A method for separating and extracting muconic acid from a fermentation broth

Abstract

The present application relates to a kind of methods for separating and extracting muconic acid from fermentation broth, comprising the following steps: separating and treating the fermentation broth containing muconic acid to remove bacterial bodies, obtaining clear fermentation broth;Remove macromolecular protein pigment from clear fermentation broth, obtain decolorized clear fermentation broth;Adjust the pH of decolorized clear fermentation broth to acidic, obtain acidified liquid;Acidified liquid is cooled and precipitated, mother liquor 1 is removed by filtration, and crude muconic acid solid is obtained;After crude muconic acid solid is redissolved, adjust the pH value, obtain crude muconic acid solution;Activated activated carbon is added to crude muconic acid solution for decolorization, stir evenly, then filter, obtain decolorized muconic acid solution;Adjust the pH of decolorized muconic acid solution to acidic, precipitate, remove mother liquor 2 by filtration, dry, and obtain pure muconic acid product.The method for separating and extracting muconic acid from fermentation broth is simple in separation and purification process, without organic solvent, high in product purity, low in cost, and meets the requirements of industrialization.

Description

Technical Field

[0001] This invention belongs to the field of biochemical separation technology and relates to a method for separating and extracting mucoconic acid from fermentation culture broth. Background Technology

[0002] Mucoconic acid (MA), also known as hexadienoic acid or 2,4-hexadienoic acid, is an important intermediate in the degradation of aromatic compounds. Mucoconic acid can absorb 260nm ultraviolet light, and is therefore widely used as an anti-ultraviolet protectant and a UV collector in cosmetics, coatings, fibers, plastics, and military special products. In particular, cis,cis-muconic acid (CCMA) can be hydrogenated to adipic acid in a reducing atmosphere in the presence of a heterogeneous catalyst. Adipic acid is an organic dicarboxylic acid with biodegradability, low toxicity, and good chemical stability, and is widely used in pharmaceuticals, coatings, food processing, and especially in organic synthesis (e.g., nylon 6.6, engineering plastics, and biodegradable plastic PBAT).

[0003] To date, biocompatible mucoconic acid has primarily been separated and purified using precipitation methods, and mainly with organic solvents. For example, patent CN 102985537 A (Amyris) discloses a method for isomerization, separation, and purification of cis-trans-mucoconic acid from a fermentation broth containing cis-cis-mucoconic acid obtained from microbial fermentation of a renewable carbon source (such as glucose). This method has a low recovery rate of approximately 60% in the precipitation stage of separating cis-trans-mucoconic acid, requiring concentration and reprecipitation of the residual portion in the supernatant, or the addition of solvent extraction to improve the recovery rate. Furthermore, the precipitation process also requires the use of organic solvents to remove salts involved in the separation process by precipitating cis-trans-mucoconic acid.

[0004] For another example, Vardon, RD et al. A method for separating and purifying cis-cis-mucoconic acid was proposed in 2016, 18(11):3397-3413. This method separates and purifies cis-cis-mucoconic acid through protein removal, activated carbon adsorption, acid treatment precipitation, and ethanol recrystallization. The total recovery rate of mucoconic acid is 81.5%, but it does not mention the proportion of cis-cis-mucoconic acid in the product. The disadvantage of this separation process is that the amount of activated carbon used is large, and the activated carbon will also adsorb a small amount of mucoconic acid, reducing the recovery rate of mucoconic acid. At the same time, this separation process requires the use of organic solvent ethanol to improve the yield, but the use of ethanol, a flammable and explosive organic solvent, increases the safety risk, and the recovery of organic solvent increases the separation cost.

[0005] As can be seen from the above, existing mucoconic acid separation processes suffer from low yield and purity. The extensive use of activated carbon during separation further reduces the mucoconic acid yield, while adding ethanol for recrystallization increases safety risks and the recovery of organic solvents raises separation costs. Furthermore, cis-cis-mucoconic acid is easily isomerized into cis-trans-mucoconic acid and / or trans-trans-mucoconic acid during separation due to the influence of pH, temperature, and light, resulting in unsatisfactory cis-cis-mucoconic acid yield and purity, which is detrimental to subsequent adipic acid production. Therefore, how to separate mucoconic acid, especially cis-cis-mucoconic acid, simply, efficiently, safely, environmentally friendly, and at low cost has become a technical problem that those skilled in the art have long desired to solve but have yet to successfully resolve. Summary of the Invention

[0006] The technical problem this invention aims to solve is that existing separation processes require the use of organic solvents such as ethanol to improve yield and product purity. However, the use of ethanol, a flammable and explosive organic solvent, increases safety risks. Furthermore, the recovery of organic solvents increases separation costs, and the low proportion of cis-cis-mucoconic acid is detrimental to adipic acid production. This invention provides a method for separating and extracting mucoconic acid from fermentation broth. This method does not use any organic solvents, including ethanol, during the separation and purification of bio-mucoconic acid. It achieves high product yield and purity, and the product contains a high proportion of cis-cis-mucoconic acid.

