Method for purifying bio-fermented gallic acid and application thereof

Abstract

The application provides a biological fermentation gallic acid purification method and application thereof, and relates to the technical field of biological fermentation engineering. The purification method removes bacteria and a large amount of protein in high-titer gallic acid fermentation liquor obtained through biological fermentation by screening a suitable filter pore size, and improves the yield and purity of gallic acid by optimizing a decolorization and washing mode, and is suitable for the purification of fermentation liquor with high-titer gallic acid obtained through biological fermentation. The technical problem that a large number of crystal impurities, color difference and difficulty in reaching the actual production application standard are caused when a traditional plant extraction method is used to extract biological gallic acid is solved, and the application has a good industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation engineering technology, specifically relating to a bio-fermentation method for purifying gallic acid and its application. Background Technology

[0002] Gallic acid (GA) is an important phenolic compound with wide applications in medicine, food, and cosmetics. To increase gallic acid production, scientists typically employ methods that systematically optimize metabolic flux and dynamically regulate the expression of key enzymes. This approach involves a deep understanding and precise regulation of microbial metabolic networks to ensure the efficient operation of biosynthetic pathways.

[0003] Currently, the biosynthesis of gallic acid (GA) is a green and sustainable production method. However, previous biosynthetic GA titers were generally low, hindering industrial-scale production. The applicant previously systematically controlled key metabolic nodes in the GA production process, such as increasing hydroxylase activity, regulating pyruvate flow, and enhancing the supply of aromatic amino acid precursors, achieving a dynamic balance between engineered bacterial growth and GA product synthesis. This resulted in a modified strain, WJ54. Experiments showed that the modified genetically engineered strain WJ54 achieved a GA yield of 12.5 g / L in shake-flask experiments and 109.8 g / L in a 3 L fermenter; the carbon yield was increased to 0.64 g / g glucose, the highest level previously reported in the literature (refer to Chinese Patent CN121271770A). However, existing technologies for bio-fermentation of GA titers do not reach the 100 g / L required for industrial production, while laboratory bio-fermentation of GA titers is relatively high. Therefore, using traditional plant extraction methods for bio-gallic acid fermentation results in numerous crystalline impurities and poor color, making it difficult to meet the standards for practical production applications.

[0004] In summary, there is an urgent need in this field to develop a purification method for gallic acid in the fermentation products of engineered bacteria that efficiently biosynthesize gallic acid. The purified gallic acid product should have high crystal purity, good color, and high yield, and have good prospects for industrial application. Summary of the Invention

[0005] The purpose of this invention is to provide a method for purifying gallic acid through bio-fermentation and its application, which significantly improves the purification effect of gallic acid and achieves high purity, good color, and high yield of the purified gallic acid product crystals.

[0006] This invention provides a method for purifying gallic acid through bio-fermentation, comprising the following steps: (1) Obtain a fermentation broth rich in gallic acid; the fermentation broth is heated and then filtered with a pore size of 45 nm to 55 nm, and filtrate 1 is collected; filtrate 1 is heated and then filtered with a pore size of 8 nm to 15 nm, and filtrate 2 is collected; the heating temperature is 80°C to 90°C. (2) The filtrate 2 is concentrated to obtain a concentrate. Activated carbon with a mass-to-volume ratio of 0.5~1.5g:10mL is added to the concentrate for decolorization. The decolorization temperature is 65℃~75℃. After decolorization, the pH is adjusted to 1~2, the temperature is lowered to 0℃, and the solution is maintained for 36h~60h. The solution is filtered, and solid precipitate 1 is collected to obtain gallic acid crystals containing activated carbon. The gallic acid crystals containing activated carbon are dried and then dissolved at a water temperature of 60℃~70℃. The solution is filtered, and filtrate 3 is collected. (3) Repeat step (2) to decolorize filtrate 3 and collect filtrate 4; (4) Concentrate filtrate 4, adjust pH to 1.5~2.5, cool to 0 ℃, maintain for 36 h~60 h, filter, collect solid precipitate 2, dry, and obtain gallic acid.

[0007] Preferably, in step (1), hot water at 80℃~90℃ is continuously added during the filtration process.

[0008] Preferably, in step (1), when preparing filtrate 1, the amount of hot water added is 2 to 4 times the volume of the fermentation liquid; when preparing filtrate 2, the amount of hot water added is 1 to 3 times the volume of filtrate 1.

[0009] Preferably, in step (1), the fermentation liquid and the hot water are added simultaneously during filtration, with the same flow rate of 80~120 mL / min; the filter membrane used for filtration is a ceramic membrane.

