Separation and purification method for removing sulfate in gamma-aminobutyric acid fermentation liquor

Abstract

The invention provides a separation and purification method for removing sulfate in gamma-aminobutyric acid fermentation broth, and belongs to the technical field of separation and purification, the separation and purification method for removing sulfate in gamma-aminobutyric acid fermentation broth comprises the following steps: providing gamma-aminobutyric acid fermentation broth; performing solid-liquid separation and first concentration on the gamma-aminobutyric acid fermentation liquor to obtain first concentrated liquor; performing chromatographic separation on the first concentrated solution to obtain a separated solution; purifying the separated liquid through anion exchange resin to obtain a purified liquid; and carrying out secondary concentration and drying on the purified liquid to obtain a gamma-aminobutyric acid product. According to the separation and purification method for removing the sulfate in the gamma-aminobutyric acid fermentation liquor, through chromatographic separation and anion exchange resin purification, sulfate residues in the gamma-aminobutyric acid product can be reduced, the purity of the gamma-aminobutyric acid product can be improved, the separation and purification process is simple, and the method is suitable for industrial production. The method is suitable for industrial continuous batch production and application.

Description

Technical Field

[0001] The present application relates to the technical field of separation and purification, and in particular to a separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth. Background Art

[0002] γ-Aminobutyric acid (GABA), also known as 4-aminobutyric acid, has a relative molecular mass of 103.12 and a chemical formula of C₄H₄NO₂. As an amino acid, it is widely found in microorganisms, plants, and vertebrates. GABA is an important inhibitory neurotransmitter in the central nervous system, with good water solubility and thermal stability, and possesses a variety of physiological regulatory functions. As a small, non-protein amino acid, GABA has been shown to be safe for consumption and can be used in the production of beverages and other foods.

[0003] The microbial method for producing γ-aminobutyric acid has the advantages of mild reaction conditions, no pollution, and high yield. It is widely used in the production of γ-aminobutyric acid. The microbial strains used to synthesize GABA mainly include lactic acid bacteria, Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, etc. However, since the amino acid fermentation broth is an extremely complex multiphase system containing microbial cells, metabolites, and unexhausted culture medium, it causes difficulties in downstream separation and purification. In the related art, common separation and purification methods are centrifugation or membrane filtration or plate and frame filtration for solid-liquid separation to remove microbial cells and impurities, electrodialysis, cross-linking, activated carbon, etc. for desalting, decolorization and impurity removal, adding anti-solvents such as methanol, ethanol, etc. or evaporation, concentration and cooling for crystallization to obtain GABA finished products. However, the process flow of these methods is relatively complicated and easily leads to sulfate residues, affecting product quality. Summary of the Invention

[0004] The embodiments of the present application provide a separation and purification method for removing sulfate from a γ-aminobutyric acid fermentation broth, which can reduce sulfate residue and simplify the separation and purification process.

[0005] The embodiments of the present application provide a separation and purification method for removing sulfate from a γ-aminobutyric acid fermentation broth, comprising: Providing γ-aminobutyric acid fermentation broth; The γ-aminobutyric acid fermentation liquid is subjected to solid-liquid separation and a first concentration to obtain a first concentrated liquid; subjecting the first concentrated liquid to chromatographic separation to obtain a separated liquid; Purifying the separated liquid through an anion exchange resin to obtain a purified liquid; The purified solution is concentrated and dried for the second time to obtain the γ-aminobutyric acid product.

[0006] Optionally, the first concentrate is subjected to chromatographic separation, comprising: The first concentrated solution is loaded onto a chromatographic separation column, and then eluted with 1-3 times the volume of the chromatographic separation column of pure water, and the eluate with a conductivity lower than 1.0 mS / cm is collected; Each time the sample is loaded, the ratio of the volume of the first concentrated liquid to the volume in the chromatographic separation column is 10%-20%.

[0007] Optionally, a first filler is provided in the chromatographic separation column, and the first filler includes at least one of LX1850NH filler and LX1880NH filler.

[0008] Optionally, the conductivity of the purified liquid is 10 μS / cm-50 μS / cm.

[0009] Optionally, the separated solution is purified by anion exchange resin, comprising: The separated liquid is passed through an anion exchange resin column, the first permeate is collected, and then eluted with pure water 1-3 times the volume of the anion exchange resin column to obtain a first eluate, and the first permeate and the first eluate are combined.

[0010] Optionally, a second filler is provided in the anion exchange resin column, and the second filler includes at least one of a D315 filler and a D311 filler.

