A method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts.

Abstract

This invention belongs to the field of biochemical technology and relates to a method for separating and purifying pyrroloquinoline quinone (PQQ) from fermentation broth using soluble strontium salts. The fermentation broth containing PQQ is subjected to membrane filtration to remove solid particles and macromolecular impurities with a molecular weight of not less than 10,000 Daltons. A strontium salt solution is added to the filtrate after membrane filtration, reacting to produce an organic strontium precipitate. The strontium salt used in the strontium salt solution is a water-soluble strontium salt. The organic strontium precipitate is added to a carbonate solution, and the pH of the solution is adjusted to alkaline, causing the organic strontium precipitate to convert into strontium carbonate precipitate. Solid-liquid separation is then performed to obtain a separated solution containing pyrroloquinoline quinone anions. The pH of the separated solution containing pyrroloquinoline quinone anions is adjusted to acidity, and then filtered to obtain the final product. This method is entirely based on an aqueous phase system, achieving the separation and purification of PQQ from fermentation broth through precipitation-to-aqueous phase extraction, simplifying the separation and purification process and reducing costs.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology and relates to a method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pyrroloquinoline quinone (PQQ) is a natural compound with mitochondrial, neuroprotective, and cardioprotective functions, widely found in natural foods such as parsley, green bell peppers, and green tea. However, the content of PQQ in natural foods is extremely low (only in the nanogram to microgram / kg range), making it difficult to meet the human body's needs through dietary means. Therefore, developing technologies for the large-scale production of PQQ has significant application value.

[0004] Currently, industrial production of PQQ mainly relies on chemical synthesis and microbial fermentation. Compared to chemical synthesis, microbial fermentation has become the mainstream technology due to its advantages such as low cost and environmental friendliness. However, the core bottleneck of this process lies in the efficient separation and purification of PQQ from the fermentation broth. Currently, the main methods for separating and purifying PQQ from the fermentation broth are adsorption-desorption and extraction.

[0005] The adsorption-desorption method selectively enriches PQQ with adsorbents (such as DEAE dextran gel, macroporous resin, polyamide resin, etc.) and then desorbs the target product using an eluent. A typical process involves: after the fermentation broth is adsorbed by the resin, impurities are washed sequentially with buffer and water, and then PQQ is eluted with a specific solution; the eluent is then concentrated, crystallized, or purified a second time using a chromatography column (such as a C18 reversed-phase column) to obtain the final product. However, this method has significant drawbacks: the chromatography process is lengthy (requiring multi-stage column separation), resulting in high equipment investment; the resin regeneration process is complex, and adsorption efficiency decreases after repeated use; the eluent consumption is high, generating high-concentration organic wastewater, leading to high overall costs.

[0006] Extraction methods utilize extractants (such as ion-pair reagent / phosphate aqueous two-phase systems, trioctylamine complexation systems, etc.) to separate PQQ from fermentation broth. A typical process involves mixing the PQQ-containing filtrate with the extractant, collecting the PQQ-rich organic phase, purifying it with anion exchange resin or back-extracting it with ammonia, and finally concentrating and drying it to obtain the crude product. However, this method relies on large amounts of organic solvents, making the operation cumbersome; the extractant is prone to remain in the product, posing a solvent contamination risk; and the back-extraction and concentration steps further increase energy consumption and the difficulty of safety management. Summary of the Invention

[0007] To address the problems of complex processes, high costs, poor environmental performance, or product purity risks in the separation and purification of PQQ from fermentation broth in existing technologies, the present invention aims to provide a method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts. This method is entirely based on an aqueous phase system, achieving the separation and purification of PQQ from fermentation broth through precipitation-to-aqueous phase extraction, thus simplifying the separation and purification process and reducing costs.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts, comprising the following steps:

[0010] The fermentation broth containing PQQ was subjected to membrane filtration to remove solid particles and macromolecular impurities with a molecular weight of not less than 10,000 Daltons.

[0011] A strontium salt solution was added to the filtrate after membrane filtration, and an organic strontium precipitate was produced by the reaction; the strontium salt used in the strontium salt solution was a water-soluble strontium salt.