[0007] Therefore, the present invention provides a method for separating and extracting mucoconic acid from fermentation broth, comprising:

[0008] Step A: Separate and process the fermentation broth containing mucoconic acid to remove the bacterial cells and obtain a clear fermentation broth;

[0009] Step B: Remove macromolecular protein pigments from the clarified fermentation broth to obtain decolorized clarified fermentation broth;

[0010] Step C: Adjust the pH of the decolorized and clarified fermentation broth to acidic to obtain an acidified broth;

[0011] Step D: Cool the acidified solution and let it stand to precipitate. Filter to remove mother liquor 1 and obtain crude viscous solid.

[0012] Step F: After redissolving the crude mucocomic acid solid, adjust the pH value to obtain a crude mucocomic acid solution;

[0013] Step G: Add activated carbon to the crude mucoaconic acid solution for decolorization, stir and mix well, then filter to obtain a decolorized mucoaconic acid solution.

[0014] Step H: Adjust the pH of the decolorized mucoacin solution to acidic, allow it to stand and precipitate, filter to remove mother liquor 2, and dry to obtain pure mucoacin.

[0015] According to the present invention, in step B, nanofiltration is used to remove macromolecular protein pigments. Preferably, the nanofiltration membrane is an 800-1000 dalc membrane, more preferably an 800 dalc organic membrane, and the operating pressure is 3-4 MPa.

[0016] According to the present invention, in steps C and H, the pH is adjusted to 1.0–3.0 using an acid solution, wherein the acid solution is a 1–6 mol / L hydrochloric acid aqueous solution or a 50%–98% sulfuric acid aqueous solution by mass fraction; preferably, the dropping rate of the acid solution is 0.5–2.5 × 10⁻⁶. -2 vvm.

[0017] In some embodiments of the present invention, in step A, the separation process includes filtration and / or centrifugation; preferably, a ceramic membrane with a pore size of 100 nm is used for filtration, and the operating pressure is 0.2 MPa; and / or, the centrifugation rate is 6000 to 8000 rpm.

[0018] In some embodiments of the present invention, in step D, the temperature is lowered to 0-10°C, and the settling time is 0.5-2 hours; and / or, a microporous membrane with a pore size of 0.45 μm is used for filtration.

[0019] In some embodiments of the present invention, in step F, the crude mucoaconic acid solid is reconstituted with water to a mucoaconic acid concentration of 80-110 g / L; and / or, the pH value is adjusted to 6.0-8.0.

[0020] In some embodiments of the present invention, in step G, the amount of activated carbon used is 0.003 to 0.005 g / mL, based on the crude viscosic acid solution; and / or, the stirring and mixing temperature is 30 to 40°C, and the stirring and mixing time is 60 to 90 min; and / or, filtration is performed using a microporous membrane with a pore size of 0.45 μm.

[0021] In some embodiments of the present invention, in step H, the pH value of the decolorized mucoacin solution is adjusted to 1.0 to 3.0; and / or, the solution is filtered using a microporous membrane with a pore size of 0.45 μm; and / or, the settling temperature is 0 to 10°C, and the settling time is 0.5 to 2 h; and / or, the drying temperature is 30 to 60°C.

[0022] According to some preferred embodiments of the present invention, the method further includes step E, adjusting the pH value of mother liquor 1 to neutral, concentrating it by rotary evaporation 10 to 20 times, filtering, and repeating steps C and D to obtain crude viscous solid.

[0023] According to some preferred embodiments of the present invention, the method further includes step I, adjusting the pH value of the mother liquor 2 to neutral, concentrating it by rotary evaporation 10 to 20 times, filtering, and repeating steps G and H to obtain pure mucoconic acid.

[0024] As can be seen from the above technical solution, compared with the prior art, the method for separating and extracting mucoconic acid from fermentation broth provided by the present invention has a simple process flow, does not require the use of organic solvents such as ethanol, eliminates the safety risks of using flammable and explosive organic solvents such as ethanol, has low cost, high product recovery rate, and product purity meets industry requirements. In particular, the proportion of cis-cis-mucoconic acid in the product is very high, reaching up to 99%, which is beneficial to adipic acid production.

[0025] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0026] The above and other objects, features, and advantages of the present invention will become more apparent from the more detailed description of the embodiments of the invention in conjunction with the accompanying drawings. The drawings are provided to further illustrate the embodiments of the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0027] Figure 1 This is a schematic diagram of the process flow for separating and extracting mucoconic acid from fermentation broth in this invention.