[0011] Optionally, the γ-aminobutyric acid fermentation broth includes Corynebacterium glutamicum fermentation broth.

[0012] Optionally, the γ-aminobutyric acid fermentation broth is subjected to solid-liquid separation and a first concentration, comprising: The γ-aminobutyric acid fermentation broth is filtered using a ceramic membrane, the second permeate is collected, and then the concentrated phase is top-washed with pure water, the water wash is collected, and the second permeate and the water wash are combined to obtain a filtrate; The filtrate is concentrated to a transmittance of 50%-70% to obtain a first concentrated solution.

[0013] Optionally, the pore size of the ceramic membrane is 50 nm, 100 nm or 200 nm.

[0014] Optionally, the purified solution is subjected to a second concentration and drying step, comprising: Concentrating the purified solution to a light-shielding rate of 10%-25% to obtain a second concentrated solution; The second concentrated liquid is spray-dried to obtain a γ-aminobutyric acid product.

[0015] Optionally, the second concentrate is spray-dried, comprising: The second concentrated liquid is dried using a spray dryer, wherein the inlet air temperature of the spray dryer is set to 150°C-180°C, the outlet air temperature is set to 100°C-110°C, the feed speed is set to 25rpm-40rpm, and the fan frequency is set to 30Hz-50Hz.

[0016] Beneficial effects of the embodiments of the present application: The separation and purification method for removing sulfate from a γ-aminobutyric acid (GABA) fermentation broth provided in an embodiment of the present application comprises: providing a GABA fermentation broth; subjecting the GABA fermentation broth to solid-liquid separation and a first concentration to obtain a first concentrated liquid; subjecting the first concentrated liquid to chromatographic separation to obtain a separated liquid; purifying the separated liquid through an anion exchange resin to obtain a purified liquid; and subjecting the purified liquid to a second concentration and drying to obtain a GABA product. Pure water is used as the elution solvent in both the chromatographic separation and anion exchange resin purification processes. Impurities in the fermentation broth can be removed by solid-liquid separation. After concentration, the broth is subjected to chromatographic separation for decolorization and impurity removal. The conductivity is then reduced through an anion exchange resin. After further concentration and drying, the GABA product can be obtained. The separation and purification method for removing sulfate from a GABA fermentation broth provided in an embodiment of the present application, through chromatographic separation and anion exchange resin purification, can reduce residual sulfate in the GABA product and improve the purity of the GABA product. The separation and purification process is simple and suitable for industrial continuous batch production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 This is the detection spectrum of the γ-aminobutyric acid product in Example 1 of the present application; Figure 2 This is a comparison chart of the sulfate content in the γ-aminobutyric acid products prepared in Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of this application. In addition, it should be understood that the specific embodiments described herein are only used to illustrate and explain the present application and are not used to limit the present application.

[0020] γ-Aminobutyric acid (GABA) is found in relatively low concentrations in naturally occurring food-derived plants and animals. Large-scale GABA extraction and production primarily rely on chemical synthesis and microbial fermentation. There are two main chemical methods for preparing GABA: one involves ring-opening hydrolysis of 2-pyrrolidone with sodium hydroxide or calcium hydroxide to produce 4-aminobutyric acid sodium salt. This is then treated with ammonium bicarbonate for precipitation or sodium ion exchange with ion exchange resins before further concentration and crystallization to produce GABA. The other involves reacting γ-butyrolactone with thionyl chloride, followed by ring-opening and chlorination, to produce 4-chlorobutyryl chloride. This esterification yields methyl 4-chlorobutyrate, which, under the influence of a catalyst, undergoes amination and hydrolysis with ammonia to produce GABA. Both preparation methods involve numerous hazardous chemicals, including organic reagents that are potentially harmful to the human body and are expensive.

[0021] The microbial method for producing γ-aminobutyric acid has the advantages of mild reaction conditions, no pollution, and high yield. It is widely used in the production of γ-aminobutyric acid. The microbial strains used to synthesize GABA mainly include lactic acid bacteria, Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, etc. However, since the amino acid fermentation broth is an extremely complex multiphase system containing microbial cells, metabolites, and unexhausted culture medium, it poses difficulties for downstream separation and purification. Common separation and purification methods include centrifugation, membrane filtration, or plate and frame filtration for solid-liquid separation to remove microbial cells and impurities, electrodialysis, cross-linking, activated carbon, etc. for desalting, decolorization, and impurity removal, and adding antisolvents such as methanol and ethanol or evaporation, concentration, and cooling for crystallization to obtain the finished GABA product.

