A method for extracting 3,4-dihydroxybenzoic acid from a fermentation broth

Abstract

The application discloses a method for extracting 3,4-dihydroxybenzoic acid from fermentation liquor, and relates to the technical field of 3,4-dihydroxybenzoic acid extraction. The method comprises the following steps: adjusting the pH of the fermentation liquor containing 3,4-dihydroxybenzoic acid to 5.0-6.0; filtering to obtain a clear liquid; continuously separating the clear liquid through a simulated moving bed chromatography to obtain an extraction liquid with high purity; wherein the simulated moving bed chromatography is a four-section SMB system, the four sections are connected in series, each section comprises two or more than two identical chromatographic columns connected in series, the fixed phase in the chromatographic column is hydrogen type weak alkaline anion exchange resin D301; the eluent is a dilute hydrochloric acid solution with a mass fraction of 0.8-1.2%; and after the extraction liquid is concentrated, crystallization and solid-liquid separation are carried out to obtain 3,4-dihydroxybenzoic acid pure product. The application realizes continuous and efficient separation of 3,4-dihydroxybenzoic acid, saves the consumption of acid, alkali and other raw material reagents, and can realize large-scale and automatic continuous production.

Description

Technical Field

[0001] This invention relates to the field of 3,4-dihydroxybenzoic acid extraction technology, and more specifically, to a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth. Background Technology

[0002] 3,4-Dihydroxybenzoic acid, also known as protocatechuic acid, is an important natural polyphenol compound widely found in commonly used traditional Chinese medicines such as Acanthopanax senticosus, Eucommia ulmoides, and Phyllanthus urinaria. Modern medical research indicates that 3,4-dihydroxybenzoic acid and its derivatives possess high physiological activity value, including antiplatelet aggregation, reduced myocardial oxygen consumption, increased myocardial oxygen tolerance, slowed heart rate, antibacterial activity, and analgesic effects. It also exhibits antioxidant, antitumor, and neuroprotective properties, thus finding wide application in medicine, food, and animal feed.

[0003] Currently, there are few reports on the industrial production of 3,4-dihydroxybenzoic acid. As market demand increases daily, its production methods are gradually shifting from traditional plant extraction to modern bio-enzyme catalysis. The former's advantage lies in the widespread availability of 3,4-dihydroxybenzoic acid in natural resources. Extracting 3,4-dihydroxybenzoic acid from abundant traditional Chinese medicinal materials greatly enhances the comprehensive utilization value of these materials. For example, CN107266308A discloses a process for preparing 3,4-dihydroxybenzoic acid from longan shells, CN104098465A discloses a process for extracting 3,4-dihydroxybenzoic acid from *Artemisia argyi*, and CN114573446A discloses a method for preparing 3,4-dihydroxybenzoic acid from *Sanghuang*. However, the scarcity of plant resources makes large-scale industrial production of 3,4-dihydroxybenzoic acid from plants difficult to achieve, and the subsequent purification process is cumbersome, requiring a large amount of organic reagents and posing a significant negative impact on the environment. The latter method uses engineered bacteria constructed using bioengineering technology as its core to rapidly convert target compounds into large quantities of 3,4-dihydroxybenzoic acid, greatly alleviating the limitations of plant resources and providing a stable source for the extraction of 3,4-dihydroxybenzoic acid. For example, invention CN113717994A introduces the quiC gene into E. coli to prepare a whole-cell catalyst, which is then applied to the fermentation broth rich in 3-dehydroshikimic acid to convert 3,4-dihydroxybenzoic acid. Public data shows that the content of 3,4-dihydroxybenzoic acid in its whole-cell conversion broth reaches 88.5 g / L, laying an important foundation for the industrial production of 3,4-dihydroxybenzoic acid. However, the cell fermentation process and the introduction of the whole-cell catalyst result in a complex composition of the conversion broth, which to some extent increases the difficulty of downstream separation and extraction. Furthermore, 3-dehydroshikimic acid is easily catalyzed by phosphate to form gallic acid under aerobic conditions, resulting in insufficient purity of 3,4-dihydroxybenzoic acid, which cannot meet market demand.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, comprising the following steps:

[0008] The pH of the fermentation broth containing 3,4-dihydroxybenzoic acid was adjusted to 5.0-6.0;

[0009] The insoluble impurities and macromolecular substances in the fermentation broth are removed to obtain a clear liquid;

[0010] The clarified liquid is continuously separated by simulated moving bed chromatography to obtain a high-purity extract. The simulated moving bed chromatography is a four-segment SMB system, with the four segments connected in series. Each segment includes two or more identical chromatographic columns connected in series. The stationary phase in each column is a hydroxyl-type weakly basic anion exchange resin D301. Each segment is sequentially provided with an inlet, a raffinate outlet, an eluent inlet, and an extract outlet. The clarified liquid enters through the inlet, and the eluent enters through the eluent inlet. The eluent is a 0.8-1.2% (w / w) dilute hydrochloric acid solution. The inlet, raffinate outlet, eluent inlet, and extract outlet are periodically switched in a clockwise direction until the clarified liquid feeding is completed.