[0028] Figure 2 This is a schematic diagram of the main process flow for the traditional separation and extraction of mucoconic acid.

[0029] Figure 3 A schematic diagram of the process flow for separating and extracting mucoconic acid from Derek R.

[0030] Figure 4 This is a liquid chromatogram of the original fermentation broth of this invention.

[0031] Figure 5 This is a liquid chromatogram of the mucoconic acid aqueous solution in Example 1 of the present invention.

[0032] Figure 6 This is a liquid chromatogram of the mucoconic acid aqueous solution in Example 2 of the present invention.

[0033] Figure 7 This is a liquid chromatogram of the mucoconic acid aqueous solution in Example 3 of the present invention.

[0034] Figure 8 This is a liquid chromatogram of the mucoconic acid aqueous solution in Example 4 of the present invention.

[0035] Figure 9 The image shows the nuclear magnetic resonance (NMR) spectrum of the mucoconic acid product of this invention. Detailed Implementation

[0036] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0037] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0038] I. Implementation Plan

[0039] As mentioned earlier, existing mucoconic acid separation processes suffer from low yield and purity. The extensive use of activated carbon during separation further reduces the mucoconic acid yield, while recrystallization with ethanol increases safety risks and increases separation costs due to the recovery of organic solvents. Furthermore, cis-cis-mucoconic acid is easily isomerized into cis-trans-mucoconic acid and / or trans-trans-mucoconic acid during separation due to the influence of pH, temperature, and light, resulting in unsatisfactory cis-cis-mucoconic acid yield and purity, which is detrimental to subsequent adipic acid production. Therefore, how to separate mucoconic acid, especially cis-cis-mucoconic acid, simply, efficiently, safely, environmentally friendly, and at low cost has become a technical problem that those skilled in the art have long desired to solve but have yet to successfully resolve.

[0040] To address these issues, the inventors conducted extensive research on the separation and purification technology of biocompatible mucoconic acid. Through this research, the inventors discovered that a method involving nanofiltration decolorization followed by precipitation and then further decolorization can reduce the use of activated carbon and minimize product loss while improving the recovery rate and purity of mucoconic acid without the use of any organic solvents (including ethanol) during the separation and purification process. This successfully solved the aforementioned technical challenges, leading to this invention. The advantages of this invention are that it eliminates the use of organic solvents, has lower equipment requirements, significantly reduces equipment costs, is relatively safe, eliminates organic solvent recovery and loss, lowers production costs, and has greater potential for industrial scale-up.

[0041] Therefore, the process flow of the method for separating and extracting mucoconic acid from fermentation broth provided by the present invention is as follows: Figure 1 As shown, from Figure 1 It can be seen that the method for separating and extracting mucoconic acid from the fermentation broth includes:

[0042] Step A: Separate and process the fermentation broth containing mucoconic acid to remove the bacterial cells and obtain a clear fermentation broth;

[0043] Step B involves removing macromolecular protein pigments from the obtained clarified fermentation broth to obtain a decolorized clarified fermentation broth.

[0044] Step C: Adjust the pH of the decolorized and clarified fermentation broth to 1.0–3.0 to obtain an acidified broth;

[0045] Step D: Cool the acidified solution to 0-10℃, let it stand to precipitate for 0.5-2 hours, and filter it with a microporous membrane with a pore size of 0.45μm to remove the mother liquor 1, and obtain crude viscous solid.

[0046] Step F: The crude mucocomic acid solid is reconstituted with water to a mucocomic acid concentration of 80-110 g / L, and then a sodium hydroxide solution with a concentration of 1-3 mol / L is added to adjust the pH value to 6.0-8.0 to obtain a crude mucocomic acid solution.

[0047] Step G: Add activated carbon to the crude mucoaconic acid solution for decolorization, stir at 30-40℃ for 60-90 min to mix, and then filter with a microporous membrane with a pore size of 0.45 μm to remove the activated carbon, and obtain the decolorized mucoaconic acid solution.

[0048] Step H: Adjust the pH of the decolorized mucoacin solution to 1.0–3.0, let it stand at 0–10°C for 0.5–2 h to allow it to fully precipitate, filter it with a microporous membrane with a pore size of 0.45 μm to remove the mother liquor 2, and dry it at 30–60°C to obtain pure mucoacin.

[0049] In this invention, the term "water" refers to deionized water, distilled water, or ultrapure water unless otherwise specified or limited.

[0050] In this invention, the fermentation broth containing mucoconic acid is obtained by fermentation culture of a strain that produces mucoconic acid (refer to patent CN 117004547 A).

[0051] In some embodiments of the present invention, in step A, the separation process includes filtration and / or centrifugation; preferably, a ceramic membrane with a pore size of 100 nm is used for filtration, and the operating pressure is 0.2 MPa; the centrifugation rate is 6000-8000 rpm.