[0022] Among them, electrodialysis has high requirements for the clarity of the fermentation broth, and its ability to reduce conductivity is limited, and the energy consumption is high. The use of activated carbon for decolorization will make the regeneration and recovery of activated carbon more difficult, resulting in higher consumables costs. In the crystallization process, organic reagents are used for dissolution crystallization. On the one hand, the recycling of organic solvents is difficult and there is a certain loss. On the other hand, there will be 10%-20% γ-aminobutyric acid residues in the crystallization mother liquor, which affects the recovery rate. In addition, in the process of preparing γ-aminobutyric acid fermentation broth through fermentation technology, sulfate ions will be introduced into the culture medium or feeding process, but the relevant technology does not test the sulfate residues in the γ-aminobutyric acid product. If the γ-aminobutyric acid product with sulfate residues is used in food additives or health products, it is easy to cause gastrointestinal disorders and diarrhea.

[0023] The embodiments of the present application provide a separation and purification method for removing sulfate from a γ-aminobutyric acid fermentation broth, comprising: Providing γ-aminobutyric acid fermentation broth; The γ-aminobutyric acid fermentation liquid is subjected to solid-liquid separation and a first concentration to obtain a first concentrated liquid; subjecting the first concentrated liquid to chromatographic separation to obtain a separated liquid; Purifying the separated liquid through an anion exchange resin to obtain a purified liquid; The purified solution is concentrated and dried for the second time to obtain the γ-aminobutyric acid product.

[0024] The separation and purification method for removing sulfate from a γ-aminobutyric acid fermentation broth provided in the embodiments of the present application can remove impurities from the fermentation broth by solid-liquid separation, and after concentration, decolorize and remove impurities by chromatographic separation, and then reduce the conductivity by an anion exchange resin. After reconcentration and drying, a γ-aminobutyric acid product can be obtained. In the separation and purification method for removing sulfate from a γ-aminobutyric acid fermentation broth provided in the embodiments of the present application, chromatographic separation and anion exchange resin purification can reduce the residual sulfate in the γ-aminobutyric acid product and improve the purity of the γ-aminobutyric acid product. The separation and purification process is simple and suitable for industrial continuous batch production applications.

[0025] Specifically, the separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth provided in the embodiment of the present application can produce a γ-aminobutyric acid product with higher purity, the γ-aminobutyric acid content can reach more than 99%, and the yield of γ-aminobutyric acid can reach more than 90%. The residual sulfate in the γ-aminobutyric acid product can be reduced by chromatographic separation and anion exchange resin purification, so that the mass percentage of sulfate ions in the γ-aminobutyric acid product is less than 0.03%.

[0026] In some embodiments, subjecting the first concentrate to chromatographic separation comprises: The first concentrated solution is loaded onto a chromatographic separation column, and then eluted with 1-3 times the volume of the chromatographic separation column of pure water, and the eluate with a conductivity of less than 1.0 mS / cm is collected; Each time the sample is loaded, the ratio of the volume of the first concentrated liquid to the volume in the chromatographic separation column is 10%-20%.

[0027] Chromatographic separation is a highly efficient and rapid method of separating substances based on differences in the distribution coefficients between the stationary and mobile phases. During the chromatographic separation process, precise separation and purification of GABA can be achieved by controlling the sample load and elution conditions.

[0028] In this embodiment, during the chromatographic separation process, the volume ratio of the first concentrated liquid to the filler in the chromatographic separation column is 10%-20% for each sample loading. This can avoid overloading the filler in the chromatographic separation column, reduce the risk of clogging the chromatographic separation column, and help extend the service life of the chromatographic separation column. Using 1-3 times the volume of the chromatographic separation column for elution with pure water can ensure that the γ-aminobutyric acid in the chromatographic separation column is fully eluted, improve the purity of the γ-aminobutyric acid in the separated liquid, and reduce the process difficulty and cost of subsequent treatments such as concentration.

[0029] For example, each time the sample is loaded, the volume ratio of the first concentrate to the filler in the chromatographic separation column can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. During elution, the volume ratio of the purified water used to the chromatographic separation column can be 1, 1.5, 2, 2.5, or 3. The eluate having a conductivity lower than 1.0 mS / cm, 0.9 mS / cm, 0.8 mS / cm, 0.7 mS / cm, 0.6 mS / cm, 0.5 mS / cm, 0.4 mS / cm, 0.3 mS / cm, 0.2 mS / cm, or 0.1 mS / cm is collected.

[0030] In some embodiments, a first filler is provided in the chromatographic separation column, and the first filler includes at least one of LX1850NH filler and LX1880NH filler.