[0011] The extract was concentrated and crystallized, followed by solid-liquid separation to obtain 3,4-dihydroxybenzoic acid.

[0012] In an optional embodiment, the flow rate V1 of the feed inlet is 0-100 mL / min, the flow rate V2 of the raffinate outlet is 1-1000 mL / min, the flow rate V3 of the eluent inlet is 1-1000 mL / min, the flow rate V4 of the extract outlet is 1-1000 mL / min, and the switching time of the periodic switching is 10-20 min.

[0013] Preferably, V3 ≥ V2 + V4 - V1.

[0014] In an optional implementation, before the clarified liquid is fed from the feed inlet, the eluent inlet, the extract outlet, and the raffinate outlet are set to run for 20-40 minutes according to the set values.

[0015] In an optional embodiment, after the clear liquid is fed from the inlet, the system further includes replacing the clear liquid with pure water and continuing to run for 20-40 minutes.

[0016] In an optional embodiment, after separation by simulated moving bed chromatography, the content of 3,4-dihydroxybenzoic acid in the extract is ≥99.5%.

[0017] In an optional embodiment, the concentration of 3,4-dihydroxybenzoic acid in the fermentation broth is 60-85 g / L, the purity is 95%-98%, and the pH of the fermentation broth system is 5.0-9.0.

[0018] In an optional embodiment, removing insoluble impurities and macromolecular substances from the fermentation broth to obtain a clear liquid includes: firstly, separating the fermentation broth using a ceramic membrane to remove insoluble impurities such as bacterial cells, and then filtering the filtrate using a nanofiltration membrane to remove soluble proteins, sugars, pigments, and oxidized components from the filtered clear liquid.

[0019] In an optional embodiment, the ceramic membrane has a pore size of 80-120 nm and the nanofiltration membrane has an average molecular weight of 250-500 Da.

[0020] In an optional embodiment, the extract is concentrated and crystallized, followed by solid-liquid separation, which includes: first, the extract is concentrated under vacuum to a mass fraction of 20%-30%, then transferred to a crystallization container, cooled and stirred until the liquid temperature is less than or equal to 15°C, and then the solid and liquid are separated and vacuum dried.

[0021] In an optional embodiment, the vacuum concentration process is performed with a vacuum degree not exceeding 100 Pa, a water bath temperature of 90-100°C, and a material temperature not lower than 40°C.

[0022] The present invention has the following beneficial effects:

[0023] The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth provided in this application achieves a high purity of 3,4-dihydroxybenzoic acid through the synergistic effect of each step, and also results in a high yield of the target product. In this invention, the simulated moving bed chromatography stationary phase is a hydroxyl-type weakly basic anion exchange resin D301, and the eluent is a 0.8-1.2% (w / w) dilute hydrochloric acid solution. This method can initially separate the finished product from incompletely converted 3-dehydroshikimic acid and its byproducts (such as gallic acid), while simultaneously acidifying the 3,4-dihydroxybenzoic acid component.

[0024] The method of this invention achieves continuous and efficient separation of 3,4-dihydroxybenzoic acid, saving the consumption of raw materials and reagents such as acids and alkalis. It is conducive to the large-scale and automated continuous production of 3,4-dihydroxybenzoic acid, greatly improving its industrial production efficiency and laying an important theoretical foundation for its large-scale industrial production. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram of the structure of the simulated moving bed chromatography provided in this application. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0028] This invention provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, comprising the following steps:

[0029] S1, fermentation broth.

[0030] In the fermentation broth of this invention, the concentration of 3,4-dihydroxybenzoic acid is 60-80 g / L, the liquid phase purity is 95% to 98%, and the pH of the fermentation broth system is 5.0-9.0.

[0031] Fermentation broths containing 3,4-dihydroxybenzoic acid prepared using *Escherichia coli* as the engineered strain can all be used as the fermentation broth of this application. The concentration of 3,4-dihydroxybenzoic acid in the fermentation broth is 60-80 g / L, the purity is 95% to 98%, and the pH of the fermentation broth system is 5.0-9.0. The method for preparing the fermentation broth containing 3,4-dihydroxybenzoic acid using *Escherichia coli* as the engineered strain can refer to existing conventional techniques; for example, the fermentation broth in invention CN113717994A can be extracted to obtain the target product 3,4-dihydroxybenzoic acid.

[0032] Of course, fermentation broth containing 3,4-dihydroxybenzoic acid prepared by other engineered bacteria can also be used as the fermentation broth of this application.

[0033] S2, Adjust the pH of the system.

[0034] Adjusting the pH of the system to 5.0-6.0 creates a weakly acidic environment. The 3,4-dihydroxybenzoic acid structure contains catechol, and the phenolic hydroxyl groups at the 3 and 4 positions of its benzene ring are easily oxidized or form quinones under alkaline conditions. Adjusting the pH to make the system weakly acidic can ensure its stability to a certain extent.

[0035] Adjustments are made using either acids or bases. Acids include, but are not limited to, hydrochloric acid, sulfuric acid, and phosphoric acid. Bases include, but are not limited to, sodium hydroxide, with a concentration of 100-300 g / L.

[0036] S3, Filtering.

[0037] The fermentation broth is purified by removing insoluble impurities and macromolecules. In this application, the fermentation broth is filtered sequentially through a ceramic membrane and a nanofiltration membrane. Specifically, a ceramic membrane with a pore size of 80-120 nm is first used to separate the fermentation broth and remove the bacterial cells. Then, a nanofiltration membrane with an average molecular weight of 250-500 Da is used to filter the filtrate to remove soluble proteins, pigments produced by biocatalysis, and a small amount of 3,4-dihydroxybenzoic acid oxidation components from the filtered filtrate. Studies have shown that omitting the nanofiltration step results in lower purity and a pale yellow color.

[0038] S4, simulated moving bed chromatography for continuous separation.

[0039] The clear liquid was continuously separated by simulated moving bed chromatography to obtain a high-purity extract.

[0040] Please refer to Figure 1 The simulated moving bed chromatography is a four-segment SMB system, with four segments connected in series. Each segment includes 2-4 identical columns connected in series. The stationary phase in the columns is a hydroxyl-type weakly basic anion exchange resin D301. Each segment is connected sequentially by an inlet, a raffinate outlet, an eluent inlet, and an extract outlet. The clear liquid enters through the inlet, and the eluent enters through the eluent inlet. The eluent is a 0.8-1.2% (w / w) dilute hydrochloric acid solution. The acidified 3,4-dihydroxybenzoic acid component and its salts flow out through the raffinate outlet, while other impurities flow out through both the raffinate and extract outlets. The inlet, raffinate outlet, eluent inlet, and extract outlet are periodically switched in a clockwise direction until the clear liquid feeding is completed.

[0041] The flow rate V1 at the feed inlet is 0-100 mL / min, and the flow rate V2 at the raffinate outlet is 1-1000 mL / min; the flow rate V3 at the eluent inlet is 1-1000 mL / min, and the flow rate V4 at the extract outlet is 1-1000 mL / min. The switching time for periodic switching is 10-20 min.

[0042] Preferably, V3 ≥ V2 + V4 - V1.

[0043] Before feeding the clarified liquid into the feed inlet, set the eluent inlet, extract outlet, and raffinate outlet to the set values ​​and run for 20-40 minutes.

[0044] After the clear liquid is fed from the inlet, the process also includes replacing the clear liquid with pure water and continuing to run for 20-40 minutes.

[0045] After separation by simulated moving bed chromatography, the content of 3,4-dihydroxybenzoic acid in the extract was 99.5%-99.9%.

[0046] S5. Concentration, crystallization, solid-liquid separation.

[0047] The extract was concentrated and crystallized, followed by solid-liquid separation to obtain the 3,4-dihydroxybenzoic acid product. Specifically, the extract was first concentrated under vacuum to a mass fraction of 20%-30%, with a vacuum degree not exceeding 100 Pa, a water bath temperature of 90-100℃, and a material temperature not lower than 40℃. It was then transferred to a crystallization container, cooled and stirred until the liquid temperature was less than or equal to 15℃, followed by solid-liquid separation and vacuum drying.

[0048] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0049] Example 1

[0050] This embodiment provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, which includes the following steps:

[0051] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 80g / L, adjust the pH to 5.0 with concentrated hydrochloric acid, and filter it sequentially through a ceramic membrane with a pore size of 100nm and a nanofiltration membrane with an average molecular weight of 250Da to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0052] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used instead of feed liquid and the system was run for 30 minutes. The purity of the 3,4-dihydroxybenzoic acid component in the extract was then detected by high performance liquid chromatography, which showed that it was 99.9%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 95%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0053] The extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid) mass fraction of 25%. During this process, most of the 3,4-dihydroxybenzoic acid was observed to precipitate slowly as relatively regular crystals. The resulting solid-liquid mixture was transferred to a crystallization container and cooled and stirred until the solution temperature reached 15°C. After solid-liquid separation and vacuum drying, approximately 722g of the final 3,4-dihydroxybenzoic acid product was obtained, with a purity of 101.2%. HPLC analysis showed a liquid phase purity of 99.97% and a product recovery rate of 90.25%.

[0054] Example 2

[0055] This embodiment provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, which includes the following steps:

[0056] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 60g / L, adjust the pH to 6.0 with concentrated hydrochloric acid, and filter it sequentially through a ceramic membrane with a pore size of 100nm and a nanofiltration membrane with an average molecular weight of 250Da to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0057] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 100 mL / min, the eluent flow rate to 1000 mL / min, the extract flow rate to 500 mL / min, and the raffinate flow rate to 510 mL / min. The switching time was set to 10 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used to replace the feed solution and the system was run for 30 minutes. Then, the purity of the 3,4-dihydroxybenzoic acid component in the extract was detected by high performance liquid chromatography, which showed that it was 99.85%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 97%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0058] The above extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 20%. During this process, it was observed that most of the 3,4-dihydroxybenzoic acid precipitated slowly as relatively regular crystals. After obtaining the solid-liquid mixture of 3,4-dihydroxybenzoic acid, it was transferred to a crystallization container and cooled and stirred until the temperature of the liquid reached 10°C. After solid-liquid separation and vacuum drying, approximately 549g of 3,4-dihydroxybenzoic acid product was obtained. The purity of the liquid phase was 99.90% by HPLC, the product recovery rate was 91.5%, and the mass purity was 100.6%.

[0059] Example 3

[0060] This embodiment provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, which includes the following steps:

[0061] Take 20L of 3,4-dihydroxybenzoic acid fermentation broth and determine that the 3,4-dihydroxybenzoic acid content is 82g / L. Adjust the pH to 5.0 with concentrated hydrochloric acid and filter it sequentially through a ceramic membrane with a pore size of 100nm and a nanofiltration membrane with an average molecular weight of 250Da to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0062] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 100 mL / min, the eluent flow rate to 800 mL / min, the extract flow rate to 450 mL / min, and the raffinate flow rate to 450 mL / min. The switching time was set to 10 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used to replace the feed solution and the system was run for 30 minutes. Then, the purity of the 3,4-dihydroxybenzoic acid component in the extract was detected by high performance liquid chromatography, which showed that it was 99.89%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 96.5%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0063] The above extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 30%. During this process, it was observed that most of the 3,4-dihydroxybenzoic acid precipitated slowly as relatively regular crystals. After obtaining the solid-liquid mixture of 3,4-dihydroxybenzoic acid, it was transferred to a crystallization container and cooled and stirred until the temperature of the liquid reached 10°C. After solid-liquid separation and vacuum drying, approximately 1505g of 3,4-dihydroxybenzoic acid product was obtained. HPLC analysis showed that its liquid phase purity was 99.95%, its mass purity was 100.8%, and the product recovery rate was 91.76%.

[0064] Example 4

[0065] This embodiment provides a method for extracting 3,4-dihydroxybenzoic acid from fermentation broth, which includes the following steps:

[0066] Take 20L of 3,4-dihydroxybenzoic acid fermentation broth and determine that the 3,4-dihydroxybenzoic acid content is 82g / L. Adjust the pH to 5.0 with concentrated hydrochloric acid and filter it sequentially through a ceramic membrane with a pore size of 120nm and a nanofiltration membrane with an average molecular weight of 500Da to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0067] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 50 mL / min, the eluent flow rate to 500 mL / min, the extract flow rate to 275 mL / min, and the raffinate flow rate to 300 mL / min. The switching time was set to 30 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used to replace the feed solution and the system was run for 30 minutes. The purity of the 3,4-dihydroxybenzoic acid component in the extract was then detected by high performance liquid chromatography (HPLC), which showed that it was 99.86%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 96.8%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0068] The above extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 20%. During this process, it was observed that most of the 3,4-dihydroxybenzoic acid precipitated slowly as relatively regular crystals. After obtaining the solid-liquid mixture of 3,4-dihydroxybenzoic acid, it was transferred to a crystallization container, cooled and stirred until the temperature of the liquid reached 10°C. After solid-liquid separation and vacuum drying, approximately 1488g of 3,4-dihydroxybenzoic acid product was obtained. HPLC analysis showed that its liquid phase purity was 99.93%, its mass purity was 101.5%, and the product recovery rate was 90.73%.

[0069] Comparative Example 1

[0070] Compared with the scheme designed in this invention, Comparative Example 1 uses ceramic membrane separation, nanofiltration membrane filtration, acidification crystallization to separate crude product, and pure water redissolution and recrystallization to separate and extract 3,4-dihydroxybenzoic acid. The specific operation and experimental results are as follows:

[0071] 20 L of 3,4-dihydroxybenzoic acid fermentation broth was taken, and the 3,4-dihydroxybenzoic acid content was determined to be 80 g / L. HPLC analysis showed that the purity of the 3,4-dihydroxybenzoic acid component was 98%. The filtrate containing 3,4-dihydroxybenzoic acid was obtained by sequential filtration through a ceramic membrane and a nanofiltration membrane. The pH of the system was adjusted to 1.0 with concentrated hydrochloric acid. After the 3,4-dihydroxybenzoic acid was almost completely precipitated, the temperature was lowered to 15 °C, and a certain amount of crude 3,4-dihydroxybenzoic acid was obtained by solid-liquid separation.

[0072] The crude product was completely dissolved in pure water at a mass fraction of 30% within a temperature range of 80-100℃. Then, under magnetic stirring and water bath conditions, the temperature was slowly reduced to 15℃ at a rate of 5℃ / h. After solid-liquid separation and vacuum drying, approximately 1288g of 3,4-dihydroxybenzoic acid was obtained. HPLC analysis showed that its liquid phase purity was 99.20%, mass purity was 100.8%, and the product recovery rate was 80.5%.

[0073] This comparative example obtained a high-purity 3,4-dihydroxybenzoic acid product through crystallization and recrystallization, which differs significantly from the scheme designed in this invention. Because the initial separation of impurities via column chromatography was not involved, the quality and recovery rate of the obtained 3,4-dihydroxybenzoic acid differed considerably. Specifically, the purity of the finished product in the liquid phase decreased, and the product recovery rate significantly decreased from approximately 90% to 80%.

[0074] Comparative Example 2

[0075] Compared with the scheme designed in this invention, Comparative Example 2 uses single-column chromatography instead of the SMB chromatographic separation system for the separation and extraction of 3,4-dihydroxybenzoic acid. The specific operation and experimental results are as follows:

[0076] Take 1L of the filtrate of 3,4-dihydroxybenzoic acid after membrane separation for later use. The content of 3,4-dihydroxybenzoic acid in the filtrate was determined to be 75g / L. The purity of the 3,4-dihydroxybenzoic acid component was 95% by HPLC analysis.

[0077] A sufficient quantity of D301 hydroxyl-type weakly basic anion exchange resin was packed into a single column with a resin bed volume of 800 mL. The filtered supernatant of 3,4-dihydroxybenzoic acid was slowly and uniformly passed through the chromatography column from top to bottom at a flow rate of 1 BV / h. HPLC analysis of the 3,4-dihydroxybenzoic acid concentration in the eluent revealed that a detection signal for 3,4-dihydroxybenzoic acid began to appear when the eluent volume was 1 L. The adsorbed 3,4-dihydroxybenzoic acid was then eluted with 1% hydrochloric acid at a rate of 2 BV / h and an elution volume of 4 BV. The eluent was collected for later use. HPLC analysis showed that the 3,4-dihydroxybenzoic acid fraction was 99.95%, with a total recovery rate of approximately 90%.

[0078] The eluent was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 25%. During this process, most of the 3,4-dihydroxybenzoic acid was observed to precipitate slowly as relatively regular crystals. After obtaining the solid-liquid mixture of 3,4-dihydroxybenzoic acid, it was transferred to a crystallization container and cooled and stirred until the temperature of the liquid reached 15°C. After solid-liquid separation and vacuum drying, approximately 62.5 g of 3,4-dihydroxybenzoic acid product was obtained. The purity of the liquid phase was determined by HPLC to be 99.98%, and the product recovery rate was 83.3%.

[0079] This comparative example demonstrates the separation and purification of 3,4-dihydroxybenzoic acid using single-column chromatography, yielding a highly pure 3,4-dihydroxybenzoic acid product. Compared to the scheme designed in this invention, both methods can obtain high-purity 3,4-dihydroxybenzoic acid products. The difference lies in the significantly reduced resin utilization rate of single-column chromatography. According to the experimental results of Comparative Example 2, the processing capacity of 3,4-dihydroxybenzoic acid per unit volume of the same type of resin is approximately 93.75 g per kilogram of wet resin. However, using the SMB chromatographic separation system, based on the results of Examples 1-4, the processing capacity can reach at least 400 g per kilogram of wet resin. Furthermore, single-column chromatography requires a larger amount of acid and alkali solutions for resin regeneration and recovery to meet the needs of subsequent production processes.

[0080] In conclusion, SMB chromatographic separation maximizes resin utilization and reduces the consumption of acid and alkali reagents. Furthermore, its continuous operation greatly ensures the feasibility of continuous generation of 3,4-dihydroxybenzoic acid.

[0081] Comparative Example 3

[0082] Compared with the scheme designed in this invention, in Comparative Example 3, the pH value of the system was not adjusted to weakly acidic before membrane separation. Other implementation methods were the same as in Example 1. The specific operations and experimental results are as follows:

[0083] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 80g / L, and the initial pH value to be 8.2. Filter the broth through a ceramic membrane and a nanofiltration membrane to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0084] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used to replace the feed solution and the system was run for 30 minutes. The purity of the 3,4-dihydroxybenzoic acid component in the extract was then detected by high performance liquid chromatography (HPLC), which showed that it was 99.85%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 94.8%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0085] The above extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 25%, with no crystal precipitation. However, as the concentration increased, crystals began to precipitate slowly. The resulting solid-liquid mixture of 3,4-dihydroxybenzoic acid was transferred to a crystallization container and cooled and stirred until the solution temperature reached 15°C. After solid-liquid separation and vacuum drying, approximately 751g of the finished 3,4-dihydroxybenzoic acid product was obtained. HPLC analysis showed a liquid phase purity of 99.93% and a mass purity of 95.6%. The net yield of 3,4-dihydroxybenzoic acid was 89.75%, and the dried product was a light yellow color.

[0086] The lack of pH adjustment resulted in a weakly alkaline system, causing some instability in the structure of catechol under these conditions, leading to a darker color in the 3,4-dihydroxybenzoic acid product. Simultaneously, insufficient acidification of the raw material solution resulted in some 3,4-dihydroxybenzoic acid remaining in its metal salt form. This delayed crystallization during concentration and crystallization, and reduced the final product recovery rate, liquid phase purity, and mass purity. While increasing the pickling solution concentration led to thorough acidification, some impurities were washed out, resulting in lower liquid phase purity and mass purity.

[0087] Comparative Example 4

[0088] Compared with the scheme designed in this invention, in Comparative Example 4, the simulated moving bed packing was replaced with conventional octadecylsilane-bonded silica gel ODS as the stationary phase, and the eluent was replaced with 50% ethanol aqueous solution. Other implementation methods were the same as in Example 1. The specific operations and experimental results are as follows:

[0089] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth and determine that the 3,4-dihydroxybenzoic acid content is 78g / L. Adjust the pH to 5.0 with concentrated hydrochloric acid, and then filter it through a ceramic membrane and a nanofiltration membrane to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0090] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. The eluent was a 50% ethanol aqueous solution pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After the feeding was completed, pure water was used to replace the feed solution and the system was run for 30 minutes. Then, the purity of the 3,4-dihydroxybenzoic acid component in the extract was detected by high performance liquid chromatography, which showed that it was 99.95%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 95.6%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0091] The extract was concentrated under vacuum until the ethanol solvent completely evaporated. Then, 37% concentrated hydrochloric acid was slowly added dropwise until the pH of the system reached approximately 3.0. Vacuum concentration continued until the mass fraction of 3,4-dihydroxybenzoic acid reached 25%, at which point crystals began to slowly precipitate. The resulting solid-liquid mixture of 3,4-dihydroxybenzoic acid was transferred to a crystallization container. The mixture was cooled and stirred until the temperature reached 15°C. After solid-liquid separation and vacuum drying, approximately 717.6 g of the finished 3,4-dihydroxybenzoic acid product was obtained. HPLC analysis showed a liquid phase purity of 99.98% and a mass purity of 100.5%. The net yield of 3,4-dihydroxybenzoic acid was 92%, and it appeared as pure white crystals after drying.

[0092] The final results show that using octadecylsilane-bonded silica gel ODS as the stationary phase and 50% ethanol aqueous solution as the eluent can also yield 3,4-dihydroxybenzoic acid with high purity. However, octadecylsilane-bonded silica gel ODS packing material is expensive, and the eluent requires organic reagents such as ethanol and methanol, which can easily cause safety and environmental hazards, placing extremely high demands on the investment and requirements of the downstream processing equipment. In contrast, this invention uses D301 resin as the stationary phase and 1% dilute hydrochloric acid as the eluent. This avoids the introduction of organic reagents, making it more environmentally friendly, and significantly reduces the investment cost of purification equipment. Furthermore, D301 resin has a lower raw material cost than octadecylsilane-bonded silica gel ODS, and can also obtain 3,4-dihydroxybenzoic acid products of equivalent quality based on the technical solution described in this invention.

[0093] Comparative Example 5

[0094] Compared with the scheme designed in this invention, this Comparative Example 5 did not use a nanofiltration membrane to filter and separate the ceramic separation liquid. The specific operation and experimental results are as follows:

[0095] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 80g / L, adjust the pH to 5.0 with concentrated hydrochloric acid, and obtain a filtered clear liquid containing 3,4-dihydroxybenzoic acid through ceramic membrane separation for later use.

[0096] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 minutes. The 3,4-dihydroxybenzoic acid ceramic membrane supernatant was pumped into the system through the feed inlet. After feeding, pure water was used instead of feed liquid and the system was run for 30 minutes. The purity of the 3,4-dihydroxybenzoic acid component in the extract was then detected by high performance liquid chromatography (HPLC), which showed that it was 99.71%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 96.5%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0097] The above extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 25%, and crystals slowly precipitated out. The resulting solid-liquid mixture of 3,4-dihydroxybenzoic acid was transferred to a crystallization container and cooled and stirred until the solution temperature reached 15°C. After solid-liquid separation and vacuum drying, the final product of 3,4-dihydroxybenzoic acid was obtained. HPLC analysis showed a liquid phase purity of 99.85% and a mass purity of 96.5%. The net yield of 3,4-dihydroxybenzoic acid was 91.67%, and it appeared as pale yellow crystals after drying.

[0098] In this comparative example, removing the nanofiltration membrane filtration process has little impact on the subsequent simulated moving bed chromatography separation results. However, because soluble proteins and biocatalytic pigments in the feed solution cannot be effectively removed through other processes, the final product has a low purity and the crystal color becomes darker, turning pale yellow.

[0099] Comparative Example 6

[0100] Compared with the scheme designed in this invention, in Comparative Example 6, the simulated moving bed process is placed between the separation of the ceramic membrane and the nanofiltration membrane. The specific operation and experimental results are as follows:

[0101] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 70g / L, adjust the pH to 5.0 with concentrated hydrochloric acid, and obtain a filtered clear liquid containing 3,4-dihydroxybenzoic acid through ceramic membrane separation for later use.

[0102] Before feeding into the simulated moving bed, the feed flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% (w / w) dilute hydrochloric acid solution was pumped into the system through the eluent inlet. The system was run for 30 minutes according to the set values. After that, the feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 minutes. The 3,4-dihydroxybenzoic acid nanofiltration solution was pumped into the system through the feed inlet. After feeding, pure water was used instead of feed liquid for 30 minutes. The purity of the 3,4-dihydroxybenzoic acid component in the extract was then detected by high performance liquid chromatography (HPLC), which showed that it was 98.0%, and the recovery rate of the total 3,4-dihydroxybenzoic acid was 96.2%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0103] The extract was filtered through a nanofiltration membrane and then concentrated under vacuum to a 3,4-dihydroxybenzoic acid mass fraction of 25%. Crystals slowly precipitated out, and the resulting solid-liquid mixture of 3,4-dihydroxybenzoic acid was transferred to a crystallization container. The mixture was cooled and stirred until the temperature reached 15°C. After solid-liquid separation and vacuum drying, the final product of 3,4-dihydroxybenzoic acid was obtained. HPLC analysis showed a liquid phase purity of 99.1% and a mass purity of 99.86%. The net yield of 3,4-dihydroxybenzoic acid was 91.5%, and it appeared as pure white crystals after drying.

[0104] In this comparative example, the simulated moving bed separation step was placed between the ceramic membrane and nanofiltration membrane separation steps. Due to the presence of more impurities such as protein and salt in the clear liquid separated by the ceramic membrane, the simulated moving bed separation effect deteriorated. Under the same conditions, the HPLC purity of 3,4-dihydroxybenzoic acid in the separated liquid decreased. Although the purity and color of the product after crystallization met the standard requirements (the standard is a purity of 99.8%-102% and a pure white crystal color), the liquid phase purity of the obtained 3,4-dihydroxybenzoic acid crystals was only 99.1%, which could not meet the production requirements (the liquid phase purity required for production is greater than 99.7%).

[0105] Comparative Example 7

[0106] Compared with the scheme designed in this invention, the simulated moving bed in Comparative Example 7 also has 8 chromatographic columns, but every 4 chromatographic columns form an SMB system, and a total of 2 SMB separations are performed. The specific operation and experimental results are as follows:

[0107] Take 10L of 3,4-dihydroxybenzoic acid fermentation broth, determine the 3,4-dihydroxybenzoic acid content to be 80g / L, adjust the pH to 5.0 with concentrated hydrochloric acid, and filter it sequentially through a ceramic membrane with a pore size of 100nm and a nanofiltration membrane with an average molecular weight of 250Da to obtain a clear filtrate containing 3,4-dihydroxybenzoic acid for later use.

[0108] Before feeding the simulated moving bed, the feed liquid flow rate was set to 0, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 100 mL / min. A 1% hydrochloric acid solution was pumped into the system through the eluent inlet, and the system was run for 30 minutes according to the set values.

[0109] Then, the first SMB separation was performed. The feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 min. The 3,4-dihydroxybenzoic acid sodium filtrate was pumped into the system through the feed inlet. After the feeding was completed, pure water was used to replace the feed solution and the system was run continuously for 30 min.

[0110] The extract was used as the feed liquid for the second SMB separation. The feed rate was adjusted to 20 mL / min, the eluent flow rate to 200 mL / min, the extract flow rate to 100 mL / min, and the raffinate flow rate to 120 mL / min. The switching time was set to 15 min. After the extract was pumped into the system through the feed inlet, pure water was used to replace the feed liquid and the system was run for 30 min.

[0111] The purity of 3,4-dihydroxybenzoic acid in the extract was then determined by high performance liquid chromatography (HPLC), and was found to be 99.65%. The total recovery rate of 3,4-dihydroxybenzoic acid was 92%. The 3,4-dihydroxybenzoic acid extract was collected for later use.

[0112] The extract was concentrated under vacuum to a 3,4-dihydroxybenzoic acid (3,4-dihydroxybenzoic acid) mass fraction of 25%. During this process, most of the 3,4-dihydroxybenzoic acid was observed to precipitate slowly as relatively regular crystals. The resulting solid-liquid mixture was transferred to a crystallization container and cooled and stirred until the solution temperature reached 15°C. After solid-liquid separation and vacuum drying, approximately 680.14 g of the final 3,4-dihydroxybenzoic acid product was obtained, with a purity of 100.8%. HPLC analysis showed a liquid phase purity of 99.97% and a product recovery rate of 85.17%.

[0113] After splitting the simulated moving bed (SMB) system into two SMB systems according to the comparative method, the experimental results showed that, under the condition of achieving the same experimental results, the split led to increased water consumption and a significant dilution of the concentration of 3,4-dihydroxybenzoic acid in the final chromatographic separation solution. This required more energy and concentration time. From the perspective of resource conservation in industrial production, the two SMB systems resulted in a significant increase in both equipment investment and production costs. Furthermore, after the split, the loss of 3,4-dihydroxybenzoic acid increased significantly with the number of chromatographic separations, leading to a certain degree of reduction in the final product recovery rate, which was only 85.17%.

[0114] In summary, the method for extracting 3,4-dihydroxybenzoic acid from fermentation broth provided in this application achieves continuous and efficient separation of 3,4-dihydroxybenzoic acid using simulated moving bed chromatography. This saves on the consumption of raw materials and reagents such as acids and alkalis, which is beneficial for the large-scale, automated, and continuous production of 3,4-dihydroxybenzoic acid, greatly improving its industrial production efficiency and laying an important theoretical foundation for its large-scale industrial production. In this invention, the simulated moving bed chromatography serves to initially separate the finished product from incompletely converted 3-dehydroshikimic acid and its byproducts (such as gallic acid), while also acidifying the 3,4-dihydroxybenzoic acid component.

[0115] 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 extracting 3,4-dihydroxybenzoic acid from fermentation broth, characterized in that, Includes the following steps: The pH of the fermentation broth containing 3,4-dihydroxybenzoic acid is adjusted to 5.0-6.0; the concentration of 3,4-dihydroxybenzoic acid in the fermentation broth is 60-85 g / L, the purity is 95%-98%, and the pH of the fermentation broth system is 5.0-9.

0. The insoluble impurities and macromolecular substances in the fermentation broth are removed to obtain a clear liquid; The process of removing insoluble impurities and macromolecular substances from the fermentation broth to obtain a clear liquid includes: firstly, separating the fermentation broth using a ceramic membrane, and then filtering the filtrate using a nanofiltration membrane; the ceramic membrane has a pore size of 80-120 nm, and the nanofiltration membrane has an average molecular weight of 250-500 Da; the clear liquid is then continuously separated by simulated moving bed chromatography to obtain a high-purity extract; wherein the simulated moving bed chromatography is a four-segment SMB system, with the four segments connected in series, and each segment including two or more identical chromatographs. The chromatographic columns are connected in series, and the stationary phase in the columns is a hydroxyl-type weakly basic anion exchange resin D301. Each section is sequentially provided with an inlet, a raffinate outlet, an eluent inlet, and an extract outlet. The clear liquid enters through the inlet, and the eluent enters through the eluent inlet. The eluent is a 0.8-1.2% (w / w) dilute hydrochloric acid solution. The inlet, raffinate outlet, eluent inlet, and extract outlet are periodically switched in a clockwise direction until the clear liquid feeding is completed. The extract is concentrated and crystallized, followed by solid-liquid separation to obtain 3,4-dihydroxybenzoic acid. The process of concentrating and crystallizing the extract, followed by solid-liquid separation, includes: first, concentrating the extract under vacuum to a mass fraction of 20%-30%, then transferring it to a crystallization container, cooling and stirring until the liquid temperature is less than or equal to 15°C, followed by solid-liquid separation and vacuum drying.

2. The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth according to claim 1, characterized in that, The flow rate V1 at the feed inlet is 0-100 mL / min, the flow rate V2 at the raffinate outlet is 1-1000 mL / min, the flow rate V3 at the eluent inlet is 1-1000 mL / min, the flow rate V4 at the extract outlet is 1-1000 mL / min, and the switching time for the periodic switching is 10-20 min. V3≥V2+V4﹣V1.

3. The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth according to claim 1, characterized in that, Before the clarified liquid is fed into the feed inlet, the eluent inlet, the extract outlet, and the raffinate outlet are set to run for 20-40 minutes according to the set values.

4. The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth according to claim 1, characterized in that, After the clear liquid finishes feeding from the inlet, the process also includes replacing the clear liquid with pure water and continuing to run for 20-40 minutes.

5. The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth according to claim 1, characterized in that, After separation by simulated moving bed chromatography, the content of 3,4-dihydroxybenzoic acid in the extract is ≥99.5%.

6. The method for extracting 3,4-dihydroxybenzoic acid from fermentation broth according to claim 1, characterized in that, During vacuum concentration, the vacuum degree is not higher than 100 Pa, the water bath temperature is 90-100℃, and the material temperature is not lower than 40℃.

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