[0052] In some embodiments of the present invention, in step B, nanofiltration is used to remove macromolecular protein pigments. Preferably, the nanofiltration membrane is 800-1000 Da, more preferably an organic membrane of 800 Da, and the operating pressure is 3-4 MPa.

[0053] During the research, the inventors unexpectedly discovered that nanofiltration equipment with a membrane pore size of 800–1000 Dal can effectively remove pigments from our fermentation broth, while minimizing the loss of mucoacin. This step reduces the amount of activated carbon used in the subsequent decolorization process and reduces the loss of mucoacin caused by activated carbon adsorption, thereby improving the product recovery rate.

[0054] Further research revealed that due to the higher concentration of mucoconic acid and the osmotic pressure of impurities in the fermentation broth, the 1000Dal nanofiltration membrane was not as effective as the 800Dal nanofiltration membrane, which removed more large-molecule proteins and pigments. Meanwhile, the 500Dal membrane had too low flux and too large transmembrane pressure difference. In actual operation, the membrane flux would drop sharply or even fail to pass through, and the transmembrane pressure difference would also rise sharply, resulting in too low filtration efficiency, which was not conducive to industrial scale-up in actual operation.

[0055] In some embodiments of the present invention, in step C, the pH value of the clarified fermentation broth is adjusted to 1.0 to 3.0 using a hydrochloric acid solution with a concentration of 1 to 6 mol / L or a sulfuric acid solution with a mass fraction of 50% to 98%.

[0056] In some embodiments of the present invention, in step G, the amount of activated carbon used is 0.003 to 0.005 g / mL, based on the crude viscosic acid solution.

[0057] In some preferred embodiments of the present invention, the activated carbon is activated carbon activated with 10% to 40% nitric acid solution; the volume ratio of 10% to 40% nitric acid solution to activated carbon is 1 to 5:1; the activation time is 1 to 3 hours; and the activation temperature is 20 to 60°C.

[0058] In some embodiments of the present invention, in step H, the pH value of the decolorized mucoacinic acid solution is adjusted to 1.0 to 3.0 using a hydrochloric acid solution with a concentration of 1 to 6 mol / L or a sulfuric acid solution with a mass fraction of 50% to 98%.

[0059] It is well known in the art that it is extremely difficult to simultaneously achieve a yield of 90% and a purity of over 99% when separating and extracting mucoconic acid from fermentation broth. This is especially true during the separation process, where cis-cis-mucoconic acid easily transforms into cis-trans-mucoconic acid and / or trans-trans-mucoconic acid under the influence of pH, temperature, and light, resulting in a low proportion of cis-cis-mucoconic acid in the product. During the research process, the inventors accidentally discovered that controlling the dropping rate of the acid solution during pH adjustment and prior acidification and concentration before decolorization can simultaneously achieve a yield of 90% and a purity of over 99% for mucoconic acid. The product contains a higher proportion of cis-cis-mucoconic acid. Specifically, the dropping rate of the acid solution needs to be controlled during pH adjustment. For example, in steps C and H, adding the acid solution too quickly (e.g., pouring it directly) will rapidly generate a large amount of precipitate, resulting in more impurities being carried over and thus reducing purity. Adding the acid solution too slowly will prolong the process, reduce space-time efficiency, increase the difficulty of industrial scale-up, and reduce economic efficiency. At the same time, adding the acid solution too slowly will also cause some impurities to slowly crystallize out, reducing product purity. When the dropping rate of the acid solution is 0.5–2.5 × 10⁻⁶... -2 At vvm, the product yield and purity are optimal, and the product has the highest proportion of cis-cis-mucoconic acid.

[0060] According to some preferred embodiments of the present invention, the method further includes step E ( Figure 1 (Not shown in the text) Add 1 mol / L sodium hydroxide solution to mother liquor 1 to adjust the pH value of mother liquor 1 to 7.0. After rotary evaporation and concentration by 10 to 20 times, filter and repeat steps C and D to obtain crude viscous solid.

[0061] According to some preferred embodiments of the present invention, the method further includes step I ( Figure 1 (Not shown in the text) Add 1 mol / L sodium hydroxide solution to mother liquor 2 to adjust the pH value of mother liquor 2 to 7.0. After rotary evaporation and concentration by 10 to 20 times, filter and repeat step H to obtain pure mucoconic acid.

[0062] The results show that using steps E and I in this invention can further improve the yield of mucoconic acid.

[0063] This invention belongs to the field of biochemical separation technology and relates to a method for separating and extracting mucoconic acid from fermentation broth. The method includes: Step A, separating and treating the fermentation broth containing mucoconic acid to remove bacterial cells, obtaining a clarified fermentation broth; Step B, removing macromolecular protein pigments from the obtained clarified fermentation broth, obtaining a decolorized clarified fermentation broth; Step C, adjusting the pH of the decolorized clarified fermentation broth to acidic, obtaining an acidified solution; Step D, allowing the acidified solution to stand, filtering to remove mother liquor 1, obtaining crude mucoconic acid solid; Step F, redissolving the crude mucoconic acid solid, adjusting the pH value, obtaining a crude mucoconic acid solution; Step G, adding activated carbon to the crude mucoconic acid solution for decolorization, stirring and mixing, filtering, obtaining a decolorized mucoconic acid solution; Step H, adjusting the pH value of the decolorized mucoconic acid solution to acidic, allowing it to stand, removing mother liquor 2, drying, obtaining mucoconic acid. Using this method, the purity of mucoconic acid extracted from fermentation broth can reach over 99%, and the yield can reach over 85%. As can be seen from the above technical solution, compared with the prior art, the method for separating and extracting mucoconic acid from fermentation broth provided by the present invention has a simple process flow, does not require the use of organic solvents such as ethanol, eliminates the safety risks of using flammable and explosive organic solvents such as ethanol, has low cost, high product recovery rate, and product purity meets industry requirements. The advantages of the method of the present invention are that the separation and purification process is simple, there are no organic solvents, the product has high purity, low cost, and meets industrial requirements. In particular, the product has a high proportion of cis-cis-mucoconic acid, which is beneficial to adipic acid production.

[0064] II. Examples

[0065] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0066] In the following examples, the fermentation broth of Corynebacterium glutamicum containing mucoconic acid was prepared with reference to patent CN 117004547 A: Specifically, a genetically engineered bacterium MA1 that synthesizes cis,cis-mucoconic acid de novo using glucose as a substrate and a fermentation method in a 5 L fermenter were used (CN 117004547 A).

[0067] Example 1: Isolation and extraction of mucoacin from the fermentation broth of Corynebacterium glutamicum

[0068] (1) Take the fermentation broth containing mucoconic acid, and perform liquid chromatography (HPLC) as shown in the figure. Figure 4 The bacterial cells were removed by filtration using a ceramic membrane with a pore size of 100 nm at an operating pressure of 0.2 MPa. 1 L of clarified fermentation broth was collected, and the concentration of mucoconic acid in the clarified fermentation broth was 81.6 g / L.

[0069] (2) Take the clarified fermentation broth and filter it with an 800 Dal nanofiltration membrane to remove macromolecular pigments, and obtain the decolorized clarified fermentation broth.

[0070] (3) Take the decolorized and clarified fermentation broth, and use 1.5×10 -2 A 50% sulfuric acid solution was added dropwise using a VVM flow rate to adjust the pH of the clarified fermentation broth to 1.5, thus obtaining an acidified broth.

[0071] (4) Take the acidified solution, cool it to 4°C and let it stand for 1 hour. Filter it with a microporous membrane with a pore size of 0.45 μm to remove the mother liquor 1 and obtain crude viscous solid.

[0072] (5) Take crude mucocomic acid solid, redissolve it in deionized water to 800 mL (mucocomic acid concentration is about 100 g / L), add 3 mol / L sodium hydroxide solution, adjust the pH value to 7.0, and obtain crude mucocomic acid solution.

[0073] (6) Take the crude mucoaconic acid solution, add 0.003 (g / mL) of activated carbon, stir and mix at 30℃ and 200 rpm for 60 min, and then filter with a microporous membrane with a pore size of 0.45 μm to obtain a decolorized mucoaconic acid solution.

[0074] The activated carbon was activated with 40% nitric acid solution; the volume ratio of 40% nitric acid solution to activated carbon was 1:1; the activation time was 3 hours and the activation temperature was 20℃.

[0075] (7) Take the decolorized mucoacin solution and dilute it with 1.5 × 10⁻⁶ ppm. -2 The vvm was fed with a 50% sulfuric acid solution to adjust the pH of the decolorized mucoacinic acid solution to 1.5. The solution was allowed to stand at 4°C for 1 hour, and the mother liquor was removed by filtration through a microporous membrane with a pore size of 0.45 μm. The solution was then dried at 30°C to obtain pure mucoacinic acid.

[0076] The final yield was 72.16 g of mucoconic acid, with a yield of 88.43%.

[0077] Mucoconic acid yield = final mass of mucoconic acid crystals / mucoconic acid content in clarified fermentation broth × 100%.

[0078] Mucoconic acid yield = 72.16g / [(81.6g / L) × 1L)] × 100% = 88.43%.

[0079] Mucoconic acid was dissolved in deionized water and detected by liquid chromatography (Thermo Vanquish high-performance liquid chromatograph). Results are shown below. Figure 5 The results showed that the mucoconic acid concentration was 99.8%; the nuclear magnetic resonance H-ray spectrum was as follows. Figure 9 The results showed that cis-mucoconic acid accounted for more than 99%.