[0012] The organic strontium precipitate was added to the carbonate solution, and the pH of the solution was adjusted to alkaline so that the organic strontium precipitate was converted into strontium carbonate precipitate. Then, solid-liquid separation was performed to obtain a separation solution containing pyrroloquinoline quinone anions.

[0013] Adjust the pH of the separation solution containing pyrroloquinoline quinone anions to make it acidic, then filter to obtain the final product.

[0014] The basic principle of this invention is as follows:

[0015]

[0016] Adding excess Sr to a weakly acidic fermentation broth 2+ In ionic solutions, strontium can form soluble complexes with amino acids and oligopeptides in the solution, while PQQ reacts with Sr. 2+ An organostrontium precipitate is formed, and this precipitate is separated to enrich PQQ. Under soluble carbonate and alkaline conditions, the organostrontium precipitate undergoes precipitation transformation to form the less soluble SrCO3, thereby releasing the alkali-soluble PQQ trivalent anion. Finally, the solution pH is adjusted to acidity, and the PQQ trivalent anion combines with a proton to form PQQ, which then precipitates out of the solution.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention introduces a method for separating and purifying pyrroloquinoline quinone using strontium salts, effectively overcoming the shortcomings of resin extraction purification or extraction-concentration methods, such as complex processes, numerous operation steps, large wastewater production, potential risks from organic solvents, and high costs. It utilizes the formation of a poorly soluble precipitate between soluble strontium salts and pyrroloquinoline quinone anions to separate and enrich pyrroloquinoline quinone from the fermentation filtrate. Further, through carbonate treatment and pH control, the strontium pyrroloquinoline quinone (organic strontium precipitate) is converted into even less soluble strontium carbonate, selectively releasing the pyrroloquinoline quinone anions. Further pH adjustment yields pyrroloquinoline quinone or its disodium salt.

[0019] This invention utilizes the formation of a precipitate and selective transformation of the precipitate with strontium to selectively separate and purify pyrroloquinoline quinone from fermentation broth using an aqueous system. This avoids the use of harmful organic solvents, achieving a pyrroloquinoline quinone yield of over 80% and a purity of up to 90%, both higher than conventional separation and purification methods (yields below 70% and purity below 80%). Furthermore, it is low-cost, produces less pollution, and is easy to operate rapidly and continuously, making it suitable for the industrial production of pyrroloquinoline quinone. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] Given that existing technologies for the separation and purification of PQQ in fermentation broth suffer from problems such as complex processes, high costs, poor environmental performance, or risks to product purity, this invention proposes a method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts.

[0023] A typical embodiment of the present invention provides a method for separating and purifying pyrroloquinoline quinone from fermentation broth using soluble strontium salts, comprising the following steps:

[0024] The fermentation broth containing PQQ was subjected to membrane filtration to remove solid particles and macromolecular impurities with a molecular weight of not less than 10,000 Daltons.

[0025] A strontium salt solution was added to the filtrate after membrane filtration, and an organic strontium precipitate was produced by the reaction; the strontium salt used in the strontium salt solution was a water-soluble strontium salt.

[0026] The organic strontium precipitate was added to the carbonate solution, and the pH of the solution was adjusted to alkaline so that the organic strontium precipitate was converted into strontium carbonate precipitate. Then, solid-liquid separation was performed to obtain a separation solution containing pyrroloquinoline quinone anions.

[0027] Adjust the pH of the separation solution containing pyrroloquinoline quinone anions to make it acidic, then filter to obtain the final product.

[0028] This invention purifies and concentrates pyrroloquinoline quinone solution using membrane filtration and water-soluble strontium salts, replacing traditional resin extraction or extraction-based purification and concentration methods. It develops a highly efficient, inexpensive, and convenient aqueous-phase pyrroloquinoline quinone extraction method with a total yield exceeding 80%. This invention utilizes membrane filtration technology to remove solid particles and large molecular protein impurities, followed by selective precipitation and release of pyrroloquinoline quinone anions through strontium salt precipitation and precipitation transformation. The operation is simple and easy to implement, using no harmful organic solvents, and the equipment and operation are simple, significantly reducing extraction costs, wastewater discharge, and improving product quality. This process has significant potential for further optimization, effectively improving the process, increasing yield, reducing costs, and enhancing product competitiveness.