[0031] LX1850NH and LX1880NH packings have specific pore sizes and surface properties that facilitate efficient distribution of GABA between the stationary and mobile phases. They also exhibit strong selectivity for GABA, minimizing interference from other impurities, thereby achieving efficient separation and improving purity. Furthermore, both LX1850NH and LX1880NH packings possess high chemical stability and mechanical strength, extending the life of chromatographic columns and reducing costs.

[0032] In some embodiments, the conductivity of the purified solution is 10 μS / cm-50 μS / cm.

[0033] Anion exchange resin is a resin material with specific ion exchange properties. When the separated liquid is purified by anion exchange resin, the anion exchange resin selectively adsorbs anions in the separated liquid, thereby separating GABA from other impurities. After purification by anion exchange resin, the resulting purified liquid has a conductivity of 10μS / cm-50μS / cm, further reducing the ion content, improving the purity of GABA in the purified liquid, and minimizing interference from other impurities, while also reducing the difficulty and cost of subsequent processing.

[0034] In some embodiments, the separation solution is purified by anion exchange resin, comprising: The separated liquid is passed through an anion exchange resin column, the first permeate is collected, and then eluted with pure water 1-3 times the volume of the anion exchange resin column to obtain a first eluate, and the first permeate and the first eluate are combined.

[0035] When the separated liquid passes through the anion exchange resin column, the anion exchange resin selectively adsorbs anions, inorganic salts, small organic molecules, and other components in the separated liquid, allowing the majority of the GABA to permeate with the separated liquid, yielding the first permeate. However, the small amount of GABA remaining in the anion exchange resin column, due to its weak adsorption to the anion exchange resin, can be eluted with 1-3 times the volume of the anion exchange resin column in pure water, yielding the first eluate. By combining the first permeate and the first eluate, the concentration of GABA can be increased and its loss reduced.

[0036] By using pure water as the elution solvent in both the chromatographic separation and the anion exchange resin purification process, the use of reagents such as hydrochloric acid and ethanol can be reduced, and the cost of consumables can be reduced.

[0037] In some embodiments, a second filler is disposed in the anion exchange resin column, and the second filler includes at least one of a D315 filler and a D311 filler.

[0038] Among them, D315 filler and D311 filler have a high adsorption capacity for anions, can effectively adsorb anions and other impurities in the separation liquid, and improve the purification efficiency. In addition, both D315 filler and D311 filler have high selectivity, which helps to improve the purity of γ-aminobutyric acid in the purified liquid.

[0039] In certain embodiments, the gamma-aminobutyric acid fermentation liquid comprises a Corynebacterium glutamicum fermentation liquid. Corynebacterium glutamicum itself lacks the glutamate decarboxylase system. Through genetic modification, Corynebacterium glutamicum can obtain the ability of converting glutamate into gamma-aminobutyric acid. Corynebacterium glutamicum is a safe strain that is guaranteed in terms of the safety of fermentation-producing gamma-aminobutyric acid. Corynebacterium glutamicum has significant advantages in synthesizing glutamate, and glutamate is the main prerequisite for gamma-aminobutyric acid. It can efficiently utilize substrates to synthesize glutamate, and then convert it into gamma-aminobutyric acid. In addition, Corynebacterium glutamicum has strong fermentation process adaptability and can improve the output of gamma-aminobutyric acid by optimizing fermentation conditions. The fermentation process of Corynebacterium glutamicum is relatively stable, which is conducive to realizing industrialized production.

[0040] For example, the Corynebacterium glutamicum can be selected from Corynebacterium glutamicum FF10 independently developed by our company (Senruisi Biotechnology Co., Ltd.). For materials related to Corynebacterium glutamicum FF10, reference can be made to the patent publication number CN114752544A.

[0041] In some embodiments, the γ-aminobutyric acid fermentation broth is subjected to solid-liquid separation and a first concentration, comprising: The γ-aminobutyric acid fermentation broth is filtered using a ceramic membrane, the second permeate is collected, and then the concentrated phase is top-washed with pure water, the water wash is collected, and the second permeate and the water wash are combined to obtain a filtrate; The filtrate is concentrated to a transmittance of 50%-70% to obtain a first concentrated solution.