[0080] Example 2:

[0081] (1) Take the fermentation broth containing mucoconic acid, filter it with a ceramic membrane with a pore size of 100 nm to remove the cells, operate at a pressure of 0.2 MPa, collect 1 L of the clarified fermentation broth, and the concentration of mucoconic acid in the clarified fermentation broth is 81.6 g / L.

[0082] (2) Take the clarified fermentation broth and filter it with an 800 Dal nanofiltration membrane to remove macromolecular pigments, and obtain the decolorized clarified fermentation broth.

[0083] (3) The decolorized and clarified fermentation broth was prepared at 2.5 × 10⁻⁶. -2 The pH of the clarified fermentation broth was adjusted to 3.0 by adding 98% sulfuric acid to the VVM to obtain an acidified broth.

[0084] (4) Take the acidified solution, cool it to 4°C and let it stand for 1 hour. Filter it with a microporous membrane with a pore size of 0.45 μm to remove the mother liquor and obtain crude viscous solid.

[0085] (5) Take mother liquor 1, add 1 mol / L sodium hydroxide solution, adjust the pH of mother liquor 1 to 8.0, evaporate and concentrate it 20 times using a rotary vacuum evaporator, filter, and repeat steps (3) and (4).

[0086] (6) Take crude mucocomic acid solid, add deionized water to redissolve to 900 mL (mucocomic acid concentration is about 90 g / L), add 2 mol / L sodium hydroxide solution, adjust the pH value to 7.0, and obtain crude mucocomic acid solution.

[0087] (7) Take the crude mucoaconic acid solution, add 0.004 (g / mL) of activated carbon, stir and mix at 37℃ and 200 rpm for 75 min, and then filter with a microporous membrane with a pore size of 0.45 μm to obtain a decolorized mucoaconic acid solution.

[0088] The activated carbon was activated with 20% nitric acid solution; the volume ratio of 20% nitric acid solution to activated carbon was 3:1; the activation time was 1 hour; and the activation temperature was 40℃.

[0089] (8) Take the decolorized mucoconic acid solution and dilute it with 2.5 × 10⁻⁶ ppm. -2The vvm was fed with 98% sulfuric acid to adjust the pH of the decolorized mucoacin solution to 3.0. The solution was allowed to stand at 10°C for 1.5 hours. The mother liquor was removed by filtration through a microporous membrane with a pore size of 0.45 μm and dried at 40°C to obtain mucoacin.

[0090] (9) Take mother liquor 2, add 1 mol / L sodium hydroxide solution, adjust the pH value of mother liquor 2 to 8.0, evaporate and concentrate it 20 times using a rotary vacuum evaporator, filter, and repeat steps (7) and (8) to obtain solid mucoconic acid.

[0091] The final yield was 75.54g of mucoconic acid, with a yield of 92.57%.

[0092] Mucoconic acid was dissolved in deionized water and detected by liquid chromatography. The results are shown in the figure. Figure 6 The results showed that the mucoconic acid concentration was 99.1%.

[0093] Example 3:

[0094] (1) Take the fermentation broth containing mucoconic acid, centrifuge it at 8000 rpm to remove the cells, collect 1 L of clear fermentation broth, and the concentration of mucoconic acid in the clear fermentation broth is 81.6 g / L.

[0095] (2) Take the clarified fermentation broth and filter it with an 800 Dal nanofiltration membrane to remove macromolecular pigments, and obtain the decolorized clarified fermentation broth.

[0096] (3) Take the clarified fermentation broth and add 0.5×10 -2 The vvm was fed with a 1 mol / L hydrochloric acid solution to adjust the pH of the clarified fermentation broth to 1.5, thus obtaining an acidified broth.

[0097] (4) Take the acidified solution, cool it to 4°C and let it stand for 0.5 h. Filter it with a microporous membrane with a pore size of 0.45 μm to remove the mother liquor and obtain crude viscous solid.

[0098] (5) Take the mother liquor, add 1 mol / L sodium hydroxide solution, adjust the pH of the mother liquor to 7.0, evaporate and concentrate it 15 times using a rotary vacuum evaporator, filter it, and repeat steps (3) and (4).

[0099] (6) Take crude mucocomic acid solid, redissolve it in deionized water to 850 mL (mucocomic acid concentration is 95 g / L), add 1 mol / L sodium hydroxide solution, adjust the pH value to 6.0, and obtain crude mucocomic acid solution.

[0100] (7) Take the crude mucoaconic acid solution, add 0.005 (g / mL) of activated carbon, stir and mix at 40℃ and 200 rpm for 60 min, and then filter with a microporous membrane with a pore size of 0.45 μm to obtain a decolorized mucoaconic acid solution.