[0029] In membrane filtration, when using an ultrafiltration membrane, the molecular weight of large molecular impurities can be controlled by adjusting the membrane's specifications. In some embodiments, a ceramic membrane and an ultrafiltration membrane are used sequentially to filter fermentation broth containing PQQ. Since ultrafiltration requires pressure, and the presence of large impurity particles and bacteria in the fermentation broth affects ultrafiltration efficiency, this invention first uses a ceramic membrane to remove larger impurity particles and bacteria from the fermentation broth, thereby improving the efficiency of subsequent ultrafiltration.

[0030] Specifically, the pore size of the ceramic membrane is 10~100 nm; further, the pore size of the ceramic membrane is 40~60 nm. The filtration effect is better under these pore size conditions.

[0031] Specifically, when using ceramic membranes for filtration, the temperature is 0~30℃; further, the temperature is 10~15℃. The filtration effect is better under these temperature conditions.

[0032] Ultrafiltration membranes are primarily used to remove large molecular impurities. Specifically, the molecular weight cutoff of ultrafiltration membranes is 10,000 to 100,000 Daltons; further, the molecular weight cutoff of ultrafiltration membranes is 30,000 to 50,000 Daltons.

[0033] Specifically, when using an ultrafiltration membrane for ultrafiltration, the temperature is 0~30℃; further, the temperature is 10~15℃. Ultrafiltration effect is better under these temperature conditions.

[0034] Specifically, the ultrafiltration membrane is a spiral wound membrane or a tubular membrane.

[0035] The water-soluble strontium salt described in this invention can be an inorganic strontium salt such as strontium chloride or strontium nitrate, or an organic strontium salt such as strontium citrate or strontium acetate. In some embodiments, the mass concentration of the strontium salt solution is 15-25%.

[0036] In some embodiments, the volume ratio of PQQ fermentation broth to strontium salt solution is 19~21:1.

[0037] To better convert pyrroloquinoline quinone and strontium into an organostrontium precipitate, in some embodiments, the pH of the filtrate after membrane filtration is adjusted to acidic, and then a strontium salt solution is added.

[0038] Specifically, adjust the pH of the filtrate after membrane filtration to 5-6. The effect is better under these pH conditions.

[0039] After the reaction produces an organostrontium precipitate, the precipitate can be obtained from the reaction solution by filtration or centrifugation. The filtration method can be membrane filtration, plate and frame filtration, pressure filtration, etc. This invention does not further limit the specific method for obtaining the organostrontium precipitate.

[0040] The carbonate solution described in this invention can be a sodium carbonate solution, a potassium carbonate solution, an ammonium carbonate solution, etc.

[0041] After the organostrontium precipitate is added to the carbonate solution, the pH can be adjusted using an alkali metal hydroxide, such as sodium hydroxide or potassium hydroxide, or ammonia. The organostrontium precipitate is washed with water before being added to the carbonate solution. In some embodiments, after adding the organostrontium precipitate to the carbonate solution, the pH of the solution is adjusted to be not lower than 13.

[0042] After the organostrontium precipitate is converted into strontium carbonate precipitate, solid-liquid separation can be performed by filtration or centrifugation. In some embodiments, filtration is used for solid-liquid separation after the organostrontium precipitate is converted into strontium carbonate precipitate.

[0043] In some embodiments, the pH of the separation solution containing the pyrroloquinolinequinone anion is adjusted to below 4. The pyrroloquinolinequinone product obtained by pH adjustment is pyrroloquinolinequinone or pyrroloquinolinequinone disodium salt. For example, when the pH is 2-3, pyrroloquinolinequinone disodium salt is obtained; when the pH is 1, pyrroloquinolinequinone is obtained.

[0044] In some embodiments, the pH of the separation solution containing the pyrroloquinoline quinone anion is adjusted to be acidic, and the reaction is carried out for 20-40 minutes, followed by filtration. Specifically, the pyrroloquinoline quinone product obtained after filtration is washed with water. More specifically, ice water is used for washing.

[0045] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0046] Unless otherwise specified, the techniques or conditions described in the following examples shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0047] In this invention, the content of disodium pyrroloquinoline quinone is determined according to the 2022 Announcement No. 1 of the National Health Commission of the People's Republic of China on 32 kinds of "three new foods" such as Guanshan cherry blossoms, regarding the detection method of pyrroloquinoline quinone. Pyrroloquinoline quinone is measured by the above method and then converted into the pyrroloquinoline quinone content based on stoichiometry.

