Separation and purification method of pyrroloquinoline quinone disodium salt

Abstract

The invention belongs to the technical field of compound purification, and relates to a method for separating and purifying pyrroloquinoline quinone disodium salt, which comprises the following steps: sequentially carrying out large-particle impurity removal, pH value regulation, filter aid and adsorbent treatment, nanofiltration and salting-out on pyrroloquinoline quinone fermentation liquor; crystallizing the obtained salting-out substance by taking the pyrroloquinoline quinone disodium salt crystal form A with a specific characteristic peak as a seed crystal; the pyrroloquinoline quinone disodium salt crystal form A is characterized in that an X-ray diffraction pattern obtained by adopting Cu-K alpha radioactive ray radiation has characteristic peaks at the following 2 theta angles: 7.0 degrees + / -0.2 degrees, 8.4 degrees + / -0.2 degrees, 9.0 degrees + / -0.2 degrees, 17.8 degrees + / -0.2 degrees, 19.2 degrees + / -0.2 degrees, 20.8 degrees + / -0.2 degrees, 23.4 degrees + / -0.2 degrees, 26.9 degrees + / -0.2 degrees, 28.2 degrees + / -0.2 degrees and 29.2 degrees + / -0.2 degrees. By adopting the method, the purity of the pyrroloquinoline quinone disodium salt can be effectively improved.

Description

Technical Field

[0001] The invention belongs to the technical field of compound purification, and specifically relates to a method for separating and purifying pyrroloquinoline quinone disodium salt. Background Art

[0002] Pyrroloquinoline quinone is a redox enzyme cofactor, and its biological functions are mainly concentrated in two aspects. First, it can support the growth and development of mitochondria and stimulate the rapid growth of human cells. Second, it has good antioxidant properties, which can help remove free radicals and reduce cell damage. Pyrroloquinoline quinone can be prepared by chemical synthesis and fermentation. However, the chemical synthesis method not only requires the use of more organic solvents, but also has a complex process, many by-products, and low yield, so it is gradually replaced by the fermentation method.

[0003] CN110698472A discloses a method for purifying pyrroloquinoline quinone, which comprises the following steps: pre-treating the pyrroloquinoline quinone fermentation broth to obtain a filtrate; passing the obtained filtrate through a polyamide resin, and then washing it with an acidic aqueous solution, and discarding the leaked liquid; desorbing it with a mixed aqueous solution of acid and salt, and collecting a desorbed liquid with a chromatographic purity of more than 90%; adjusting the pH of the desorbed liquid, and then performing ultrafiltration and nanofiltration in sequence, and finally crystallizing and drying. The purity of the pyrroloquinoline quinone crystals obtained by this method is ≥98%, which is still relatively low and cannot meet the needs of the high-end market. In addition, although this method can extract pyrroloquinoline quinone from the pyrroloquinoline quinone fermentation broth, this method requires the use of an aqueous solution of acid and salt as a desorbed liquid for desorption, and the solubility of pyrroloquinoline quinone in the desorbed liquid is low. Therefore, precipitation is prone to occur during the desorption process, which is not suitable for mass production.

[0004] CN111440167A discloses a method for extracting pyrroloquinoline quinone without the need for a chromatography step. The method comprises pre-treating the pyrroloquinoline quinone fermentation broth, performing two adsorptions with an adsorbent and filtering, and then subjecting the filtrate to ultrafiltration and nanofiltration and low-temperature concentration and crystallization. The purity of the final product is ≥98%, which is still relatively low and cannot meet the needs of the high-end market.

[0005] In addition, the prior art also has a technology for obtaining pyrroloquinoline quinone disodium salt by chromatography. Although this technology can obtain pyrroloquinoline quinone disodium salt with higher purity, the process of extracting pyrroloquinoline quinone disodium salt from the fermentation broth by chromatography usually has many steps and cannot effectively improve the production efficiency. In addition, the chromatography step is prone to produce a large amount of salt-containing wastewater, and the treatment cost is high. Summary of the invention

[0006] The object of the present invention is to provide a novel separation and purification method of pyrroloquinoline quinone disodium salt, which can obtain high-purity pyrroloquinoline quinone disodium salt.

[0007] Specifically, the separation and purification method of pyrroloquinoline quinone disodium salt provided by the present invention comprises: removing large particle impurities in pyrroloquinoline quinone fermentation broth, adjusting the pH value of the obtained fermentation broth clear liquid to 3-4 and then mixing it evenly with a filter aid and an adsorbent, performing solid-liquid separation on the obtained mixture, and then performing nanofiltration concentration on the obtained filtrate using a nanofiltration membrane, adding salt to the obtained concentrated liquid for salting out, filtering after the salting out is completed, and crystallizing the obtained salted out product using pyrroloquinoline quinone disodium salt crystal form A as a seed crystal. The pyrroloquinoline quinone disodium salt crystal form The X-ray diffraction pattern of A obtained by irradiating with Cu-Kα radiation has characteristic peaks at the following 2θ angles: 7.0°±0.2°, 8.4°±0.2°, 9.0°±0.2°, 17.8°±0.2°, 19.2°±0.2°, 20.8°±0.2°, 23.4°±0.2°, 26.9°±0.2°, 28.2°±0.2°, and 29.2°±0.2°. After the crystallization is completed, the steps of washing, filtering and drying are carried out in sequence to obtain pyrroloquinoline quinone disodium salt.

[0008] The key to the present invention is to use pyrroloquinoline quinone fermentation broth as the starting material, and to sequentially remove large particle impurities, adjust the pH value, treat with filter aids and adsorbents, nanofilter and salt out the pyrroloquinoline quinone fermentation broth, and then crystallize the resulting salt out with pyrroloquinoline quinone disodium salt crystal form A having a specific characteristic peak as a seed, so that the purity of pyrroloquinoline quinone disodium salt can be effectively improved. The reason for this is speculated to be that: after the pyrroloquinoline quinone fermentation broth is sequentially subjected to large particle impurities removal, pH adjustment, filter aid and adsorbent treatment, nanofiltration and salt out, impurities that are easy to be embedded in the crystal lattice and easy to be embedded in the crystal can be effectively removed, and the solubility curve of pyrroloquinoline quinone disodium salt crystal form A is relatively gentle, and using it as a seed for crystallization is very beneficial to improve the purity of the product. The present invention introduces pyrroloquinoline quinone disodium salt form A as a seed crystal into the separation and purification process of pyrroloquinoline quinone fermentation broth, which can not only improve the purity of the product, but also simplify the extraction steps, reduce energy consumption in the extraction process, shorten the construction period, reduce costs, reduce the discharge of wastewater in the extraction process, and reduce the pollution to the environment and the waste of energy in the production process.

[0009] Furthermore, if the crystal growth rate is too fast during the crystallization process, impurities may be embedded in the product lattice, or impurities may be embedded in the crystal in the form of inclusions, which will reduce the purity of the product; if the crystal growth rate is too slow, the construction period will be extended and the production efficiency will be reduced. In other words, when the crystallization method is used to extract the product, improving the product purity and crystallization efficiency are actually a contradiction. How to balance purity and crystallization efficiency is a major production problem. In a preferred embodiment of the present invention, the crystallization method is semi-continuous crystallization, and the semi-continuous crystallization step includes dissolving the salting out in a solvent at 45°C~55°C, adding pyrroloquinoline quinone disodium salt crystal form A, and then cooling to 15°C~25°C for crystallization. The cooling adopts gradient cooling, and the gradient cooling method includes first cooling to 40°C±2°C at a rate of 1~2°C / min and keeping warm for 30s~2min, then cooling to 30°C±2°C at a rate of 2~4°C / min and keeping warm for 20s~1min, and then cooling to 15°C~25°C at a rate of 4~6°C / min. At this time, high-purity pyrroloquinoline quinone disodium salt can be obtained with high efficiency, perfectly solving the contradiction between product purity and crystallization efficiency. It is speculated that the reason may be that: when crystallizing in the above-mentioned specific manner, the crystallization mother liquor contains a large number of microcrystals after adding the seed crystals. These microcrystals can provide a larger specific surface area for the crystallization process. Therefore, although the crystal growth process is relatively slow when crystal form A is used as the seed crystal, the microcrystals contained in the crystallization mother liquor itself can be efficiently deposited on the surface of the seed crystal to make up for the defect of the low crystallization rate. The overall crystallization efficiency can be maintained at a high level, maintaining a faster crystallization rate under the premise of slow growth of the crystal nucleus itself.

[0010] In a preferred embodiment, the solvent used in the semi-continuous crystallization is a composite solvent composed of water, dimethyl sulfoxide and N,N-dimethylformamide in a volume ratio of 1:(0.5~0.8):(0.1~0.3), in which case pyrroloquinoline quinone disodium salt with higher purity can be obtained with higher efficiency.

[0011] The separation and purification method of pyrroloquinoline quinone disodium salt provided by the present invention has at least one of the following beneficial effects: (1) It can effectively improve the purity of pyrroloquinoline quinone disodium salt; (2) It can effectively improve the crystallization efficiency of pyrroloquinoline quinone disodium salt; (3) It can streamline the extraction steps, reduce energy consumption during the extraction process, shorten the construction period, reduce costs, reduce the discharge of wastewater during the extraction process, and reduce environmental pollution and energy waste during the production process. DETAILED DESCRIPTION

[0012] The separation and purification method of pyrroloquinoline quinone disodium salt provided by the present invention comprises: removing large particle impurities in pyrroloquinoline quinone fermentation broth, adjusting the pH value of the obtained fermentation broth clear liquid to 3-4, and then uniformly mixing it with a filter aid and an adsorbent, performing solid-liquid separation on the obtained mixture, then performing nanofiltration concentration on the obtained filtrate using a nanofiltration membrane, adding salt to the obtained concentrated liquid for salting out, filtering after the salting out is completed, crystallizing the obtained salting out product using pyrroloquinoline quinone disodium salt crystal form A as a seed crystal, and washing, filtering and drying in sequence after the crystallization is completed to obtain pyrroloquinoline quinone disodium salt.

[0013] In the present invention, the X-ray diffraction pattern obtained by Cu-Kα radiation of the pyrroloquinoline quinone disodium salt crystal form A has characteristic peaks at the following 2θ angles: 7.0°±0.2°, 8.4°±0.2°, 9.0°±0.2°, 17.8°±0.2°, 19.2°±0.2°, 20.8°±0.2°, 23.4°±0.2°, 26.9°±0.2°, 28.2°±0.2°, 29.2°±0.2°. The error of the above characteristic peaks is ±0.2°, such as -0.2°, -0.15°, -0.1°, -0.05°, 0.05°, 0.1°, 0.15°, 0.2°, etc. The pyrroloquinoline quinone disodium salt crystal form A corresponds to the crystal of pyrroloquinoline quinone disodium salt trihydrate.

[0014] In the present invention, the X-ray diffraction pattern of the pyrroloquinoline quinone disodium salt crystal form A is obtained by scanning X-ray diffraction (XRPD). The test conditions include a target material of Cu-Kα (30KV, 10mA), a scanning range of 3°-40° (2θ value), and a scanning step of 0.1s.

[0015] In a preferred embodiment, the pyrroloquinoline quinone disodium salt crystal form A is prepared according to the following method: dissolving pyrroloquinoline quinone disodium salt in water at a temperature of 55°C to 80°C (such as 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or any value therebetween) and a pH value of 7 to 9 (such as 7, 7.2, 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9 or any value therebetween), and then cooling the resulting solution to 0°C to 10°C (such as 0°C, 2°C, 5°C, 8°C, 10°C or any value therebetween), add lower alcohol to the system under stirring, the volume proportion of lower alcohol is 40-70% (such as 40%, 45%, 50%, 55%, 60%, 65%, 70% or any value therebetween), solid is precipitated into a suspension, and then the pH value of the system is adjusted to 3-4 (such as 3, 3.2, 3.4, 3.6, 3.8, 4 or any value therebetween), filtered and dried to obtain pyrroloquinoline quinone disodium salt crystal form A.

[0016] In the preparation process of the above-mentioned pyrroloquinoline quinone disodium salt crystal form A, the pyrroloquinoline quinone disodium salt (PQQ disodium salt) can be obtained commercially or prepared according to various existing methods, for example, it can be prepared according to the method disclosed in CN102471336B or CN108026091B.

[0017] In the preparation process of the above-mentioned pyrroloquinoline quinone disodium salt crystal form A, the dosage ratio of the pyrroloquinoline quinone disodium salt to water is preferably 1g: (15~30) mL. In a specific embodiment, the method of dissolving the pyrroloquinoline quinone disodium salt in water at a temperature of 55°C~80°C and a pH value of 7~9 can be to first disperse the pyrroloquinoline quinone disodium salt in water, then heat to raise the temperature to 55°C~80°C, and then adjust the pH value to 7~9; it can also be to dissolve the pyrroloquinoline quinone disodium salt in water at a temperature of 55°C~80°C, and then adjust the pH value to 7~9. The method of adjusting the pH value of the solution to 7~9 is to add a base to the system. Among them, specific examples of the base include but are not limited to: at least one of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate and ammonia water. The method of adjusting the pH value of the system to 3~4 is to add an acid to the system. Specific examples of the acid include, but are not limited to, at least one of phosphoric acid, sulfuric acid, hydrochloric acid and nitric acid.

[0018] In the preparation process of the above-mentioned pyrroloquinoline quinone disodium salt crystal form A, the lower alcohol can be a C1-C5 monohydric alcohol, dihydric alcohol or trihydric alcohol, and its specific examples include but are not limited to: at least one of methanol, ethanol, n-propanol, isopropanol, 1-butanol, isobutanol, ethylene glycol, 1,3-propylene glycol, 2,3-butanediol and glycerol. From the perspective of raw material availability and environmental protection, the lower alcohol is preferably ethanol and / or isopropanol, and ethanol is particularly preferred.

[0019] In the preparation process of the above-mentioned pyrroloquinoline quinone disodium salt crystal form A, the stirring conditions preferably include a rotation speed of 40 to 80 rpm, such as 40 rpm, 50 rpm, 60 rpm, 70 rpm, 80 rpm or any value therebetween; the time is 4 to 10 h, such as 4 h, 6 h, 8 h, 10 h or any value therebetween. The drying conditions preferably include a temperature of 40 to 60 ° C, such as 40 ° C, 45 ° C, 50 ° C, 55 ° C, 60 ° C or any value therebetween; the time is 10 to 24 h, such as 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 24 h or any value therebetween.

[0020] The present invention has no particular limitation on the source of the pyrroloquinoline quinone fermentation broth, which can be obtained by commercial purchase, donation, etc., or prepared according to various existing methods. The pyrroloquinoline quinone fermentation broth is preferably a denitrifying mycelial microorganism fermentation broth, more preferably a methanol-utilizing denitrifying mycelial microorganism ( Hyphomicrobium denitrificans ) fermentation broth. Wherein, the methanol-utilizing denitrifying mycelial microbe ( Hyphomicrobium denitrificans ) For example, it can be CGMCC1.15943.

[0021] The present invention has no specific limitation on the step of removing large particle impurities in the pyrroloquinoline quinone fermentation broth, and preferably includes using a ceramic membrane to separate the pyrroloquinoline quinone fermentation broth into solid and liquid, and then using an ultrafiltration membrane to ultrafilter the obtained ceramic clear liquid, and the obtained ultrafiltration clear liquid is the fermentation broth clear liquid. Wherein, the pore size of the ceramic membrane is preferably 0.1 μm~0.5 μm, such as 0.1 μm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm or any value therebetween. The molecular weight cutoff of the ultrafiltration membrane is preferably 10 kDa~20 kDa, such as 10 kDa, 12 kDa, 14 kDa, 16 kDa, 18 kDa, 20 kDa or any value therebetween. In addition, the temperature for solid-liquid separation using a ceramic membrane and ultrafiltration using an ultrafiltration membrane is preferably independently 10~20 ° C, and specifically can be 10 ° C, 12 ° C, 14 ° C, 16 ° C, 18 ° C, 20 ° C or any value therebetween.

[0022] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, before the fermentation broth is uniformly mixed with the filter aid and the adsorbent, the pH value thereof needs to be adjusted to 3-4, specifically 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, etc. The pH adjuster used can generally be an acidic solution, specifically at least one of a hydrochloric acid solution, a phosphoric acid solution, a sulfuric acid solution and an acetic acid solution, preferably a phosphoric acid solution and / or a sulfuric acid solution.

[0023] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the filter aid is preferably diatomaceous earth and / or perlite. The addition amount of the filter aid is preferably 0.5-5% of the weight of the fermentation liquid, such as 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value therebetween. The adsorbent is preferably at least one of activated carbon, kaolin, bentonite and zeolite. The addition amount of the adsorbent is preferably 0.1-2% of the weight of the fermentation liquid, such as 0.1%, 0.3%, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2% or any value therebetween.

[0024] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the molecular weight cutoff of the nanofiltration membrane is preferably 100Da~300Da, such as 100Da, 120Da, 150Da, 180Da, 200Da, 220Da, 250 Da, 280Da, 300Da or any value therebetween. The concentration conditions are preferably such that the content of pyrroloquinoline quinone disodium salt in the obtained concentrated solution is 4~10g / L, such as 4g / L, 6g / L, 8g / L, 10g / L or any value therebetween.

[0025] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the salting out salt is preferably selected from at least one of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate and potassium carbonate. The amount of the salt added is preferably such that the concentration of the salt in the system is 0.8 to 1.2 mol / L, specifically 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2 mol / L, etc.

[0026] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the crystallization method is preferably semi-continuous crystallization, and the semi-continuous crystallization step preferably includes dissolving the salting out in a solvent at 45°C to 55°C, adding pyrroloquinoline quinone disodium salt crystal form A, and then reducing the temperature of the resulting salting out solution to 15°C to 25°C for crystallization. Among them, the method of dissolving the salting out in the solvent can, for example, be to first control the temperature of the solvent at 45°C to 55°C, and then add the salting out to dissolve; or the salting out can be first added to the solvent, and then the whole temperature is raised to 45°C to 55°C for dissolution. The temperature at which the salting out is dissolved in the solvent can, for example, be 45°C, 48°C, 50°C, 52°C, 55°C, or any value therebetween. The temperature of the crystallization can specifically be 15°C, 18°C, 20°C, 22°C, 25°C, or any value therebetween.

[0027] In the separation and purification process of the disodium salt of pyrroloquinoline quinone provided by the present invention, the process of reducing the temperature of the obtained salting-out solution can be cooled in one step or in steps, preferably by gradient cooling in the following manner: first, the salting-out solution is cooled to 40°C ± 2°C at a rate of 1 to 2°C / min and kept warm for 30s to 2min, then cooled to 30°C ± 2°C at a rate of 2 to 4°C / min and kept warm for 20s to 1min, and then cooled to 15°C to 25°C at a rate of 4 to 6°C / min. Among them, the cooling rate of the first stage can be specifically 1, 1.2, 1.4, 1.6, 1.8, 2°C / min or any value therebetween. The insulation time of the first stage can be specifically 30s, 40s, 50s, 60s, 80s, 90s, 100s, 110s, 120s or any value therebetween. The cooling rate of the second stage can be 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4°C / min or any value therebetween. The holding time of the second stage can be 20s, 30s, 40s, 50s, 60s or any value therebetween. The cooling rate of the third stage can be 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6°C / min or any value therebetween.

[0028] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the washing detergent used is preferably an aqueous solution of alcohol. Wherein, the alcohol can be listed as: at least one of methanol, ethanol, n-propanol and isopropanol. The concentration of the aqueous solution of alcohol is preferably 10~90(v / v)%, such as 10(v / v)%, 15(v / v)%, 20(v / v)%, 25(v / v)%, 30(v / v)%, 35(v / v)%, 40(v / v)%, 45(v / v)%, 50(v / v)%, 55(v / v)%, 60(v / v)%, 65(v / v)%, 70(v / v)%, 75(v / v)%, 80(v / v)%, 85(v / v)%, 90(v / v)% or any value therebetween.

[0029] In the separation and purification process of pyrroloquinoline quinone disodium salt provided by the present invention, the drying conditions preferably include a temperature of 40°C to 60°C, such as 40°C, 42°C, 45°C, 48°C, 50°C, 52°C, 55°C, 58°C, 60°C or any value therebetween; and a time of 10h to 24h, such as 10h, 12h, 15h, 18h, 20h, 22h, 24h or any value therebetween.

[0030] The present invention will be described in detail below through examples.

[0031] In the following examples and comparative examples, the pyrroloquinoline quinone fermentation broth is the same fermentation broth, specifically, methanol is used as the carbon source and the fermentation broth is prepared from denitrifying mycelial microorganisms ( Hyphomicrobium denitrificans , CGMCC1.15943) was obtained by fermentation.

[0032] In the following examples and comparative examples, the conditions of the analytical methods involved are as follows: (1) Purity analysis: Instrument model: Agilent Technologies 1260 Infinity (with UV detector); Chromatographic column: ZORBAX SB-C18, 4.6 mm × 250 mm, 5 μm; Detection wavelength: 250nm; Eluent: A water (containing 0.01 M potassium dihydrogen phosphate and 0.015 M tetrabutylammonium bromide): B acetonitrile solution (water: acetonitrile (v:v) = 75:25); Elution rate: 1 mL / min.

[0033] (2) XRPD analysis: Instrument model: D2 PHASER BRUKER; Target: Cu-Kα (30KV, 10mA); Scanning range: 3°-40° (2θ value); Scanning step: 0.1s.

[0034] (3) Analysis of sodium ion content: Instrument model: Agilent Technologies 1260 Infinity (with CAD detector); Chromatographic column: Atlantis HILIC Silica (4.6*100mm, 5um); Column temperature: 40°C; Detection wavelength: 250nm; Eluent: 0.05 M ammonium acetate aqueous solution-acetonitrile, volume ratio 1:1; Elution rate: 1 mL / min; CAD parameters: atomization temperature is 50°C and acquisition frequency is 20 Hz.

[0035] Preparation Example 1: Preparation of Pyrroloquinoline Quinone Disodium Salt Form A 10g of pyrroloquinoline quinone disodium salt raw material was dispersed in 200mL of pure water, heated to 60°C, and the pH of the resulting solution was adjusted to 9 with sodium hydroxide. After stirring until the solid was completely dissolved, the temperature was lowered to 5°C, and ethanol was slowly added to make the volume fraction of ethanol reach 50%. At this time, a large amount of solid was generated. The pH of the suspension was adjusted to 3.5 with phosphoric acid, and the pH was filtered after stabilization. The filter residue was dried at 40°C for 16h to obtain the crystal form A of PQQ disodium salt. Crystal form A was detected by X-ray diffractometer (XRPD), and the obtained X-ray diffraction spectrum showed that the crystal form A showed characteristic peaks at 2θ angles at 7.0°, 8.4°, 9.0°, 17.8°, 19.2°, 20.8°, 23.4°, 26.9°, 28.2°, and 29.2°. The purity was 99.9% as determined by HPLC; the proportion of sodium ions detected by HPLC-CAD was 10.2%.

[0036] Preparation Example 2: Preparation of Pyrroloquinoline Quinone Disodium Salt Form A 10g of pyrroloquinoline quinone disodium salt was dispersed in 250mL of pure water, heated to 60°C, and the pH of the resulting solution was adjusted to 7.5 with sodium carbonate. After stirring until the solid was completely dissolved, the temperature was lowered to 5°C, and isopropanol was slowly added to make the volume fraction of isopropanol reach 50%. At this time, solid was generated, and the pH of the suspension was adjusted to 4 with sulfuric acid. After the pH was stabilized, the filter cake was dried at 50°C for 20h to obtain the crystal form A of PQQ disodium salt. The XRPD detection of the crystal form A showed that it showed characteristic peaks at 2θ angles at 7.0°, 8.4°, 9.0°, 17.8°, 19.2°, 20.8°, 23.4°, 26.9°, 28.2°, and 29.2°. The purity was 99.9% as determined by HPLC, and the proportion of sodium ions was 10.3% as determined by HPLC-CAD.

[0037] Comparative Preparation Example 1: Preparation of Reference Pyrroloquinoline Quinone Disodium Salt Crystals According to the method provided in CN102471336B, pyrroloquinoline quinone disodium salt crystals were prepared, and the specific steps included: adding 20 g of pyrroloquinoline quinone trisodium salt solid to 1 L of ethanol / water 1:1 (v:v) solution, slowly adding hydrochloric acid to make the pH value 3.5, filtering after the pH value stabilized, and obtaining PQQ disodium salt crystals, which were recorded as crystals B. XRPD detection of the PQQ disodium salt crystals showed that it showed characteristic peaks at 2θ angles at 9.1°, 10.3°, 13.8°, 17.7°, 18.3°, 24.0°, 27.4°, 31.2°, and 39.2°.

[0038] Comparative Preparation Example 2: Preparation of Reference Pyrroloquinoline Quinone Disodium Salt Crystals According to the method provided in CN108026091B, pyrroloquinoline quinone disodium salt crystals were prepared, and the specific steps included: dissolving 10 g of pyrroloquinoline quinone crude product in 1 L of water / ethanol 1:1 (v:v) solution, heating to 50°C and stirring for 30 min, filtering after the solid was dissolved, cooling to 10°C, adjusting the pH to 3.5, and then filtering and vacuum drying to obtain PQQ disodium salt crystals, which were recorded as crystals C. XRPD detection of the PQQ disodium salt crystals showed that it showed characteristic peaks at 2θ angles at 7.4°, 8.6°, 14.0°, 14.6°, 19.9°, 21.4°, 26.0°, 27.3°, and 28.5°.

[0039] Example 1: Extraction of pyrroloquinoline quinone disodium salt from fermentation broth S1: cooling the pyrroloquinoline quinone fermentation broth to 15°C and performing solid-liquid separation through a ceramic membrane with a pore size of 0.1 μm, filtering the obtained ceramic clear liquid at 15°C using a 20 kDa ultrafiltration membrane to obtain a fermentation clear liquid; S2: Adjust the pH of the fermentation liquid to 3.5, add 0.03 times the weight of the fermentation liquid, diatomaceous earth and 0.005 times the weight of the fermentation liquid, and filter after stirring for 30 minutes. Concentrate the obtained filtrate to 6 g / L of pyrroloquinoline quinone disodium salt using a nanofiltration membrane with a molecular weight cutoff of 150 Da. Add sodium chloride to the obtained concentrated solution until the sodium chloride concentration reaches 1.0 mol / L, stir for 1 hour, and filter to obtain a salting out product; S3: putting the salting out material into a dissolving tank, which contains a composite solvent composed of water, dimethyl sulfoxide and N,N-dimethylformamide in a volume ratio of 1:0.5:0.1, and the amount ratio of the salting out material to the composite solvent is 1kg:50L, the temperature in the dissolving tank is controlled at 50°C, the obtained salting out material solution is poured into a crystallization tank, and the crystal A obtained in Preparation Example 1 is added as a seed crystal, and the seed crystal addition amount is 0.1%, and then the temperature is reduced to 40°C±0.5°C at a rate of 2°C / min and kept warm for 30s, and then the temperature is reduced to 30°C±0.5°C at a rate of 4°C / min and kept warm for 1min, and then the temperature is reduced to 20°C at a rate of 6°C / min, and the suspension in the crystallization tank is continuously filtered through a cross-flow filter, and the filtrate is returned to the dissolving tank to continue to dissolve the salting out material; S4: The crystals in the crystallization tank are filtered and then put into a 50% ethanol aqueous solution, and then pulped and washed at 5°C. After washing for 1 hour, the filter cake is dried at 40°C for 24 hours using a double cone dryer to obtain pyrroloquinoline quinone disodium salt.

[0040] The yield of pyrroloquinoline quinone disodium salt product is 72.3%, the purity is 99.8%, and the crystallization efficiency is 27 g / min / L.

[0041] Example 2: Extraction of pyrroloquinoline quinone disodium salt from fermentation broth S1: cooling the pyrroloquinoline quinone fermentation broth to 10°C and performing solid-liquid separation through a ceramic membrane with a pore size of 0.5 μm, filtering the obtained ceramic clear liquid at 10°C using a 10 kDa ultrafiltration membrane to obtain a fermentation clear liquid; S2: Adjust the pH of the fermentation liquid to 4.0, add 0.01 times the weight of the fermentation liquid, diatomaceous earth and 0.002 times the weight of the fermentation liquid, stir for 30 minutes and filter. Concentrate the obtained filtrate to 7 g / L of pyrroloquinoline quinone disodium salt using a nanofiltration membrane with a molecular weight cutoff of 300 Da. Add sodium chloride to the obtained concentrated solution until the sodium chloride concentration reaches 0.8 mol / L, stir for 1 hour and filter to obtain a salting out product; S3: putting the salting out material into a dissolving tank, which contains a composite solvent composed of water, dimethyl sulfoxide and N,N-dimethylformamide in a volume ratio of 1:0.8:0.3, and the amount ratio of the salting out material to the composite solvent is 1kg:80L, the temperature in the dissolving tank is controlled at 55°C, the obtained salting out material solution is poured into a crystallization tank, and the crystal A obtained in Preparation Example 2 is added as a seed crystal, and the seed crystal addition amount is 0.1%, and then the temperature is reduced to 40°C±0.5°C at a rate of 1.5°C / min and kept warm for 2min, and then the temperature is reduced to 30°C±0.5°C at a rate of 3°C / min and kept warm for 1min, and then the temperature is reduced to 15°C at a rate of 5°C / min, and the suspension in the crystallization tank is continuously filtered through a cross-flow filter, and the filtrate is returned to the dissolving tank to continue to dissolve the salting out material; S4: The crystals in the crystallization tank are filtered and then put into a 50% ethanol aqueous solution, and then pulped and washed at 5°C. After washing for 1 hour, the filter cake is dried at 60°C for 10 hours using a double cone dryer to obtain pyrroloquinoline quinone disodium salt.

[0042] The yield of pyrroloquinoline quinone disodium salt product is 76.9%, the purity is 99.5%, and the crystallization efficiency is 20 g / min / L.

[0043] Example 3: Extraction of pyrroloquinoline quinone disodium salt from fermentation broth S1: cooling the pyrroloquinoline quinone fermentation broth to 20°C and performing solid-liquid separation through a ceramic membrane with a pore size of 0.3 μm, filtering the obtained ceramic clear liquid at 20°C using a 20 kDa ultrafiltration membrane to obtain a fermentation clear liquid; S2: Adjust the pH of the fermentation liquid to 3.0, add 0.05 times the weight of the fermentation liquid perlite and 0.01 times the weight of the fermentation liquid white clay, stir for 30 minutes and filter. Concentrate the obtained filtrate to 6 g / L of pyrroloquinoline quinone disodium salt using a nanofiltration membrane with a molecular weight cutoff of 200 Da. Add sodium chloride to the obtained concentrated solution until the sodium chloride concentration reaches 1.2 mol / L, stir for 1 hour and filter to obtain a salting out product; S3: putting the salting out material into a dissolving tank, which contains a composite solvent composed of water, dimethyl sulfoxide and N,N-dimethylformamide in a volume ratio of 1:0.6:0.2, and the amount ratio of the salting out material to the composite solvent is 1kg:60L, the temperature in the dissolving tank is controlled at 45°C, the obtained salting out material solution is poured into a crystallization tank, and the crystal A obtained in Preparation Example 1 is added as a seed crystal, and the seed crystal addition amount is 0.1%, and then the temperature is reduced to 40°C±0.5°C at a rate of 1°C / min and kept warm for 60s, and then the temperature is reduced to 30°C±0.5°C at a rate of 2°C / min and kept warm for 20s, and then the temperature is reduced to 25°C at a rate of 4°C / min, and the suspension in the crystallization tank is continuously filtered through a cross-flow filter, and the filtrate is returned to the dissolving tank to continue to dissolve the salting out material; S4: The crystals in the crystallization tank are filtered and then put into a 50% ethanol aqueous solution, and then pulped and washed at 5°C. After washing for 1 hour, the filter cake is dried at 50°C for 18 hours using a double cone dryer to obtain pyrroloquinoline quinone disodium salt.

[0044] The yield of pyrroloquinoline quinone disodium salt product is 74.3%, the purity is 99.6%, and the crystallization efficiency is 24 g / min / L.

[0045] Example 4: Extraction of pyrroloquinoline quinone disodium salt from fermentation broth Pyrroloquinoline quinone disodium salt was separated and extracted from the fermentation broth according to the method of Example 1, except that in step S3, the composite solvent in the dissolution tank was replaced by water of the same volume, and the other conditions were the same as those in Example 1 to obtain pyrroloquinoline quinone disodium salt. The final yield of pyrroloquinoline quinone disodium salt product was 63.5%, the purity was 98.1%, and the crystallization efficiency was 19 g / min / L.

[0046] Example 5: Extraction of pyrroloquinoline quinone disodium salt from fermentation broth Pyrroloquinoline quinone disodium salt was separated and extracted from the fermentation broth according to the method of Example 4, except that in step S3, the salting-out solution was not cooled by gradient in the crystallization tank, but was directly cooled from 50°C to 20°C at a rate of 4°C / min, and the other conditions were the same as those of Example 4 to obtain pyrroloquinoline quinone disodium salt. The yield of pyrroloquinoline quinone disodium salt product was 60.2%, the purity was 97.2%, and the crystallization efficiency was 18 g / min / L.

[0047] Comparative Example 1 Pyrroloquinoline quinone disodium salt was separated and extracted from the fermentation broth according to the method of Example 4, except that in step S3, crystal A was replaced by crystal B of the same weight, and the other conditions were the same as those of Example 4 to obtain pyrroloquinoline quinone disodium salt. The yield of pyrroloquinoline quinone disodium salt product was 58.4%, the purity was 95.2%, and the crystallization efficiency was 13 g / min / L.

[0048] Comparative Example 2 The pyrroloquinoline quinone disodium salt was separated and purified according to the method of Example 4, except that in step S3, the crystal A was replaced by the crystal C of the same weight, and the other conditions were the same as those of Example 4 to obtain the pyrroloquinoline quinone disodium salt. The yield of the pyrroloquinoline quinone disodium salt product was 57.9%, the purity was 93.6%, and the crystallization efficiency was 17 g / min / L.

[0049] Comparative Example 3 The pyrroloquinoline quinone disodium salt was separated and purified according to the method of Example 4, except that in step S2, the pH of the fermentation supernatant was adjusted to 4.5, and the other conditions were the same as those of Example 4 to obtain pyrroloquinoline quinone disodium salt. The yield of the pyrroloquinoline quinone disodium salt product was 54.3%, the purity was 95.4%, and the crystallization efficiency was 17 g / min / L.

[0050] As can be seen from the above results, the present invention uses pyrroloquinoline quinone disodium salt crystal form A as a seed for separation and purification to obtain high-purity pyrroloquinoline quinone disodium salt, while the same effect cannot be achieved by using seeds of other crystal forms for crystallization. From the comparison of Example 1 and Example 4, it can be seen that when the semi-continuous crystallization adopts a preferred solvent, a higher purity pyrroloquinoline quinone disodium salt can be obtained. From the comparison of Example 4 and Example 5, it can be seen that when the semi-continuous crystallization adopts a gradient method to cool, a high-purity pyrroloquinoline quinone disodium salt can be obtained with a higher efficiency, which perfectly solves the contradiction between product purity and crystallization efficiency. From the comparison of Example 4 and Comparative Examples 1 to 3, it can be seen that when a seed different from the present invention is used for crystallization or the pH value is not adjusted to the scope of the present invention before using a filter aid and an adsorbent for treatment, it is not conducive to the improvement of the yield and / or purity of pyrroloquinoline quinone disodium salt.

[0051] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for separating and purifying pyrroloquinoline quinone disodium salt, characterized in that: The method comprises: removing large particle impurities in pyrroloquinoline quinone fermentation broth, adjusting the pH value of the obtained fermentation broth clear liquid to 3-4, and then mixing it evenly with a filter aid and an adsorbent, performing solid-liquid separation on the obtained mixture, and then performing nanofiltration concentration on the obtained filtrate using a nanofiltration membrane, adding salt to the obtained concentrated liquid for salting out, filtering after the salting out is completed, and crystallizing the obtained salted out product using pyrroloquinoline quinone disodium salt crystal form A as a seed crystal, wherein the pyrroloquinoline quinone disodium salt crystal form A is subjected to Cu-Kα radiation. The X-ray diffraction pattern obtained by radiation has characteristic peaks at the following 2θ angles: 7.0°±0.2°, 8.4°±0.2°, 9.0°±0.2°, 17.8°±0.2°, 19.2°±0.2°, 20.8°±0.2°, 23.4°±0.2°, 26.9°±0.2°, 28.2°±0.2°, and 29.2°±0.2°. After the crystallization is completed, the method is washed, filtered, and dried in sequence to obtain pyrroloquinoline quinone disodium salt.

2. The method for separating and purifying pyrroloquinoline quinone disodium salt according to claim 1, characterized in that: The crystallization method is semi-continuous crystallization, and the semi-continuous crystallization steps include dissolving the salting out product in a solvent at 45°C-55°C, adding pyrroloquinoline quinone disodium salt crystal form A, and then cooling to 15°C-25°C for crystallization.

3. The method for separation and purification of pyrroloquinoline quinone disodium salt according to claim 2, characterized in that: The temperature is cooled by gradient cooling, and the gradient cooling method includes first cooling to 40°C±2°C at a rate of 1~2°C / min and keeping warm for 30s~2min, then cooling to 30°C±2°C at a rate of 2~4°C / min and keeping warm for 20s~1min, and then cooling to 15°C~25°C at a rate of 4~6°C / min.

4. The method for separation and purification of pyrroloquinoline quinone disodium salt according to claim 2, characterized in that: The solvent is a composite solvent composed of water, dimethyl sulfoxide and N,N-dimethylformamide in a volume ratio of 1:(0.5-0.8):(0.1-0.3).

5. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The pyrroloquinoline quinone disodium salt crystal form A is prepared by the following method: dissolving pyrroloquinoline quinone disodium salt in water at a temperature of 55°C to 80°C and a pH value of 7 to 9, cooling the resulting solution to 0°C to 10°C, adding lower alcohol under stirring until the volume proportion of the lower alcohol in the system is 40% to 70%, precipitating solid into a suspension, adjusting the pH value of the system to 3 to 4, filtering and drying, and obtaining pyrroloquinoline quinone disodium salt crystal form A.

6. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The pyrroloquinoline quinone fermentation broth is a denitrifying mycelial microorganism fermentation broth.

7. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The step of removing large particle impurities in the pyrroloquinoline quinone fermentation broth comprises: using a ceramic membrane to perform solid-liquid separation on the pyrroloquinoline quinone fermentation broth, and then using an ultrafiltration membrane to ultrafilter the obtained ceramic clear liquid, and the obtained ultrafiltration clear liquid is the fermentation broth clear liquid; the pore size of the ceramic membrane is 0.1μm~0.5μm; the retention molecular weight of the ultrafiltration membrane is 10kDa~20kDa.

8. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The filter aid is diatomaceous earth and / or perlite; the adsorbent is selected from at least one of activated carbon, clay, bentonite and zeolite.

9. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The molecular weight cutoff of the nanofiltration membrane is 100Da-300Da; the concentration conditions are such that the content of pyrroloquinoline quinone disodium salt in the obtained concentrated solution is 4-10g / L.

10. The method for separating and purifying pyrroloquinoline quinone disodium salt according to any one of claims 1 to 4, characterized in that: The salt is selected from at least one of sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium carbonate and potassium carbonate; the amount of the salt added is such that the concentration of the salt in the system is 0.8-1.2 mol / L.