[0101] The activated carbon was activated with 10% nitric acid solution; the volume ratio of 10% nitric acid solution to activated carbon was 5:1; the activation time was 2 hours and the activation temperature was 60℃.

[0102] (8) Take the decolorized mucoconic acid solution and dilute it with 0.5 × 10⁻⁶ ppm. -2 The vvm was fed with 6 mol / L hydrochloric acid to adjust the pH of the decolorized mucoacin solution to 1.5. The solution was allowed to stand at 4°C for 1.5 h, and the mother liquor was removed by filtration through a microporous membrane with a pore size of 0.45 μm. The solution was then dried at 60°C to obtain mucoacin.

[0103] (9) Take the mother liquor, add 1 mol / L sodium hydroxide solution, adjust the pH of the mother liquor to 7.0, evaporate and concentrate it 15 times using a rotary vacuum evaporator, filter, and repeat steps (7) and (8) to obtain solid mucoconic acid.

[0104] The final yield was 74.56g of mucoconic acid, with a yield of 91.37%.

[0105] Mucoconic acid was dissolved in deionized water and detected by liquid chromatography. The results are shown in the figure. Figure 7 The results showed that the mucoconic acid concentration was 99.3%.

[0106] Example 4:

[0107] (1) Take the fermentation broth containing mucoconic acid, centrifuge it at 6000 rpm to remove the cells, collect 1 L of clear fermentation broth, and the concentration of mucoconic acid in the clear fermentation broth is 81.6 g / L.

[0108] (2) Take the clarified fermentation broth and filter it with an 800 Dal nanofiltration membrane to remove macromolecular pigments, and obtain the decolorized clarified fermentation broth.

[0109] (3) Take the clarified fermentation broth and mix it with 1.0×10 -2 The flow rate of the VVM was increased by adding an 80% sulfuric acid solution to adjust the pH of the clarified fermentation broth to 1.0, thus obtaining an acidified broth.

[0110] (4) Take the acidified solution, cool it to 0℃ and let it stand for 2 hours. Filter it with a microporous membrane with a pore size of 0.45μm to remove the mother liquor and obtain crude viscous solid.

[0111] (5) Take mother liquor 1, add 1 mol / L sodium hydroxide solution, adjust the pH of mother liquor 1 to 7.0, use a rotary vacuum evaporator to evaporate and concentrate it 10 times, filter it, and repeat steps (3) and (4).

[0112] (6) Take crude mucocomic acid solid, redissolve it in deionized water to 850 mL (mucocomic acid concentration is about 90 g / L), add 2 mol / L sodium hydroxide solution, adjust the pH value to 7.0, and obtain crude mucocomic acid solution.

[0113] (7) Take the crude mucoaconic acid solution, add 0.005 (g / mL) of activated carbon, stir and mix at 37℃ and 200 rpm for 90 min, and then filter with a microporous membrane with a pore size of 0.45 μm to obtain a decolorized mucoaconic acid solution.

[0114] The activated carbon was activated with 20% nitric acid solution; the volume ratio of 20% nitric acid solution to activated carbon was 3:1; the activation time was 12 hours and the activation temperature was 40℃.

[0115] (8) Take the decolorized mucoconic acid solution and dilute it with 1.0 × 10⁻⁶ ppm. -2 The vvm was fed with an 80% sulfuric acid solution to adjust the pH of the decolorized mucoacinic acid solution to 1.0. The solution was allowed to stand at 0°C for 2 hours, and the mother liquor was removed by filtration through a microporous membrane with a pore size of 0.45 μm. The solution was then dried at 40°C to obtain mucoacinic acid.

[0116] (9) Take mother liquor 2, add 1 mol / L sodium hydroxide solution, adjust the pH value of mother liquor 2 to 7.0, evaporate and concentrate it 10 times using a rotary vacuum evaporator, filter, and repeat steps (7) and (8) to obtain solid mucoconic acid.

[0117] The final yield was 73.68g of mucoconic acid, with a yield of 90.29%.

[0118] Mucoconic acid was dissolved in deionized water and detected by liquid chromatography. The results are shown in the figure. Figure 8 The results showed that the mucoconic acid concentration was 99.6%.

[0119] This invention is combined with traditional processes ( Figure 2 ) and Derek R process ( Figure 3 The important parameters of the separation were compared, and the results are shown in Table 1.

[0120] Table 1. Comparison of key separation parameters between the Derek R process and the process of this invention.