[0048] For ease of description, pyrrolquinoline quinone is referred to as PQQ in the examples, and the fermentation broth is obtained by fermentation culture of Microbes genus.

[0049] PQQ solubility test:

[0050] Accurately weigh 10.00 g of PQQ and add it to 100 g of water. Adjust the pH of the reaction solution to 1-12 using 30% concentrated hydrochloric acid or 20% sodium hydroxide. Stir at room temperature for 2 hours. Filter under reduced pressure, dry the filter cake, and obtain the weight of insoluble PQQ. Further determine the solubility of PQQ in water at different pH values. See Table 1.

[0051] Table 1. Solubility of PQQ in water at different pH values

[0052]

[0053] Example 1

[0054] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 12 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to adjust the pH of the filtrate to 5.5. 10 L of a 20% strontium chloride solution was added, and the mixture was stirred at room temperature for 2 hours, then allowed to stand for 4 hours. The filtrate was filtered, and the precipitate was washed with deionized water and ethanol, respectively. The precipitate was transferred to 10 L of a solution containing 10% sodium carbonate, and the pH was adjusted to 13.5 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then obtained by filtration. Add 6M (M represents mol / L) hydrochloric acid to the filtrate until the pH is 3.5. At this point, the solution color gradually changes from dark green to red and a large amount of precipitate is produced. Continue stirring for 30 minutes, centrifuge and filter, wash the filter cake with ice water, and dry the wet powder under normal pressure at 80℃ for 2 hours to obtain 88.6g of PQQ disodium salt.

[0055] Example 2

[0056] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 12 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to the filtrate to adjust the pH to 5.0. 10 L of 20% strontium chloride solution was added, and the mixture was stirred at room temperature for 2 hours, then allowed to stand for 4 hours. The filtrate was filtered, and the precipitate was washed with deionized water and ethanol, respectively. The precipitate was transferred to 10 L of a solution containing 10% sodium carbonate, and the pH was adjusted to 14.1 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then filtered. 6 M hydrochloric acid was added dropwise to the filtrate until the pH reached 1. At this point, the solution color gradually changed from dark green to red, and a large amount of precipitate was produced. The mixture was stirred for another 30 minutes, centrifuged, and filtered. The filter cake was washed with ice water, and the wet powder was dried at atmospheric pressure at 80 °C for 2 hours to obtain 80.2 g of PQQ.

[0057] Example 3

[0058] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 10 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to adjust the pH of the filtrate to 6.0. 10 L of a 10% strontium citrate solution was added, and the mixture was stirred at room temperature for 2 hours, then allowed to stand for 4 hours. The filtrate was filtered, and the precipitate was washed with deionized water and ethanol, respectively. The precipitate was transferred to 10 L of a 10% potassium carbonate solution, and the pH was adjusted to 13.2 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then filtered. 6 M sulfuric acid was added to the filtrate until the pH reached 4.0. At this point, the solution color gradually changed from dark green to red, and a large amount of precipitate was produced. The mixture was stirred for another 30 minutes, centrifuged, and filtered. The filter cake was washed with ice water, and the wet powder was dried at atmospheric pressure at 80 °C for 2 hours to obtain 86.7 g of disodium PQQ.

[0059] Example 4

[0060] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 10 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to adjust the pH of the filtrate to 5.5. 10 L of 10% strontium chloride solution was added, and the mixture was stirred at room temperature for 2 hours. The filtrate was filtered, and the precipitate was washed with deionized water and ethanol, respectively. The precipitate was transferred to 10 L of a 10% potassium carbonate solution, and the pH was adjusted to 13.8 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then filtered. 6 M sulfuric acid was added to the filtrate until the pH reached 3.5. At this point, the solution color gradually changed from dark green to red, and a large amount of precipitate was produced. The mixture was stirred for another 30 minutes, centrifuged, and filtered. The filter cake was washed with ice water, and the wet powder was dried at atmospheric pressure at 80 °C for 2 hours to obtain 88.7 g of disodium PQQ.

