline quinone-producing bacterium is: demethylating bacillus, klebsiella pneumoniae, gluconobacter oxydans or pseudomonas aeruginosa.
3. The method of claim 1, wherein the quaternary ammonium salt is dimethylbenzyl ammonium chloride, trioctylmethyl ammonium chloride, or dodecyl dimethylbenzyl ammonium chloride; the alkanol is n-pentanol, n-hexanol or n-heptanol; the alkane is n-pentane, n-hexane or n-heptane.
4. The method of claim 1, wherein the quaternary ammonium salt is used in an amount of 0.1 to 2.0g, the volume of the alkane is 8 to 10mL, and the volume of the alkanol is 1 to 2 mL.
5. The method of claim 1, wherein the volume ratio of pyrroloquinoline quinone-containing fermentation broth to supramolecular solvent is 1: 1.
6. the method of claim 1, wherein the volume ratio of the sodium chloride solution to the supramolecular solvent phase comprising pyrroloquinoline quinone is from 1: 1; the mass concentration of the sodium chloride solution is 10-15%.
7. The method according to claim 1, wherein the recovered supramolecular solvent phase from the stripping separation is reused after being adsorbed by activated carbon for 4-8 h.
8. The method according to claim 7, wherein the volume ratio of the amount of activated carbon to the supramolecular solvent phase is from 0.5 to 1.5 g: 1L of the compound.
9. The method of claim 7, wherein the activated carbon is wood activated carbon.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011308354.4A CN112358478B (en) | 2020-11-20 | 2020-11-20 | Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011308354.4A CN112358478B (en) | 2020-11-20 | 2020-11-20 | Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112358478A true CN112358478A (en) | 2021-02-12 |
| CN112358478B CN112358478B (en) | 2021-07-23 |
Family
ID=74533013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011308354.4A Active CN112358478B (en) | 2020-11-20 | 2020-11-20 | Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112358478B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040067545A1 (en) * | 2002-08-09 | 2004-04-08 | Sysmex Corporation | Reagent for assaying lipid |
| CN104892597A (en) * | 2015-05-14 | 2015-09-09 | 郑州轻工业学院 | Complex extraction method for separation and purification of pyrroloquinoline quinine in fermentation broth |
| CN104987728A (en) * | 2015-07-22 | 2015-10-21 | 浙江传化股份有限公司 | Supramolecular composition with low surface energy and preparation method thereof |
| CN105294687A (en) * | 2015-12-02 | 2016-02-03 | 郑州轻工业学院 | Ion pair dual water phase method for extracting and separating pyrroloquinoline quinone |
| CN106282044A (en) * | 2015-05-20 | 2017-01-04 | 浙江海正药业股份有限公司 | A kind of Hyphomicrobium and the preparation method of pyrroloquinoline quinone |
| JP2017031126A (en) * | 2015-08-06 | 2017-02-09 | 三菱瓦斯化学株式会社 | Method for producing pyrrolo-quinoline quinone crystal |
| CN111675646A (en) * | 2020-06-24 | 2020-09-18 | 郑州轻工业大学 | Method for preparing 2-amino-3- (5-hydroxyindole) propionic acid by using gulonic acid crystallization mother liquor |
-
2020
- 2020-11-20 CN CN202011308354.4A patent/CN112358478B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040067545A1 (en) * | 2002-08-09 | 2004-04-08 | Sysmex Corporation | Reagent for assaying lipid |
| CN104892597A (en) * | 2015-05-14 | 2015-09-09 | 郑州轻工业学院 | Complex extraction method for separation and purification of pyrroloquinoline quinine in fermentation broth |
| CN106282044A (en) * | 2015-05-20 | 2017-01-04 | 浙江海正药业股份有限公司 | A kind of Hyphomicrobium and the preparation method of pyrroloquinoline quinone |
| CN104987728A (en) * | 2015-07-22 | 2015-10-21 | 浙江传化股份有限公司 | Supramolecular composition with low surface energy and preparation method thereof |
| JP2017031126A (en) * | 2015-08-06 | 2017-02-09 | 三菱瓦斯化学株式会社 | Method for producing pyrrolo-quinoline quinone crystal |
| CN105294687A (en) * | 2015-12-02 | 2016-02-03 | 郑州轻工业学院 | Ion pair dual water phase method for extracting and separating pyrroloquinoline quinone |
| CN111675646A (en) * | 2020-06-24 | 2020-09-18 | 郑州轻工业大学 | Method for preparing 2-amino-3- (5-hydroxyindole) propionic acid by using gulonic acid crystallization mother liquor |
Non-Patent Citations (3)
| Title |
|---|
| 周留柱: ""鲍曼不动杆菌生物合成吡咯喹啉醌的研究"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 徐佳: ""无盐型阴阳离子表面活性剂复配体系及其超分子溶剂萃取研究"", 《中国优秀博士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 杨雪鹏: ""吡咯喹啉醌生物合成及其应用研究"", 《2015年中国酶工程与糖生物工程学术研讨会》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112358478B (en) | 2021-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103571897B (en) | A kind of multiprenylmenaquinone and preparation method thereof | |
| CN102061327B (en) | Preparation method of sodium bacillus subtilis lipopeptide | |
| CN104892710B (en) | A kind of method for purifying reduced form β NADHs | |
| CN102747123A (en) | Process for preparing ademetionine butanedisulfonate | |
| CN102321682B (en) | Method for recycling water in separation process of succinic acid by fermentation | |
| CN104328155A (en) | Method and application for producing pyrroloquinoline quinone by using gluconobacter oxydans | |
| CN102321137A (en) | Preparation method of adenosylcobalamin | |
| CN1138776C (en) | Extraction method of pyrroquinolinequinone | |
| US20090036693A1 (en) | Method of purifying ubiquinone-10 | |
| CN112358478B (en) | Method for separating and purifying pyrroloquinoline quinone by using supramolecular solvent extraction method | |
| CN108949850B (en) | Online separation and purification method of rhamnolipid fermentation liquor | |
| CN110257451B (en) | Method for promoting accumulation of arteannuic acid | |
| CN109053638B (en) | Extraction and purification method of fumagillin | |
| CN105316372B (en) | A kind of method of fermenting and producing Arteannuic acid | |
| CN108611383B (en) | Method for improving fermentation yield of artemisinic acid | |
| CN102690333B (en) | Preparation method of high-purity teicoplanin | |
| CN103276028B (en) | A kind of method that fermentation method produces epothilone B | |
| CN103170496B (en) | Method for removing drugs in soil by utilizing magnetism molecular imprinting-electromagnetism grating combination | |
| CN1524961A (en) | Microorganism continuous catalysis method for producing acrylamide | |
| CN111424060B (en) | Biological method for simultaneously preparing D-proline and L-1-pyrroline-5-carboxylic acid | |
| CN110878260B (en) | Strain for producing D-arabitol and application thereof | |
| CN109609568B (en) | Method for preparing swainsonine by solid fermentation | |
| CN114146680A (en) | Method for separating and purifying pyrroloquinoline quinone by using reverse micelle surface modified magnetic nano solid-phase extraction material | |
| CN112029754A (en) | Imprinted lipase and application thereof in synthesis of sucrose-6-ethyl ester | |
| CN117225377B (en) | Core-shell structure covalent organic framework material, preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |