A method for separating and extracting 2,4-diacetylphloroglucinol from an aqueous solution

Abstract

The present application relates to the technical field of organic chemical product preparation, in particular to a method for separating and extracting 2,4-diacetyl resorcinol from an aqueous solution. In the present application, 2,4-diacetyl resorcinol can be directly and efficiently separated and extracted from the aqueous phase solvent of microbial culture medium, fermentation supernatant or whole fermentation broth by the method of low-temperature precipitation after acidification, the extraction efficiency is close to 100%, the bacterial bodies do not need to be removed in advance by centrifugation, the method is convenient to connect with the upstream fermentation process, and is suitable for industrial large-scale production.

Description

Technical Field

[0001] This invention relates to the field of organic chemical product preparation technology, specifically to a method for separating and extracting 2,4-diacetyl-resorcinol from an aqueous solution. Background Technology

[0002] 2,4-Diacetylphloroglucinol (DAPG) possesses broad-spectrum antiviral, antibacterial, antifungal, and antiparasitic properties. It can inhibit various plant pathogens, including fungi, worms, and nematodes. Furthermore, DAPG is a factor in inducing systemic resistance (ISR) in plants, enabling them to develop resistance to pathogens, thereby enhancing plant protection and promoting the secretion of amino acids in plant roots. In addition, DAPG helps inhibit various plant diseases such as wheat take-all, tomato black root rot, and wheat root rot, thus demonstrating broad application prospects as a broad-spectrum antibiotic in the field of biological control.

[0003] Currently, the synthesis of 2,4-diacetyl-resorcinol includes chemical synthesis and biosynthesis. The biosynthesis primarily utilizes *Pseudomonas fluorescens*. The extraction of 2,4-diacetyl-resorcinol from the fermentation broth typically employs liquid-liquid extraction: the fermentation broth is first centrifuged to obtain the supernatant, then acidified with hydrochloric acid to approximately pH 2, followed by extraction with an equal volume of ethyl acetate, and finally, the solvent is evaporated. However, this method suffers from problems such as large operating volume, high organic solvent consumption, high cost, cumbersome operation, and difficulty in scaling up the process, severely hindering the industrial production and application of 2,4-diacetyl-resorcinol. Summary of the Invention

[0004] To overcome the above problems, the present invention provides a method for separating and extracting 2,4-diacetyl-resorcinol from an aqueous solution.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for separating and extracting 2,4-diacetylphloroglucinol from an aqueous solution, comprising the following steps: An aqueous solution containing 2,4-diacetyl-resorcinol was acidified, then allowed to stand at low temperature, the precipitate was collected, and dried to obtain 2,4-diacetyl-resorcinol.

[0006] In one or more embodiments, the aqueous solution containing 2,4-diacetylphloroglucinol includes microbial culture medium, fermentation broth supernatant, or whole fermentation broth.

[0007] In one or more embodiments, the acid used in the acidification process is hydrochloric acid; Preferably, the mass fraction of hydrochloric acid is 36% to 38%.

[0008] Preferably, after acidification, the pH of the aqueous solution is less than 6, and more preferably 2 to 5.9.

[0009] In one or more embodiments, the low temperature is 0~4°C.

[0010] In one or more embodiments, the settling time is 30 to 60 minutes.

[0011] In one or more embodiments, the method for collecting the precipitate is centrifugation.

[0012] In one or more embodiments, the method further includes drying and purifying to obtain 2,4-diacetyl-resorcinol.

[0013] Preferably, the purification method includes: The dried precipitate was redissolved in an organic solvent, the supernatant was collected by centrifugation, and then dried to obtain 2,4-diacetyl-resorcinol.

[0014] More preferably, the organic solvent includes one or more of methanol or ethyl acetate.

[0015] The beneficial effects of this invention are as follows: The present invention enables the direct and efficient separation and extraction of 2,4-diacetylphloroglucinol from the aqueous solvent of microbial culture medium, fermentation broth supernatant or whole fermentation broth by acidification and low-temperature precipitation. The extraction efficiency is close to 100%, and there is no need to remove the bacterial cells by pre-centrifugation. It is easy to connect with the upstream fermentation process and is suitable for large-scale industrial production. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 This describes the process of extracting DAPG from M9 culture medium. Figure 2 The results of efficiency testing for the isolation and extraction of DAPG from M9 culture medium in Example 1 are shown; where M9-500-ex represents the calculated actual concentration of DAPG extracted. Figure 3 The results of efficiency testing for separating and extracting DAPG from the fermentation broth supernatant in Example 2 are shown; where culture-sup-ex represents the calculated actual DAPG concentration. Figure 4The results of efficiency testing for separating and extracting DAPG from the whole fermentation broth in Example 3 are shown; where culture-all-ex represents the calculated concentration of DAPG actually extracted. Figure 5 Comparison of liquid chromatography results for different samples (fermentation broth, supernatant, and extract precipitate reconstituted solution); the orange line represents the liquid chromatography of the supernatant, the blue line represents the liquid chromatography of the whole fermentation broth, and the purple line represents the liquid chromatography of the fermentation broth supernatant; Figure 6 To compare the efficiency of DAPG separation and extraction after acidification to different pH levels. Detailed Implementation

[0018] 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 in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0019] 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.

[0020] Currently, the synthesis of 2,4-diacetyl-resorcinol includes chemical synthesis and biosynthesis. Chemical synthesis is subject to stringent conditions, high energy consumption, and severe pollution, while biosynthesis offers advantages such as environmental friendliness, fewer byproducts, and highly selective and reactive products. Biosynthesis primarily utilizes *Pseudomonas fluorescens*, followed by extraction of 2,4-diacetyl-resorcinol from the fermentation broth. The current method for extracting 2,4-diacetyl-resorcinol from the fermentation broth typically employs liquid-liquid extraction: the fermentation broth is first centrifuged to obtain the supernatant, then acidified with hydrochloric acid to approximately pH 2, followed by extraction with an equal volume of ethyl acetate, and finally, the solvent is evaporated. However, this method suffers from large operating volume, high organic solvent consumption, high cost, cumbersome operation, and difficulty in scaling up the process, severely hindering the industrial production and application of 2,4-diacetyl-resorcinol.

[0021] To overcome the above problems, the present invention provides a method for separating and extracting 2,4-diacetyl-resorcinol from an aqueous solution.

[0022] This invention involves directly acidifying the supernatant or whole fermentation broth or microbial culture medium containing 2,4-diacetylresorcinol, followed by standing at low temperature. 2,4-diacetylresorcinol precipitates out as a precipitate. The precipitate is collected by centrifugation to obtain crude 2,4-diacetylresorcinol. Then, by redissolving it in an organic solvent, centrifuging to collect the supernatant, and drying, pure 2,4-diacetylresorcinol can be obtained.

[0023] The method provided by this invention achieves nearly 100% extraction efficiency for 2,4-diacetylphloroglucinol, eliminates the need for pre-centrifugation to remove bacterial cells, facilitates integration with upstream fermentation processes, and is suitable for large-scale industrial production. Furthermore, it replaces traditional organic solvent extraction, significantly reducing the use of organic solvents such as ethyl acetate.

[0024] The definitions and abbreviations used in this invention are as follows: 2,4-Diacetylphloroglucinol: DAPG.

[0025] 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.

[0026] Basic reagents and detection methods: M9 medium formulation: Each liter of medium contains 6.8 g Na2HPO4, 3.0 g KH2PO4, 0.5 g NaCl, and 1.0 g NH4Cl. The medium is autoclaved at 115°C for 30 min, and after cooling, 2 mM MgSO4, 0.1 mM CaCl2, and 0.4% glucose are added. DAPG stock solution: concentration of 100 g / L, prepared with methanol as solvent; DAPG detection method by liquid chromatography: An Agilent 1260 Infinity II high-performance liquid chromatograph was used for detection. The chromatographic column was an Agilent ZORBAX StableBond C18 reversed-phase column, and the chromatographic signal at a wavelength of 270 nm was detected using a VWD detector. The mobile phase was 55% acetonitrile + 45% water (containing 0.1% phosphoric acid), and the flow rate was 1 mL / min. Using DAPG stock solution as a standard, a series of standard solutions of 20, 40, 60, 80, and 100 mg / L were obtained by serial dilution. A standard curve was constructed by analyzing the peak areas detected by liquid chromatography to calculate the actual concentration of DAPG in the sample.

[0027] Example 1 Figure 1 This describes the process for extracting DAPG from M9 culture medium. (Reference) Figure 1 DAPG was extracted from M9 culture medium.

[0028] A DAPG solution with a concentration of 500 mg / L was prepared using M9 medium as the solvent and stirred thoroughly to completely dissolve the DAPG. Concentrated hydrochloric acid (37% by mass) was added to the solution to adjust the pH to 2. After thorough mixing, the solution was placed on ice and allowed to stand for 30 min, resulting in the formation of a white flocculent DAPG precipitate. The system containing the precipitate was placed in a centrifuge at 4°C and centrifuged at 13000 g for 10 min to allow the white flocculent precipitate to settle completely. The supernatant was removed to obtain the DAPG precipitate, which was then dried to obtain DAPG powder.

[0029] HPLC analysis was performed on the stock solution before extraction and the supernatant after extraction. The actual extracted DAPG concentration was calculated by subtracting the concentration of the supernatant after precipitation from the concentration before extraction. Figure 2 As shown in the figure, the concentration of DAPG detected before extraction was 555.37 mg / L. The actual extracted DAPG concentration was calculated by subtracting the concentration of the supernatant after precipitation from the concentration before extraction, and the extraction efficiency was calculated to be 99.83%.

[0030] Example 2 Extraction of DAPG from fermentation broth supernatant: The DAPG-synthesizing Escherichia coli strain Bdt03 was inoculated into M9 medium for fermentation. After fermentation, the bacterial cells were removed by centrifugation, and the supernatant of the DAPG fermentation broth was obtained. Concentrated hydrochloric acid (37% by mass) was added to the DAPG fermentation broth supernatant to adjust the pH of the solution to 2. After thorough mixing, the solution was placed on ice and allowed to stand for 30 min, resulting in the formation of a white flocculent DAPG precipitate. The system containing the precipitate was placed in a centrifuge at 4°C and centrifuged at 13000 g for 10 min to allow the white flocculent precipitate to settle completely. After removing the supernatant, the DAPG precipitate was obtained. The precipitate was dried to obtain DAPG powder.

[0031] Liquid chromatography detection results as follows Figure 3 As shown, the initial concentration of DAPG in the supernatant of the fermentation broth was 255.09 mg / L. The actual DAPG concentration extracted was calculated by subtracting the concentration of the supernatant after precipitation from the concentration before extraction, and the extraction efficiency was 98.85%.

[0032] Example 3 Extraction and purification of DAPG from the whole fermentation broth: The DAPG total fermentation broth (containing bacterial cells, etc.) of Escherichia coli strain Bdt03, which can synthesize DAPG, was used as the extraction raw material. A portion of the total fermentation broth was first taken, and an equal volume of methanol was added and ultrasonically promoted to dissolve it. The initial concentration of DAPG in the total fermentation broth was 436.13 mg / L, as determined by liquid chromatography.

[0033] Concentrated hydrochloric acid (37% by mass) was added to the whole fermentation broth to adjust the pH of the solution to 2. After thorough mixing, the solution was placed on ice and allowed to stand for 30 min, resulting in a white flocculent DAPG precipitate. The system containing the precipitate was then centrifuged at 13000 g for 10 min at 4°C to allow the white flocculent precipitate to settle completely. The supernatant was removed to obtain the DAPG precipitate, which was then dried to obtain DAPG powder. The actual extracted DAPG concentration was calculated by subtracting the concentration of the supernatant after precipitation from the concentration before extraction. In this example, the extraction efficiency of DAPG was 99.50%. Figure 4 ).

[0034] Further purification of DAPG: The DAPG precipitate extracted from the whole fermentation broth was fully reconstituted in methanol (DAPG concentration in methanol was 150 g / L). After centrifugation at 13000 g for 10 min, the supernatant was collected and analyzed by liquid chromatography. The results are as follows: Figure 5 As shown by the orange line, compared with the directly extracted whole fermentation broth sample (blue line) and the fermentation broth supernatant sample (purple line), the chromatographic impurity peaks of DAPG after methanol reconstitution and purification were significantly reduced, indicating that the purity of the product was effectively improved.

[0035] Example 4 Effect of different pH acidification conditions on DAPG extraction efficiency Following the operating steps of Example 1, the prepared DAPG solution was acidified to different pH values ​​with hydrochloric acid. After standing on ice for 30 minutes and centrifuging at 13000g for 10 minutes, the DAPG extraction efficiency under different pH conditions was detected and calculated.

[0036] Test results as follows Figure 6 As shown, when the acidification pH was 5.9, 5.3, 3.4, and 2.6, the corresponding DAPG extraction efficiencies were 79.21%, 96.34%, 99.87%, and 99.45%, respectively. The results indicate that when acidified to a pH less than 5.9, efficient DAPG extraction can be achieved, and the lower the pH, the closer the extraction efficiency is to 100%.

[0037] 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 extracting 2,4-diacetylphloroglucinol from an aqueous solution, characterized in that, Includes the following steps: An aqueous solution containing 2,4-diacetyl-resorcinol was acidified, then allowed to stand at low temperature, the precipitate was collected, and dried to obtain 2,4-diacetyl-resorcinol.

2. The method as described in claim 1, characterized in that, Aqueous solutions containing 2,4-diacetylphloroglucinol include microbial culture media, fermentation broth supernatant, or whole fermentation broth.

3. The method as described in claim 1, characterized in that, The acid used in the acidification process is hydrochloric acid.

4. The method as described in claim 1, characterized in that, After acidification, the pH of the aqueous solution is less than 6, preferably 2 to 5.

9.

5. The method as described in claim 1, characterized in that, The low temperature range is 0~4℃.

6. The method as described in claim 1, characterized in that, The settling time is 30-60 minutes.

7. The method as described in claim 1, characterized in that, The precipitate was collected by centrifugation.

8. The method as described in claim 1, characterized in that, The method also includes drying and purifying to obtain 2,4-diacetyl-resorcinol.

9. The method as described in claim 8, characterized in that, Purification methods include: The dried precipitate was redissolved in an organic solvent, the supernatant was collected by centrifugation, and then dried to obtain 2,4-diacetyl-resorcinol.

10. The method as described in claim 9, characterized in that, The organic solvent includes one or more of methanol or ethyl acetate.

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