A separation and purification method for removing sulfate from a gamma-aminobutyric acid fermentation broth

Abstract

The application provides a separation and purification method for removing sulfate in a gamma-aminobutyric acid fermentation liquor, and belongs to the technical field of separation and purification. The separation and purification method for removing sulfate in the gamma-aminobutyric acid fermentation liquor comprises the following steps: providing a gamma-aminobutyric acid fermentation liquor; subjecting the gamma-aminobutyric acid fermentation liquor to solid-liquid separation and first concentration to obtain a first concentrated liquor; subjecting the first concentrated liquor to chromatographic separation to obtain a separated liquor; subjecting the separated liquor to anion exchange resin purification to obtain a purified liquor; and subjecting the purified liquor to second concentration and drying to obtain a gamma-aminobutyric acid product. According to the separation and purification method for removing sulfate in the gamma-aminobutyric acid fermentation liquor provided in the application, chromatographic separation and anion exchange resin purification can reduce the residual sulfate in the gamma-aminobutyric acid product, improve the purity of the gamma-aminobutyric acid product, and the separation and purification process is simple and suitable for industrialized continuous batch production.

Description

TECHNICAL FIELD

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

[0002] Gamma-aminobutyric acid, also known as 4-aminobutyric acid (GABA for short), has a relative molecular mass of 103.12 and a chemical formula of C4H9NO2. As an amino acid, GABA is widely present in microorganisms, plants and vertebrates. GABA is an important central nervous system inhibitory neurotransmitter, has good water solubility and thermal stability, and has various physiological regulation functions. It has been confirmed that GABA, as a small molecule non-protein amino acid, is safe for eating and can be used in the production of food such as beverages.

[0003] Microbial production of gamma-aminobutyric acid has the advantages of mild reaction conditions, no pollution and high yield, and is widely used in the production of gamma-aminobutyric acid. The microbial strains used to synthesize GABA mainly include lactic acid bacteria, Escherichia coli, Saccharomyces cerevisiae and Corynebacterium glutamicum. However, due to the extremely complex multiphase system of the amino acid fermentation liquor, which contains microbial cells, metabolites and unspent culture medium, etc., it is difficult to separate and purify downstream. In related technologies, common separation and purification methods include centrifugation or membrane filtration or plate and frame filtration for solid-liquid separation to remove microbial cells and impurities, electrodialysis, ion exchange, activated carbon and other methods for desalting and decolorization, addition of antisolvents such as methanol and ethanol or evaporation and concentration for crystallization to obtain GABA products. However, these methods have a complex process flow and are prone to residual sulfate, which affects the quality of the product. SUMMARY

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

[0006] A gamma-aminobutyric acid fermentation liquor is provided.

[0007] The gamma-aminobutyric acid fermentation liquor is subjected to solid-liquid separation and first concentration to obtain a first concentrated liquor.

[0008] The first concentrated liquor is subjected to chromatographic separation to obtain a separated liquor.

[0009] The separated liquor is subjected to anion exchange resin purification to obtain a purified liquor.

[0010] The purified liquid is subjected to second concentration and drying to obtain the gamma-aminobutyric acid product.

[0011] Optionally, the first concentrated liquid is subjected to chromatographic separation, comprising:

[0012] The first concentrated liquid is loaded into a chromatographic separation column, and then eluted with 1-3 times the volume of the chromatographic separation column of pure water, and eluate with a conductivity of less than 1.0 mS / cm is collected.

[0013] Optionally, the ratio of the first concentrated liquid to the volume of the chromatographic separation column is 10%-20% for each loading.

[0014] Optionally, the chromatographic separation column is provided with a first filler, and the first filler comprises at least one of LX1850NH filler and LX1880NH filler.

[0015] Optionally, the conductivity of the purified liquid is 10-50 μS / cm.

[0016] Optionally, the separated liquid is subjected to purification by anion exchange resin, comprising:

[0017] The separated liquid is passed through an anion exchange resin column, and a first permeate is collected, and then 1-3 times the volume of the anion exchange resin column of pure water is used for elution to obtain a first eluate, and the first permeate and the first eluate are combined.

[0018] Optionally, the anion exchange resin column is provided with a second filler, and the second filler comprises at least one of D315 filler and D311 filler.

[0019] Optionally, the gamma-aminobutyric acid fermentation liquid comprises a corynebacterium glutamicum fermentation liquid.