[0042] The ceramic membrane's fine pore structure effectively separates small molecular impurities, soluble proteins, colloids, polysaccharides, and small insoluble particles from the GABA fermentation broth. The filtrate's high transmittance reduces interference from impurities, facilitates subsequent processes, and improves production efficiency. The ceramic membrane's separation and filtration process can be performed at room temperature, eliminating the need for high-temperature heating or chemical additives, thereby minimizing loss of active ingredients and saving energy. Topwashing the concentrated phase with pure water removes small amounts of GABA adsorbed in the ceramic membrane's pores, forming a wash solution and increasing GABA yield. The concentration process removes water and other soluble impurities from the filtrate, improving GABA purity. By controlling the transmittance after concentration between 50% and 70%, the concentration level can be controlled within a certain range. The reduced volume of the concentrated primary concentrate reduces the workload for subsequent processing, and the high GABA concentration facilitates the extraction and purification of subsequent products.

[0043] In some embodiments, the ceramic membrane has a pore size of 50 nm to 200 nm. When the ceramic membrane has a pore size of 50 nm to 200 nm, it can accurately intercept bacteria and other impurities in the γ-aminobutyric acid fermentation broth, thereby ensuring the filtration effect and reducing the impact of impurities.

[0044] Illustratively, the membrane pore size of the ceramic membrane may be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm.

[0045] In some embodiments, the purified solution is subjected to a second concentration and drying step, comprising: Concentrating the purified solution to a light-shielding rate of 10%-25% to obtain a second concentrated solution; The second concentrated liquid is spray-dried to obtain a γ-aminobutyric acid product.

[0046] By concentrating the purified solution and achieving a transmittance of 10%-25% after concentration, water and other soluble impurities can be removed from the purified solution, increasing the concentration and purity of GABA. This facilitates subsequent processes, reduces material loss during subsequent handling, and improves overall production efficiency. Spray drying can disperse the second concentrated solution into tiny droplets through an atomizer, allowing for full contact with hot air for rapid drying. The drying efficiency is high, and the spray drying process is continuous and stable, making it easy to automate and control, shortening the preparation cycle. The GABA product prepared by spray drying, due to its short heating time, can maintain good biological activity, improve the purity of the GABA product, and maintain a uniform morphology.

[0047] In some embodiments, spray drying the second concentrate comprises: The second concentrated liquid is dried using a spray dryer, wherein the inlet air temperature of the spray dryer is set to 150°C-180°C, the outlet air temperature is set to 100°C-110°C, the feed speed is set to 25rpm-40rpm, and the fan frequency is set to 30Hz-50Hz.

[0048] By setting the spray dryer's inlet air temperature within the range of 150°C-180°C, the hot air provides sufficient heat for rapid evaporation and drying of the second concentrated liquid, shortening drying time. Controlling the outlet air temperature between 100°C-110°C helps ensure the proper dryness of the GABA product, while avoiding quality degradation caused by overheating and ensuring the stability and consistency of the GABA product. Appropriate feed speed and fan frequency ensure the continuity and stability of spray drying, ensuring uniform distribution and sufficient drying of the second concentrated liquid within the drying chamber.

[0049] Illustratively, the inlet air temperature of the spray dryer can be set to 150°C, 160°C, 170°C or 180°C, the outlet air temperature can be set to 100°C, 105°C or 110°C, the feed speed can be set to 25rpm, 30rpm, 35rpm or 40rpm, and the fan frequency can be set to 30Hz, 35Hz, 40Hz, 45Hz or 50Hz.

[0050] The following examples are further described in conjunction with specific embodiments. It should be understood that these embodiments are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally based on the conditions recommended by the manufacturer.

[0051] In the examples, the γ-aminobutyric acid fermentation broth was produced using fermentation broth of Corynebacterium glutamicum FF10, which was obtained from Senruisi Biotechnology Co., Ltd. (for information on Corynebacterium glutamicum FF10, please refer to Chinese Patent Publication No. CN114752544B). The fermentation process of Corynebacterium glutamicum FF10 is as follows: Corynebacterium glutamicum FF10 was cultured in BHIS medium at 30°C for 24 hours to obtain seed liquid. 500 mL of fermentation medium was added to a 1 L fermentor, and the seed liquid was inoculated into the fermentor at a 10% inoculation rate. The fermentation temperature was 30°C, the dissolved oxygen was 30%, and ammonia was used to adjust the pH to 7.0±0.5 to produce glutamate. As the fermentation progressed, the pH was controlled to 5.5 after 76 hours.

[0052] The BHIS medium formula is: 0.74g BHI (brain heart infusion broth) and 3.64g sorbitol dissolved in 40ml of water and filtered. The fermentation medium formula is as follows: 100g / L glucose, 12g / L ammonium sulfate, 0.87g / L magnesium sulfate, 3ml / L corn steep liquor, 0.4ml / L phosphoric acid, 0.53g / L potassium chloride, 120mg / L ferrous sulfate, 120mg / L manganese sulfate, 42mg / L nicotinamide, 6.3mg / L calcium pantothenate, 6.3mg / L vitamin B1, and 0.5mg / L biotin.