[0061] Example 5

[0062] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 10 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to adjust the pH of the filtrate to 5.5. 10 L of a 20% strontium acetate solution was added, and the mixture was stirred at room temperature for 2 hours. The filtrate was filtered, and the precipitate was washed with deionized water. The precipitate was transferred to 10 L of a 5% potassium carbonate solution, and the pH was adjusted to 13.5 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then filtered. 6 M sulfuric acid was added to the filtrate until the pH reached 1.0. At this point, the solution color gradually changed from dark green to red, and a large amount of precipitate was produced. The mixture was stirred for another 30 min, centrifuged, and filtered. The filter cake was washed with ice water, and the wet powder was dried at atmospheric pressure at 80 °C for 2 hours to obtain 89.1 g of PQQ.

[0063] Example 6

[0064] 200 L of PQQ fermentation broth, with a PQQ content of 500 mg / L, was filtered through a 50 nm ceramic membrane at 10 °C. The resulting filtrate was then ultrafiltered through a spiral wound membrane with a molecular weight cutoff of 30,000. 6 M hydrochloric acid was added to adjust the pH of the filtrate to 5.5. 10 L of 20% strontium nitrate solution was added, and the mixture was stirred at room temperature for 2 hours. The filtrate was filtered, and the precipitate was washed with deionized water. The precipitate was transferred to 10 L of a 5% potassium carbonate solution, and the pH was adjusted to 13.5 with NaOH. The mixture was stirred at room temperature for 5 hours, at which point the solution turned dark green. The filtrate was then filtered. 6 M sulfuric acid was added to the filtrate until the pH reached approximately 1. At this point, the solution color gradually changed from dark green to red, and a large amount of precipitate was produced. The mixture was stirred for another 30 minutes, centrifuged, and filtered. The filter cake was washed with ice water, and the wet powder was dried at atmospheric pressure at 80 °C for 2 hours to obtain 88.9 g of PQQ.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for separating and purifying pyrroloquinoline quinone in a fermentation broth using a soluble strontium salt, characterized by, The method comprises the following steps: membrane filtration is performed on the fermentation liquor containing PQQ to remove solid particles and macromolecular impurities with a molecular weight of not less than 10,000 daltons in the fermentation liquor; a strontium salt solution is added to the filtrate after membrane filtration, and organic strontium precipitates are generated by reaction; the strontium salt in the strontium salt solution is a water-soluble strontium salt; the organic strontium precipitates are added to a carbonate solution, and the pH of the solution is adjusted to alkaline, so that the organic strontium precipitates are converted into strontium carbonate precipitates, and then solid-liquid separation is performed to obtain a separation solution containing pyrroloquinoline quinone anions; the pH of the separation solution containing pyrroloquinoline quinone anions is adjusted to be acidic, and then filtration is performed.

2. The method of claim 1 wherein, The ceramic membrane and the ultrafiltration membrane are used in sequence to perform membrane filtration on the fermentation liquor containing PQQ.

3. The method of claim 2, wherein, The pore size of the ceramic membrane is 40-60 nm. When the ceramic membrane is used for filtration, the temperature is 10-15℃. The ultrafiltration membrane has a molecular weight cut-off of 30,000-50,000 daltons. When the ultrafiltration membrane is used for ultrafiltration, the temperature is 10-15℃.

4. The method of claim 1 wherein, The mass concentration of the strontium salt solution is 15-25%.

5. The method of claim 1 wherein, The volume ratio of the PQQ fermentation liquor to the strontium salt solution is 19-21:

1.

6. The method of claim 1 wherein, The pH of the filtrate after membrane filtration is adjusted to be acidic, and then the strontium salt solution is added.

7. The method of claim 1 wherein, After the organic strontium precipitates are added to the carbonate solution, the pH of the solution is adjusted to be not less than 13.

8. The method of claim 1 wherein, After the organic strontium precipitates are converted into strontium carbonate precipitates, the solid-liquid separation is performed by filtration.

9. The method of claim 1 wherein, The pH of the separation solution containing pyrroloquinoline quinone anions is adjusted to be below 4.

10. The method of claim 1 wherein, The pH of the separation solution containing pyrroloquinoline quinone anions is adjusted to be acidic, and then the reaction is performed for 20-40 min, and then filtration is performed; further, the pyrroloquinoline quinone product obtained after filtration is washed with water.

Images