[0121] Recovery rate 66.3% More than 90% 81.4% 88.43% Activated carbon dosage 100g / L 3~5g / L 5g / L 3~5g / L purity 96.3% More than 99% 99.8% 99.8% organic solvents none none ethanol none

[0122] Note: For traditional processes, please refer to the following literature: Guokun W, Aline T, Simone S, et al. An integrated yeast-based process for cis,cis-muconic acid production. [J]. Biotechnology and bioengineering, 2021, 119(2): 376-387.

[0123] For the Derek R process, see the following reference: Vardon, R. D, Rorrer, et al. cis,cis-Muconicacid: separation and catalysis to bio-adipic acid for nylon-6,6polymerization[J]. Green chemistry, 2016,18(11):3397-3413.

[0124] It can be seen that the traditional process includes a mucoaconic acid recovery step, corresponding to Process II of this invention (Examples 2, 3, and 4) in the table. In the traditional process, the yield before mucoaconic acid recovery is only 50%, and it reaches 66.3% after recovering mucoaconic acid from the filtrate. However, in this process (Examples 2, 3, and 4), the recovery rate after recovering mucoaconic acid from the filtrate is much higher than that of the traditional process, and the amount of activated carbon used is reduced by 20 times. At the same time, the purity is also higher than that of the traditional process.

[0125] The Derek R process does not include a step for recovering mucoconic acid from the filtrate, corresponding to Process I of this invention (Example 1, which does not recover mucoconic acid from the filtrate) in the table. While the Derek R process uses an ethanol recrystallization step, Process I of this invention (Example 1) achieves a higher recovery rate and the same purity without using any organic solvents.

[0126] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A method for separating and extracting mucoconic acid from a fermentation broth, comprising: Step A: Separate and process the fermentation broth containing mucoconic acid to remove the bacterial cells and obtain a clear fermentation broth; Step B: Remove the protein pigments from the clarified fermentation broth to obtain the decolorized clarified fermentation broth; Step C: Adjust the pH of the decolorized and clarified fermentation broth to acidic to obtain an acidified broth; Step D: Cool the acidified solution and let it stand to precipitate. Filter to remove mother liquor 1 and obtain crude viscous solid. Step F: After redissolving the crude mucocomic acid solid, adjust the pH value to obtain a crude mucocomic acid solution; Step G: Add activated carbon to the crude mucoaconic acid solution for decolorization, stir and mix well, then filter to obtain a decolorized mucoaconic acid solution. Step H: Adjust the pH of the decolorized mucoacin solution to acidic, allow it to stand and precipitate, filter to remove mother liquor 2, and dry to obtain pure mucoacin.

2. The method of claim 1, wherein, In step B, nanofiltration is used to remove macromolecular protein pigments. Preferably, the nanofiltration membrane is 800-1000 Da, more preferably an organic membrane of 800 Da, and the operating pressure is 3-4 MPa.

3. The method of claim 1, wherein, In steps C and H, the pH is adjusted to 1.0-3.0 using an acid solution, which is an aqueous solution of hydrochloric acid at 1-6 mol / L or an aqueous solution of sulfuric acid at a mass fraction of 50%-98%; preferably, the dropping speed of the acid solution is 0.5-2.5 x 10 -2 vvm.

4. The method of claim 1, wherein, In step A, the separation process includes filtration and / or centrifugation; preferably, a ceramic membrane with a pore size of 100 nm is used for filtration, and the operating pressure is 0.2 MPa; and / or, the centrifugation rate is 6000–8000 rpm.

5. The method of claim 1, wherein, In step D, the temperature is lowered to 0–10°C, and the settling time is 0.5–2 h; and / or, filtration is performed using a microporous membrane with a pore size of 0.45 μm.

6. The method of claim 1, wherein, In step F, the crude mucoaconic acid solid is reconstituted with water to a mucoaconic acid concentration of 80–110 g / L; and / or, the pH is adjusted to 6.0–8.

0.

7. The method according to claim 1, characterized in that, In step G, the amount of activated carbon used is 0.003–0.005 g / mL, based on the crude viscosic acid solution. And / or, the mixing temperature is 30-40°C, and the mixing time is 60-90 min; And / or, use a microporous membrane with a pore size of 0.45 μm for filtration.

8. The method according to claim 1, characterized in that, In step H, a microporous membrane with a pore size of 0.45 μm is used for filtration; And / or, the settling temperature is 0–10°C, and the settling time is 0.5–2 hours; And / or, the drying temperature is 30–60°C.

9. The method according to any one of claims 1 to 8, characterized in that, The method further includes step E, adjusting the pH of mother liquor 1 to neutral, concentrating it by rotary evaporation 10 to 20 times, filtering, and repeating steps C and D to obtain crude viscous solid.

10. The method according to any one of claims 1-9, characterized in that, The method further includes step I, adjusting the pH of mother liquor 2 to neutral, concentrating it by rotary evaporation 10 to 20 times, filtering, and repeating steps G and H to obtain pure mucoconic acid.

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