[0053] Example 1 (1) After the fermentation liquid of Corynebacterium glutamicum FF10 is inactivated, 10 L of the fermentation liquid is taken and filtered through a ceramic membrane with a pore size of 50 nm, and top washed with pure water. The permeate and the washing liquid are collected and concentrated by vacuum distillation. The refractive index of the concentrate is 57%, and the first concentrate is obtained; (2) The first concentrated liquid was loaded on a simulated moving bed, the chromatographic resin filler was LX1850NH, a single loading amount was 15% of the volume of the chromatographic separation column, 2.2 volumes of the chromatographic separation column were rinsed with pure water, and the eluate with a conductivity lower than 400 μS / cm was collected to obtain a separated liquid; (3) The sample load was calculated based on 135 g of γ-aminobutyric acid per liter of anion exchange resin D315 for conductivity reduction treatment. After washing with water for 2 volumes of the anion exchange resin column, the sample effluent and the water wash effluent were combined to obtain a purified solution with a conductivity of 17 μS / cm; (4) Concentrating the purified solution to a γ-aminobutyric acid concentration of approximately 130 g / L to obtain a second concentrated solution; (5) The second concentrated liquid was spray-dried. The spray-drying parameters were: inlet air temperature 180°C, outlet air temperature 100°C, feed speed 25 rpm, and fan frequency 35 Hz. The γ-aminobutyric acid product was collected.

[0054] Example 2 (1) After the fermentation liquid of Corynebacterium glutamicum FF10 is inactivated, 10 L of the fermentation liquid is taken and filtered through a ceramic membrane with a pore size of 50 nm, and top washed with pure water. The permeate and the washing liquid are collected and concentrated by vacuum distillation. The refractive index of the concentrate is 54%, and the first concentrate is obtained; (2) The first concentrated liquid was loaded on a simulated moving bed, the chromatographic resin filler was LX1850NH, a single loading amount was 14% of the volume of the chromatographic separation column, two volumes of the chromatographic separation column were rinsed with pure water, and the eluate with a conductivity lower than 350 μS / cm was collected to obtain a separated liquid; (3) The sample load was calculated based on 140 g of γ-aminobutyric acid per liter of anion exchange resin D315 for conductivity reduction treatment. After washing with 2 volumes of the anion exchange resin column, the sample effluent and the water wash effluent were combined to obtain a purified solution with a conductivity of 13 μS / cm; (4) Concentrating the purified solution to a γ-aminobutyric acid concentration of approximately 150 g / L to obtain a second concentrated solution; (5) The second concentrated liquid was spray-dried with the following spray drying parameters: inlet air temperature 175°C, outlet air temperature 105°C, feed speed 25 rpm, fan frequency 30 Hz, and γ-aminobutyric acid product was collected.

[0055] Example 3 (1) After the fermentation liquid of Corynebacterium glutamicum FF10 is inactivated, 10 L of the fermentation liquid is taken and filtered through a ceramic membrane with a pore size of 50 nm, and top washed with pure water. The permeate and the washing liquid are collected and concentrated by vacuum distillation. The refractive index of the concentrated liquid is 70%, and the first concentrated liquid is obtained; (2) The first concentrated liquid was loaded on a simulated moving bed, the chromatographic resin filler was LX1880NH, the single loading amount was 10% of the volume of the chromatographic separation column, pure water was rinsed for 1 volume of the chromatographic separation column, and the eluate with a conductivity lower than 1.0 mS / cm was collected to obtain a separated liquid; (3) The sample load was calculated based on 200 g of γ-aminobutyric acid per liter of anion exchange resin D311 for conductivity reduction treatment. After washing with water for 3 volumes of the anion exchange resin column, the sample effluent and the water washing effluent were combined to obtain a purified solution with a conductivity of 10 μS / cm; (4) Concentrating the purified solution to a γ-aminobutyric acid concentration of approximately 150 g / L to obtain a second concentrated solution; (5) The second concentrated liquid was spray-dried. The spray-drying parameters were: inlet air temperature 150°C, outlet air temperature 100°C, feed speed 40 rpm, and fan frequency 50 Hz. The γ-aminobutyric acid product was collected.

[0056] Example 4 (1) After the fermentation liquid of Corynebacterium glutamicum FF10 is inactivated, 10 L of the fermentation liquid is taken and filtered through a ceramic membrane with a pore size of 50 nm, and top washed with pure water. The permeate and the washing liquid are collected and concentrated by vacuum distillation. The refractive index of the concentrated liquid is 50%, and the first concentrated liquid is obtained; (2) The first concentrated liquid was loaded on a simulated moving bed, the chromatographic resin filler was LX1880NH, the single loading amount was 20% of the volume of the chromatographic separation column, three volumes of the chromatographic separation column were rinsed with pure water, and the eluent with a conductivity lower than 0.1 mS / cm was collected to obtain a separated liquid; (3) The sample load was calculated based on 100 g of γ-aminobutyric acid per liter of anion exchange resin D311 for conductivity reduction treatment. After washing with one volume of the anion exchange resin column, the sample effluent and the water washing effluent were combined to obtain a purified solution with a conductivity of 50 μS / cm; (4) Concentrating the purified solution to a γ-aminobutyric acid concentration of approximately 150 g / L to obtain a second concentrated solution; (5) The second concentrated liquid was spray dried with the following spray drying parameters: inlet air temperature 180°C, outlet air temperature 110°C, feed speed 25 rpm, fan frequency 30 Hz, and the γ-aminobutyric acid product was collected.

[0057] Comparative Example 1 (1) After the fermentation liquid of Corynebacterium glutamicum FF10 is inactivated, 10 L of the fermentation liquid is taken and filtered through a ceramic membrane with a pore size of 50 nm, and top washed with pure water. The permeate and the washing liquid are collected and concentrated by vacuum distillation. The refractive index of the concentrated liquid is 50%, and the first concentrated liquid is obtained; (2) The first concentrated liquid was loaded on a simulated moving bed, with a single loading of 11% of the volume of the chromatographic separation column, and two volumes of the chromatographic separation column were rinsed with pure water. The eluate with a conductivity lower than 370 μS / cm was collected to obtain a separated liquid; (3) Concentrating the separated liquid to a γ-aminobutyric acid concentration of approximately 110 g / L to obtain a second concentrated liquid; (4) The second concentrated liquid was spray dried with the following spray drying parameters: inlet air temperature 180°C, outlet air temperature 102°C, feed speed 30 rpm, fan frequency 37 Hz, and γ-aminobutyric acid product was collected.

[0058] Comparative Example 2 In this comparative example, the fermentation broth of Corynebacterium glutamicum FF10 was inactivated and then tanked, 10 L of fermentation broth was taken for high-speed centrifugation with a centrifugal force of 9500 x g. The supernatant after centrifugation was heated to 80 ° C and clarified and filtered through a 0.45 μm filter membrane. The collected filtrate was adjusted to pH 5.0 with 2 M acetic acid, and activated carbon powder was added in an amount of 2%. The temperature was raised to 60 ° C and kept warm for 30 min under stirring. After filtering with double-layer filter paper, it was finely filtered with a 0.45 μm filter membrane, and the filtrate was concentrated by vacuum rotary evaporation. The water bath heating temperature was set to 55 ° C and concentrated until a large amount of crystals precipitated, presenting a solid-liquid two-phase mixed state. 95% ethanol was added in a volume of 3 times the weight of the concentrated material. After cooling to 4 ° C, stirring was stopped and kept warm for 12 h. The filtered filter cake was added with 95% ethanol at a ratio of 3 mL / g crystal for pulping and washing, and the temperature was raised to 40 ° C and stirred for 1 h to filter. The filtered wet crystals were dried in an oven at 80° C. for 12 h to a constant weight, and then cooled to obtain the finished product of γ-aminobutyric acid.

[0059] The γ-aminobutyric acid content and sulfate content in the γ-aminobutyric acid products of Examples 1-2 and Comparative Example 1 were tested.

[0060] The test method for the content of γ-aminobutyric acid is determined according to the method in Appendix A of QB / T 5633.7-2022 Amino Acids, Amino Acid Salts and Their Analogues Part 7: γ-Aminobutyric Acid. The method for determining sulfate content (measured as SO4) is: 1. Reagents and Materials 1) Potassium sulfate standard solution: 0.1 mg / mL.

[0061] 2) Hydrochloric acid solution: 10%.

[0062] 3) Barium chloride solution: 1 mol / L.