[0010] Preferably, in step (2), the coloring is continuously stirred during the decolorization process, and the decolorization time is 2 h to 5 h.

[0011] Preferably, in step (2), the filtrate 2 is concentrated to 30%~40% of the fermentation liquid volume; the activated carbon is ≥200 mesh.

[0012] Preferably, in step (2), the amount of hot water used for dissolving is 2 to 4 times the mass of the gallic acid crystals containing activated carbon; after collecting solid precipitate 1, the solid precipitate is rinsed again with hot water at a temperature of 60°C to 70°C, and the rinsing volume is 2 to 4 times the mass of the solid precipitate.

[0013] Preferably, in step (3), activated carbon with a mass-to-volume ratio of 0.2~0.3g:10mL is added to the concentrate; in step (4), the filtrate 4 is concentrated to 15%~25% of the fermentation liquid volume.

[0014] Preferably, the gallic acid-rich fermentation broth has a gallic acid titer of ≥100 g / L, and the gallic acid-rich fermentation broth is obtained by fermentation using strain WJ54.

[0015] The present invention also provides the application of gallic acid prepared by the method in the preparation of products containing high-purity gallic acid.

[0016] Beneficial effects: This invention provides a method for purifying gallic acid through bio-fermentation. By selecting appropriate heating temperatures and filter pore sizes, bacteria and a large amount of protein are removed from the high-titer gallic acid fermentation broth obtained from bio-fermentation. By optimizing the decolorization and washing method (mainly through temperature control), the yield and purity of the target product are improved. This method is suitable for purifying fermentation broths with high gallic acid titers from bio-fermentation. It solves the technical problem that traditional plant extraction methods for bio-gallic acid fermentation extraction result in many crystal impurities, poor color, and difficulty in meeting the standards for practical production applications. The purified gallic acid product has high crystal purity, good color, and high yield, and has good prospects for industrial application. Attached Figure Description

[0017] Figure 1 The images shown are of gallic acid crystals obtained after purification in Example 1, and the chromatographic detection results of gallic acid crystals. In Example 1, A is the image of gallic acid crystals obtained, and B is the chromatographic detection result of gallic acid crystals.

[0018] Figure 2 The mass spectrometry results are for gallic acid obtained after purification in Example 1.

[0019] Figure 3 This is a photograph of the gallic acid crystals obtained in Comparative Example 1.

[0020] Figure 4 This is a photograph of gallic acid crystals obtained in Comparative Example 2.

[0021] Figure 5 The results are shown in LC-MS for gallic acid crystals obtained in Comparative Example 2, where A represents the mass spectrometry result of gallic acid crystals and B represents the chromatographic result of gallic acid crystals. Detailed Implementation

[0022] This invention provides a method for purifying gallic acid through bio-fermentation, comprising the following steps: (1) Obtain a fermentation broth rich in gallic acid; the fermentation broth is heated and then filtered with a pore size of 45 nm to 55 nm, and filtrate 1 is collected; filtrate 1 is heated and then filtered with a pore size of 8 nm to 15 nm, and filtrate 2 is collected; the heating temperature is 80°C to 90°C. (2) The filtrate 2 is concentrated to obtain a concentrate. Activated carbon with a mass-to-volume ratio of 0.5~1.5g:10mL is added to the concentrate for decolorization. The decolorization temperature is 65℃~75℃. After decolorization, the pH is adjusted to 1~2, the temperature is lowered to 0℃, and the solution is maintained for 36h~60h. The solution is filtered, and solid precipitate 1 is collected to obtain gallic acid crystals containing activated carbon. The gallic acid crystals containing activated carbon are dried and then dissolved at a water temperature of 60℃~70℃. The solution is filtered, and filtrate 3 is collected. (3) Repeat step (2) to decolorize filtrate 3 and collect filtrate 4; (4) Concentrate filtrate 4, adjust pH to 1.5~2.5, cool to 0 ℃, maintain for 36 h~60 h, filter, collect solid precipitate 2, dry, and obtain gallic acid.

[0023] In this invention, the gallic acid-rich fermentation broth refers to the original fermentation broth produced by biological fermentation with a GA titer greater than or equal to 100 g / L. The purification method of this invention removes bacteria and a large amount of protein from the biological fermentation process by selecting a suitable filter pore size. By optimizing the decolorization and washing method, primarily through temperature control, the gallic acid yield and purity are improved, while residual bacterial proteins and degraded pigments are removed.

[0024] In step (1) of the method of the present invention, the fermentation broth is heated to 80 ℃~90 ℃ and maintained for 25 min~40 min. The heating temperature is further preferably 82 ℃~88 ℃, more preferably any one of 83 ℃, 84 ℃, 85 ℃, 86 ℃ or 87 ℃, and most preferably 85 ℃. The maintenance time after heating is further preferably 25 min~35 min, more preferably 26 min~32 min, and most preferably 30 min. During the filtration process, hot water at 80 ℃~90 ℃ is continuously added. The hot water temperature is further preferably 82 ℃~88 ℃, more preferably any one of 83 ℃, 84 ℃, 85 ℃, 86 ℃ or 87 ℃, and most preferably 85 ℃. When preparing filtrate 1, the amount of hot water added should be 2~4 times the volume of the original fermentation broth, preferably 2.5~3.5 times the volume, and more preferably 3 times the volume. When preparing filtrate 2, the amount of hot water added is 1~3 times the volume of filtrate 1, preferably 1.5~2.5 times the volume, and more preferably 2 times the volume. During filtration, the heated fermentation broth and the hot water are added simultaneously at a consistent flow rate, preferably 80-120 mL / min, more preferably 90-110 mL / min, and even more preferably 100 mL / min. The filtration membrane used is preferably a ceramic membrane, and the filtration is preferably performed in a disposable heated ceramic membrane filtration machine.

[0025] In step (2) of the method of the present invention, continuous stirring is performed during decolorization, and the decolorization time is preferably 2 h to 5 h, more preferably 2 h to 4 h, more preferably 2 h to 3 h, and most preferably 2 h. The filtrate 2 is preferably concentrated to 30% to 40% of the original fermentation liquid volume, more preferably one-third of the original fermentation liquid volume; the activated carbon is ≥200 mesh. As an optional embodiment, the activated carbon is purchased from Xinjinhu Activated Carbon Co., Ltd., model JH-769. In the present invention, the decolorization is to remove the degraded pigments in the gallic acid produced by bio-fermentation, ensuring that the obtained gallic acid crystals have high purity and good color. After decolorization, the pH is adjusted, preferably 1 to 2, more preferably 1.2 to 1.8, more preferably 1.3, 1.4 or 1.5, and most preferably 1.5. As an optional embodiment, concentrated hydrochloric acid is used to adjust the pH, and the concentration of the concentrated hydrochloric acid is 11 mol / L. After adjusting the pH, the temperature is lowered to 0°C and maintained for 36-60 hours, more preferably 40-60 hours, more preferably 50-60 hours, and most preferably 60 hours. The cooling in this invention is to precipitate gallic acid crystals. After cooling to a fixed time, gallic acid crystals precipitate, are filtered, and solid precipitate 1 is collected to obtain gallic acid crystals containing activated carbon. The filtration uses a sand core filter funnel or a Buchner funnel; the Buchner funnel is used with filter paper, and the inner diameter of the Buchner funnel filter plate is 120 mm. When drying the gallic acid crystals containing activated carbon, the drying temperature is preferably 45°C-55°C, more preferably 48°C-52°C, and more preferably 50°C. As an optional embodiment, the gallic acid crystals containing activated carbon are dried in an oven at 50°C for 24 hours. When dissolving the gallic acid crystals containing activated carbon, the amount of hot water used is preferably 2 to 4 times the mass of the gallic acid crystals containing activated carbon, more preferably 4 times. After collecting solid precipitate 1, the solid precipitate is rinsed again with hot water at 60°C to 70°C, more preferably 65°C; the rinsing volume is 2 to 4 times the mass of the solid precipitate, more preferably 2 times. The filtrate 3 is collected after rinsing. As an optional embodiment, after collecting filtrate 3, solid precipitate 2 is collected, and solid precipitate 2 is rinsed again with hot water at 60°C to 70°C, more preferably 65°C; the rinsing volume is twice the mass of solid precipitate 2, and filtrate 3' is collected. Filtrates 3 and 3' are combined to obtain a combined filtrate, which is used as the initial filtrate 3 for the next step.

[0026] Step (3) of the method described in this invention involves repeating step (2) for further decolorization. In step (3), activated carbon is added to the concentrate. The preferred mass-to-volume ratio of activated carbon to concentrate is 0.2-0.3 g:10 mL, more preferably 0.21-0.29 g:10 mL, even more preferably 0.22-0.28 g:10 mL, more preferably 0.23-0.27 g:10 mL, and most preferably 0.25 g:10 mL. After completing step (3), the filtrate is observed to be colorless. As an optional implementation, if the color is dark, step (3) is repeated once more.

[0027] In step (4) of the method of the present invention, the filtrate 4 is concentrated to 15%~25% of the original fermentation liquid volume, preferably 16%~24%, more preferably 17%~23%, and even more preferably 18%~22%. As an optional embodiment, the filtrate 4 is concentrated to one-fifth of the original fermentation liquid volume, i.e., 20%. The pH of the concentrated liquid is preferably adjusted to 1.5~2.5, more preferably 1.8~2.2, more preferably 1.9~2.1, and most preferably 2; then it is cooled to 0 ℃ and preferably maintained for 36 h~60 h, more preferably 40 h~60 h, more preferably 50 h~60 h, and most preferably 60 h. Crystals will continue to precipitate during the stirring process after cooling to 0 ℃. As an optional embodiment, the stirring is magnetic stirring at a speed of 400 rpm. As an optional embodiment, a Buchner funnel with three layers of filter paper is used for filtration. As an optional embodiment, the drying is carried out in an oven at a drying temperature of 50 ℃ for 24 h.

[0028] In this invention, the hot water is deionized water or purified water. As an optional embodiment, the hot water is deionized water. Gallic acid has low solubility in cold water, and the gallic acid titer in the fermentation broth is high, requiring continuous elution of gallic acid to improve its yield.

[0029] In this invention, the gallic acid-rich fermentation broth is obtained by bio-fermentation using strain WJ54, which is the engineered strain WJ54 prepared in Example 5 of Chinese Patent CN121271770A.

[0030] In this invention, the steps for producing gallic acid (GA) by fermentation using gallic acid-producing strain WJ54 are as follows: Gallic acid-producing strain WJ54 was streaked on LB solid medium and cultured at 37 ℃ for 12 h to obtain single colonies. The single colonies were inoculated into 5 mL of liquid LB medium and cultured in a shaker at 37 ℃ for 12 h. Then, 1 mL of the bacterial culture was inoculated into 50 mL of fermentation medium in a shake flask at a volume ratio of 2% for fermentation. The fermentation conditions were 33 ℃, 220 rpm, and fermentation for 48 h to obtain the shake flask seed culture. The shake flask seed culture was then inoculated into a 3 L fermenter containing 1 L of fermentation medium at an inoculation rate of 8% by volume for fermentation culture at 33 °C to produce gallic acid de novo. The process parameters for fermentation in the fermenter were as follows: ventilation rate of 0.2 vvm, stirring speed correlated with dissolved oxygen, dissolved oxygen maintained at approximately 20%, a glucose solution with a concentration of 650 g / L added, and 25% (w / v) ammonia added to adjust the pH of the fermentation broth to 6.5. The fermentation time was 48 h to 56 h, yielding a fermentation broth rich in gallic acid.

[0031] As an optional implementation method, the fermentation time for producing gallic acid using gallic acid-producing strain WJ54 is 52 hours, with a fluctuation of about 2 hours, depending on whether the gallic acid titer no longer increases during testing.

[0032] The culture medium used in the fermentation production of GA in this invention is as follows: Fermentation medium: 1 L fermenter medium contains 6.78 g NaHPO4, 3 g KH2PO4, 1 g NH4Cl, 1.5 g NaCl, 10 g yeast extract, 250 mg MgSO4, 15 mg CaCl2, and 30 mg VB1. After sterilization, 15 g glucose and trace elements (0.03 mg H3BO3, 0.38 mg CuCl2, 0.4 mg Na2EDTA, 0.5 mg CoCl2, 0.94 mg ZnCl2, 1.6 mg MnCl2, and 3.6 mg FeSO4) are added, with water as the solvent.

[0033] The LB medium formula is: 5 g / L yeast extract, 10 g / L peptone and 10 g / L NaCl, with water as the solvent.

[0034] This application also provides gallic acid prepared by the method described above.

[0035] This application also provides the application of the method or the gallic acid prepared by the method in the preparation of high-purity gallic acid products.

[0036] In this invention, the gallic acid crystals obtained after extraction and purification by the method have a good color, reaching light brown; the yield is high and the purity can reach 99.449% as tested, showing good prospects for industrial application.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional experimental methods in the art; the materials and reagents used are commercially available unless otherwise specified.

[0038] The references involved in the following embodiments of the present invention are as follows: [1]. Guo, J., Ren, X., Lu, L., An, N., Li, S., Geng, M., Li, G., Shen,

[0039] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1 A method for purifying gallic acid through bio-fermentation, comprising the following steps: 1. A fermentation broth rich in gallic acid was obtained using strain WJ54 through bio-fermentation: The experimental steps for producing gallic acid (GA) by fermentation using gallic acid-producing strain WJ54 are as follows: Gallic acid-producing strain WJ54 was streaked on LB solid medium and cultured at 37 ℃ for 12 h to obtain single colonies. The single colonies were inoculated into 5 mL of LB liquid medium and cultured in a shaker at 37 ℃ for 12 h. Then, 1 mL of the bacterial culture was inoculated into a 50 mL shake flask of fermentation medium at a volume ratio of 2% for fermentation. The fermentation conditions were 33 ℃, 220 rpm, and fermentation was carried out for 48 h to obtain the shake flask seed culture.

[0041] The shake flask seed culture was inoculated at a volume fraction of 8% into a fermenter containing 1 L of fermentation medium (3 L volume) for fermentation culture at a fermentation temperature of 33 ℃, to synthesize gallic acid de novo.

[0042] The process parameters for fermentation in the fermenter were as follows: ventilation rate of 0.2 vvm, stirring speed correlated with dissolved oxygen, dissolved oxygen maintained at approximately 20%, glucose solution with a concentration of 650 g / L added at a flow rate of 8 mL / h; 25% (w / v) ammonia added to adjust the pH of the fermentation broth to 6.5 (the instrument automatically adjusts the addition based on the pH); fermentation time was approximately 52 h, specifically determined by the point at which the gallic acid titer no longer increased, resulting in a fermentation broth rich in gallic acid.

[0043] 2. Extract gallic acid and purify and decolorize it, as follows: Take 2L of the gallic acid-rich fermentation broth obtained from the above fermentation, heat it to 85 ℃ and maintain the temperature for 30 min, then add it to the storage tank of a disposable heated ceramic membrane machine (ceramic membrane / tubular membrane pilot equipment (model: CeraMem-0100), purchased from Xiamen Fumei Technology), and then filter it. Select a ceramic membrane with a pore size of 50 nm. During the filtration process, continuously add 85 ℃ deionized water. The amount of 85 ℃ deionized water added should be 3 times the volume of the original fermentation broth (i.e., 6L).

[0044] The 85℃ deionized water was added continuously: during filtration, the fermentation broth and 85℃ deionized water were added simultaneously, both continuously via a peristaltic pump. The flow rates of the 85℃ deionized water and the filtrate filtered through the ceramic membrane were the same, at 100 mL / min, and the filtrate was collected.

[0045] The collected filtrate was then reheated to 85 °C and maintained at that temperature for 30 min. It was then filtered using a ceramic membrane with a pore size of 10 nm. During the filtration process, deionized water at 85 °C was continuously added, with the volume of the added deionized water being twice the volume of the filtrate. The addition method was continuous flow: the fermentation broth and the deionized water at 85 °C were added simultaneously during filtration, both of which were continuously added using a peristaltic pump. The flow rate of the deionized water at 85 °C and the filtrate filtered through the ceramic membrane was 100 mL / min. The filtrate was then collected.

[0046] The filtrate was concentrated (rotary evaporator, 80 ℃) to one-third of the original fermentation broth volume, yielding approximately 666 mL of concentrate. This concentrate was poured into a beaker, and activated carbon (approximately 66 g of activated carbon powder) was added at a mass-to-volume ratio of 1 g:10 mL (purchased from Xinjinhu Activated Carbon Co., Ltd., model JH-769). The mixture was heated to 70 ℃ and continuously stirred with a magnetic stirrer for 2 hours to decolorize. After decolorization, the liquid was allowed to cool to room temperature (20 ℃), and the pH was adjusted to 1.5 using 11 mol / L concentrated hydrochloric acid. The temperature was then lowered to 0 ℃ and maintained at 0 ℃ for 60 hours to precipitate crystals. The precipitate was then filtered using a Buchner funnel with three layers of filter paper, and the solid precipitate was collected, yielding gallic acid crystals containing activated carbon.

[0047] Gallic acid crystals containing activated carbon were then placed in an oven and dried at 50 °C to obtain dried gallic acid crystals (containing activated carbon). The dried gallic acid crystals were dissolved in deionized water at 65 °C in a beaker, while maintaining the temperature at 65 °C and stirring (magnetic stirring, 400 rpm). The amount of deionized water used was four times the mass of the crystals. The solution was filtered using a Buchner funnel with three layers of filter paper, and the filtrate was collected. Simultaneously, the solid precipitate was collected and washed again with deionized water at 65 °C, using twice the mass of the solid precipitate. The solution was then filtered again using a Buchner funnel with three layers of filter paper, and the filtrate was collected. The two filtrates were combined to obtain a combined filtrate.

[0048] The collected combined filtrate was concentrated (rotary evaporator, 80 ℃) to one-third of the original fermentation broth volume, yielding approximately 666 mL of concentrate. The concentrate was poured into a beaker, and then 16.7 g of activated carbon powder (Xinjinhu Activated Carbon Co., Ltd., model JH-769) was added to it, resulting in a mass-to-volume ratio of activated carbon to concentrate of 0.25 g / 10 mL. The mixture was thoroughly mixed, heated to 70 ℃, and continuously stirred with a magnetic stirrer for 2 h to decolorize the mixture. The color was observed to be colorless. After decolorization, the liquid was allowed to cool to room temperature (20 °C) in a beaker. It was then concentrated (using a rotary evaporator at 60 °C) to one-fifth of the original fermentation liquid volume (i.e., 400 mL). The pH was then adjusted to 2 using 11 mol / L concentrated hydrochloric acid, with stirring using a glass stirring rod. After the pH was adjusted to 2, the mixture was allowed to stand. The mixture was then cooled to 0 °C and stirred (magnetic stirring at 400 rpm). Crystals continued to precipitate during the process. The mixture was kept at this low temperature for 60 h. The solid precipitate was collected by filtering it through a Buchner funnel with three layers of filter paper, yielding gallic acid crystals containing activated carbon.

[0049] Gallic acid crystals containing activated carbon were then placed in an oven and dried at 50 °C for 24 h to obtain dried gallic acid crystals (containing a small amount of activated carbon). The dried gallic acid crystals were dissolved in deionized water at 65 °C in a beaker, while maintaining the temperature at 65 °C and stirring (magnetic stirring, 400 rpm). The amount of deionized water used was four times the mass of the crystals. The solution was filtered using a Buchner funnel with three layers of filter paper, and the filtrate was collected. Simultaneously, the solid precipitate (decolorized activated carbon) was collected and washed again with 65 °C hot water, using twice the amount of hot water. The solution was then filtered again using a Buchner funnel with three layers of filter paper, and the filtrate was collected. The two filtrates were combined to obtain a combined filtrate.

[0050] The combined filtrate was concentrated (rotary evaporator, 60 °C) to one-fifth of the original fermentation broth volume (i.e., 400 mL), and then cooled to 0 °C. During stirring (magnetic stirring, 400 rpm), crystals continued to precipitate. The mixture was kept at the low temperature for 60 h, filtered using a Buchner funnel with three layers of filter paper, and the solid precipitate was collected to obtain the final gallic acid product.

[0051] The final gallic acid product obtained after the above purification was detected by high performance liquid chromatography-tandem mass spectrometry. The detection method was based on that of Guo Jiao et al. [1] The literature was reviewed.

[0052] Figure 1 In the image, A represents the purified gallic acid product. Figure 1 In the figure, B represents the chromatographic result of the purified gallic acid product. Figure 1 It can be seen that the purified gallic acid product has pure white crystals, and the chromatographic purity of the crystals can reach 99.449%. Figure 2 Mass spectrometry results of the purified gallic acid product confirmed that the target product was gallic acid. Using the above method, 96.4 g / L gallic acid crystals were finally prepared from 1 L of fermentation broth with a gallic acid concentration of 98 g / L, with a yield of 98.4%. In summary, the method described in this embodiment is suitable for the purification of gallic acid from fermentation products of engineered bacteria that efficiently synthesize gallic acid. The prepared gallic acid crystals have high purity, good color, and high yield, showing good prospects for industrial application.

[0053] Comparative Example 1 A method for purifying gallic acid through bio-fermentation, comprising the following steps: 1. The steps for obtaining a fermentation broth rich in gallic acid using strain WJ54 are the same as in Example 1.

[0054] The difference is: 2. Extract gallic acid and purify and decolorize it, as follows: Take 2L of the above-mentioned fermentation broth rich in gallic acid, adjust the pH to 1 with 11 mol / L concentrated hydrochloric acid, store at 0℃ for 60 h, and crystals will precipitate. Filter the crystals using a Buchner funnel and filter paper to obtain the gallic acid product.

[0055] Figure 3 The image shows the actual product of the obtained gallic acid. According to... Figure 3 As can be seen, the obtained gallic acid product is dark brown in color, indicating that the crystal purity is extremely poor.

[0056] Comparative Example 2 A method for purifying gallic acid through bio-fermentation, comprising the following steps: 1. The steps for obtaining a fermentation broth rich in gallic acid using strain WJ54 are the same as in Example 1.

[0057] The difference is: 2. Extract gallic acid and purify and decolorize it, as follows: Take 2L of the above-mentioned gallic acid-rich fermentation broth, heat it continuously to 85℃, maintain the temperature for 30 min, and then add it to the storage tank of a disposable heated ceramic membrane machine (ceramic membrane / tubular membrane pilot equipment (model: CeraMem-0100), purchased from Xiamen Fumei Technology). Then filter it, using a ceramic membrane with a pore size of 50 nm. During the filtration process, continuously add 85℃ deionized water. The amount of 85℃ deionized water added should be 3 times the volume of the original fermentation broth (6 L).

[0058] The 85℃ deionized water was added continuously: during filtration, the fermentation broth and 85℃ deionized water were added simultaneously, both continuously via a peristaltic pump. The flow rates of the 85℃ deionized water and the filtrate filtered through the ceramic membrane were the same, at 100 mL / min, and the filtrate was collected.

[0059] The filtrate was then heated to 85 °C again using a water bath and maintained at that temperature for 30 min. It was then filtered using a ceramic membrane with a pore size of 10 nm. During the filtration process, deionized water at 85 °C was continuously added, with the volume of the added deionized water being twice the volume of the filtrate. The addition method was continuous flow: the fermentation broth and the deionized water at 85 °C were added simultaneously during filtration, both of which were continuously added using a peristaltic pump. The flow rate of the deionized water at 85 °C and the filtrate filtered through the ceramic membrane was 100 mL / min. The filtrate was then collected.

[0060] The filtrate was concentrated (rotary evaporator, 85 ℃) to one-third of the original fermentation broth volume, yielding approximately 666 mL of concentrate. This concentrate was poured into a beaker, and activated carbon (approximately 66 g of activated carbon powder) was added at a mass-to-volume ratio of 1 g:10 mL (purchased from Xinjinhu Activated Carbon Co., Ltd., model JH-769). The mixture was heated to 85 ℃ and continuously stirred magnetically for decolorization over 3 hours. After decolorization, the liquid was allowed to cool to room temperature (20 ℃) ​​in the beaker. The pH was then adjusted to 1.5 using 11 mol / L concentrated hydrochloric acid. The temperature was lowered to 0 ℃ and maintained at 0 ℃ for 48 hours. The mixture was then filtered using a Buchner funnel with three layers of filter paper, and the solid precipitate was collected, yielding gallic acid crystals containing activated carbon.

[0061] Gallic acid crystals containing activated carbon were then dried in an oven at 50 °C to obtain dried gallic acid crystals. The dried gallic acid crystals were dissolved in 85 °C deionized water in a beaker while maintaining the temperature at 85 °C and stirring (magnetic stirring, 400 rpm). The amount of 85 °C deionized water used was four times the mass of the crystals. The solution was filtered using a Buchner funnel with three layers of filter paper, and the filtrate was collected. Simultaneously, the solid precipitate was collected and washed again with 85 °C deionized water, twice the mass of the solid precipitate. The solution was then filtered again using a Buchner funnel with three layers of filter paper, and the filtrate was collected. The two filtrates were combined to obtain a combined filtrate.

[0062] The collected combined filtrate was concentrated (rotary evaporator, 85 ℃) to one-third of the original fermentation broth volume, yielding approximately 666 mL of concentrate. The concentrate was poured into a beaker, and then 16.7 g of activated carbon powder (Xinjinhu Activated Carbon Co., Ltd., model JH-769) was added to it, resulting in a mass-to-volume ratio of activated carbon to concentrate of 0.25 g / 10 mL. The mixture was thoroughly mixed and heated to 85 ℃, with continuous stirring using a magnetic stirrer for 3 h to decolorize the mixture. The color was observed to be colorless.

[0063] After decolorization, the liquid was allowed to cool to room temperature (20 °C) in a beaker. It was then concentrated (rotary evaporator, 85 °C) to one-fifth of the original fermentation liquid volume (i.e., 400 mL). The pH was then adjusted to 1 using 11 mol / L concentrated hydrochloric acid, with stirring using a glass stirring rod. After the pH was adjusted to 1, the mixture was allowed to stand. The mixture was then cooled to 0 °C and stirred (magnetic stirring, 400 rpm). Crystals continued to precipitate during the process. The mixture was kept at this low temperature for 48 h. The solid precipitate was collected by filtering it through a Buchner funnel with three layers of filter paper, yielding gallic acid crystals containing activated carbon.

[0064] Gallic acid crystals containing activated carbon were then placed in an oven and dried at 50 °C for 24 h to obtain dried gallic acid crystals. The dried gallic acid crystals were dissolved in deionized water at 85 °C in a beaker while maintaining the temperature at 85 °C and stirring (magnetic stirring, 400 rpm). The amount of deionized water used was four times the mass of the crystals. The solution was filtered using a Buchner funnel with three layers of filter paper, and the filtrate was collected. Simultaneously, the solid precipitate (decolorized activated carbon) was collected and rinsed again with deionized water at 85 °C, using twice the mass of the solid precipitate. The solution was then filtered again using a Buchner funnel with three layers of filter paper, and the filtrate was collected. The two filtrates were combined to obtain a combined filtrate.

[0065] The combined filtrate was concentrated (rotary evaporator, 85 °C) to one-fifth of the original fermentation broth volume (i.e., 400 mL), and then cooled to 0 °C. During stirring (magnetic stirring, 400 rpm), crystals continued to precipitate. The mixture was kept at the low temperature for 48 h, filtered using a Buchner funnel with three layers of filter paper, and the solid precipitate was collected to obtain the final gallic acid product.

[0066] The purified gallic acid product was detected by high performance liquid chromatography-tandem mass spectrometry. The detection method was based on that of Guo Jiao et al. [1] The literature was reviewed.

[0067] Figure 4 Image of the purified gallic acid product; Figure 5 A and Figure 5 B represents the mass spectrometry and chromatographic detection results of the purified gallic acid product, respectively. Based on the above results, it was confirmed that gallic acid was indeed obtained. The purified gallic acid product was pale yellow in color, and the chromatographic purity was 97.8%, which was slightly lower than that in Example 1.

[0068] In summary, the purification method of this invention is suitable for purifying gallic acid from fermentation products of engineered bacteria that efficiently biosynthesize gallic acid. The purified gallic acid product has high crystal purity, good color, and high yield, and has good prospects for industrial application.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for purifying gallic acid through biological fermentation, characterized in that, Includes the following steps: (1) Obtain a fermentation broth rich in gallic acid; the fermentation broth is heated and then filtered with a pore size of 45 nm to 55 nm, and filtrate 1 is collected; filtrate 1 is heated and then filtered with a pore size of 8 nm to 15 nm, and filtrate 2 is collected; the heating temperature is 80°C to 90°C. (2) The filtrate 2 is concentrated to obtain a concentrate. Activated carbon with a mass-to-volume ratio of 0.5~1.5g:10mL is added to the concentrate for decolorization. The decolorization temperature is 65℃~75℃. After decolorization, the pH is adjusted to 1~2, the temperature is lowered to 0℃, and the solution is maintained for 36h~60h. The solution is filtered, and solid precipitate 1 is collected to obtain gallic acid crystals containing activated carbon. The gallic acid crystals containing activated carbon are dried and then dissolved at a water temperature of 60℃~70℃. The solution is filtered, and filtrate 3 is collected. (3) Repeat step (2) to decolorize filtrate 3 and collect filtrate 4; (4) Concentrate filtrate 4, adjust pH to 1.5~2.5, cool to 0 ℃, maintain for 36 h~60 h, filter, collect solid precipitate 2, dry, and obtain gallic acid.

2. The method according to claim 1, characterized in that, In step (1), hot water at 80℃~90℃ is continuously added during the filtration process.

3. The method according to claim 2, characterized in that, In step (1), when preparing filtrate 1, the amount of hot water added is 2 to 4 times the volume of the fermentation liquid; when preparing filtrate 2, the amount of hot water added is 1 to 3 times the volume of filtrate 1.

4. The method according to claim 2, characterized in that, In step (1), the fermentation liquid and the hot water are added simultaneously during filtration, with the same flow rate of 80~120 mL / min; the filter membrane used for filtration is a ceramic membrane.

5. The method according to claim 1, characterized in that, In step (2), the coloring process is carried out by continuous stirring, and the coloring time is 2 h to 5 h.

6. The method according to claim 1, characterized in that, In step (2), the filtrate 2 is concentrated to 30%~40% of the fermentation liquid volume; the activated carbon is ≥200 mesh.

7. The method according to claim 1, characterized in that, In step (2), the amount of hot water used for dissolution is 2 to 4 times the mass of the gallic acid crystals containing activated carbon; after collecting solid precipitate 1, the solid precipitate is rinsed again with hot water at a temperature of 60℃ to 70℃, and the rinsing volume is 2 to 4 times the mass of the solid precipitate.

8. The method according to claim 1, characterized in that, In step (3), a mass-to-volume ratio of 0.2~0.3g:10mL is added to the concentrate; in step (4), the filtrate 4 is concentrated to 15%~25% of the fermentation liquid volume.

9. The method according to claim 1, characterized in that, The gallic acid-rich fermentation broth has a gallic acid titer of ≥100 g / L, and the gallic acid-rich fermentation broth is obtained by fermentation using strain WJ54.

10. The application of gallic acid prepared according to any one of the methods described in 1 to 9 in the preparation of products containing high-purity gallic acid.

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