[0063] 2. Analysis steps 1) Weigh 0.70 g of the sample and dissolve it in water to 40 mL (if the solution is alkaline, add hydrochloric acid to neutralize it; if the solution is not clear, filter it). Place it in a 50 mL Nessler colorimetric tube, add 2 mL of hydrochloric acid solution, and shake well to obtain the sample solution. 2) Take another 2.1 mL of potassium sulfate standard solution and place it in another 50 mL Nessler colorimetric tube. Dilute it to 40 mL with water. Add 2 mL of hydrochloric acid solution and shake well to obtain the standard control solution. 3) Add 5 mL of barium chloride solution to the sample solution and standard control solution respectively, dilute to 50 mL with water, shake well, let stand for 10 minutes, place on a black background, and observe and compare from the top of the colorimetric tube.

[0064] If the turbidity of the sample solution is not higher than that of the standard control solution, the sulfate content is ≤ 0.03%.

[0065] The test results are shown in Table 1. Figure 1-Figure 2 As shown: Table 1 Comparison of test results of γ-aminobutyric acid products in different examples and comparative examples

[0066] in Figure 1 This is the detection spectrum of the γ-aminobutyric acid product in Example 1, Figure 2 This is a comparison chart of the sulfate content in the γ-aminobutyric acid products obtained in Example 1 and Comparative Example 1. Figure 2 If the turbidity of the sample solution is not higher than that of the standard control solution, it means that the sulfate content is ≤0.03%. Figure 1-Figure 2 It can be seen that the γ-aminobutyric acid product prepared in the embodiment of the present application has a higher γ-aminobutyric acid content and higher purity than that of Comparative Examples 1 and 2, and the sulfate content can be controlled below 0.3%, thereby ensuring the quality of the γ-aminobutyric acid product and having a higher yield.

[0067] The above is a detailed introduction to the embodiments of the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of the present application. At the same time, for those skilled in the art, based on the ideas of the present application, there may be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth, characterized in that: include: Providing γ-aminobutyric acid fermentation broth; subjecting the γ-aminobutyric acid fermentation broth to solid-liquid separation and a first concentration to obtain a first concentrated liquid; subjecting the first concentrated liquid to chromatographic separation to obtain a separated liquid; Purifying the separated liquid through an anion exchange resin to obtain a purified liquid; The purified solution is subjected to a second concentration and drying to obtain a γ-aminobutyric acid product.

2. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 1, characterized in that: The step of subjecting the first concentrated liquid to chromatographic separation comprises: Loading the first concentrated solution onto a chromatographic separation column, eluting the solution with 1-3 times the volume of the chromatographic separation column of pure water, and collecting the eluate having a conductivity lower than 1.0 mS / cm; Wherein, each time the sample is loaded, the ratio of the volume of the first concentrated liquid to the volume of the chromatographic separation column is 10%-20%.

3. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 2, characterized in that: The chromatographic separation column is provided with a first filler, which includes at least one of an LX1850NH filler and an LX1880NH filler.

4. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 1, characterized in that: The conductivity of the purified liquid is 10 μS / cm-50 μS / cm.

5. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 4, characterized in that: The step of purifying the separated liquid through an anion exchange resin comprises: The separated liquid is passed through an anion exchange resin column, a first permeate is collected, and then eluted with pure water 1-3 times the volume of the anion exchange resin column to obtain a first eluate, and the first permeate and the first eluate are combined.

6. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 5, characterized in that: The anion exchange resin column is provided with a second filler, and the second filler includes at least one of a D315 filler and a D311 filler.

7. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 1, characterized in that: The γ-aminobutyric acid fermentation broth includes Corynebacterium glutamicum fermentation broth.

8. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 1, characterized in that: The γ-aminobutyric acid fermentation broth is subjected to solid-liquid separation and a first concentration, comprising: The γ-aminobutyric acid fermentation broth is filtered using a ceramic membrane to collect a second permeate, and then the concentrated phase is top-washed with pure water to collect the water wash, and the second permeate and the water wash are combined to obtain a filtrate; The filtrate is concentrated to a transmittance of 50%-70% to obtain a first concentrated solution.

9. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 8, characterized in that: The pore size of the ceramic membrane is 50 nm, 100 nm or 200 nm.

10. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to any one of claims 1 to 9, characterized in that: The second concentration and drying of the purified liquid comprises: Concentrating the purified solution to a light-shielding rate of 10% to 25% to obtain a second concentrated solution; The second concentrated liquid is spray-dried to obtain a γ-aminobutyric acid product.

11. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 10, characterized in that: The spray drying of the second concentrated liquid comprises: The second concentrated liquid is dried using a spray dryer, wherein the inlet air temperature of the spray dryer is set to 150°C-180°C, the outlet air temperature is set to 100°C-110°C, the feed speed is set to 25rpm-40rpm, and the fan frequency is set to 30Hz-50Hz.

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