[0020] Optionally, the gamma-aminobutyric acid fermentation liquid is subjected to solid-liquid separation and first concentration, comprising:

[0021] The gamma-aminobutyric acid fermentation liquid is filtered using a ceramic membrane, a second permeate is collected, and the concentrated phase is washed with pure water to obtain a water washing liquid, and the second permeate and the water washing liquid are combined to obtain a filtered liquid.

[0022] The filtered liquid is concentrated to an optical transmittance of 50%-70% to obtain the first concentrated liquid.

[0023] Optionally, the membrane pore size of the ceramic membrane is 50 nm, 100 nm, or 200 nm.

[0024] Optionally, the purified liquid is subjected to second concentration and drying, comprising:

[0025] The purified liquid is concentrated to an optical transmittance of 10%-25% to obtain a second concentrated liquid.

[0026] The second concentrated solution is subjected to spray drying to obtain the GABA product.

[0027] Optionally, the second concentrated solution is subjected to spray drying, comprising:

[0028] The second concentrated solution is subjected to drying by using a spray dryer, wherein the air inlet temperature of the spray dryer is set to 150-180℃, the air outlet temperature is set to 100-110℃, the feeding rotation speed is set to 25-40rpm, and the fan frequency is set to 30-50Hz.

[0029] The embodiments of the present application have the following beneficial effects:

[0030] The separation and purification method for removing sulfate from the GABA fermentation broth provided by the embodiments of the present application comprises the following steps: providing a GABA fermentation broth; subjecting the GABA fermentation broth to solid-liquid separation and first concentration to obtain a first concentrated solution; subjecting the first concentrated solution to chromatographic separation to obtain a separated solution; subjecting the separated solution to anion exchange resin purification to obtain a purified solution; and subjecting the purified solution to second concentration and drying to obtain a GABA product. In the chromatographic separation and anion exchange resin purification processes, pure water is used as the elution solvent. The impurities in the fermentation broth can be removed through solid-liquid separation, and the concentrated solution can be decolorized and impurity-removed through chromatographic separation, and the conductivity can be reduced through anion exchange resin, and the GABA product can be obtained after second concentration and drying. In the separation and purification method for removing sulfate from the GABA fermentation broth provided by the embodiments of the present application, the chromatographic separation and anion exchange resin purification can reduce the residual sulfate in the GABA product, improve the purity of the GABA product, and the separation and purification process is simple and suitable for industrialized continuous batch production. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiment description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0032] Figure 1 is the detection spectrum of the GABA product in Example 1 of the present application;

[0033] Figure 2 is a comparison chart of the sulfate content in the GABA products prepared in Example 1 and Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0034] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work are within the scope of protection of the present application. In addition, it should be understood that the specific implementation described herein is only used to illustrate and explain the present application, and is not used to limit the present application.

[0035] The content of γ-aminobutyric acid (GABA) in natural food animals and plants is low. The main methods for realizing large-scale extraction of GABA are chemical synthesis and microbial fermentation. The preparation of GABA by chemical method mainly includes two kinds. One is that 2-pyrrolidone is hydrolyzed by sodium hydroxide or calcium hydroxide to become 4-aminobutyric acid sodium salt, and then GABA is obtained by further treatment of ammonium bicarbonate precipitation or ion exchange resin exchange of sodium ions and concentration and crystallization. The other is that γ-butyrolactone is used as a starting material, and reacts with thionyl chloride to obtain 4-chlorobutyryl chloride. The esterification of 4-chlorobutyryl chloride can generate 4-chlorobutyric acid methyl ester, which can react with ammonia water under the action of a catalyst to generate GABA after ammination and hydrolysis. The two preparation methods involve a variety of hazardous chemicals, and the organic reagents have certain invasiveness to human body, and are expensive.

[0036] The microbial method for producing γ-aminobutyric acid has the advantages of mild reaction conditions, no pollution, high yield, etc., and is widely used in the production of γ-aminobutyric acid. The microbial strains used for synthesizing GABA mainly include lactic acid bacteria, Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. However, the amino acid fermentation broth is an extremely complex heterogeneous system, which contains microbial cells, metabolites, and unspent culture medium, etc., which causes difficulties in downstream separation and purification. The common separation and purification methods include centrifugation or membrane filtration or plate and frame filtration for solid-liquid separation to remove microbial cells and impurities, electrodialysis, ion exchange, activated carbon, and other methods for desalting and decolorizing, adding antisolvents such as methanol and ethanol, or evaporation and concentration to obtain GABA products by crystallization.

[0037] The electrodialysis has a high requirement for the clarity of the fermentation liquor, and has a limited ability to reduce the conductivity, and has a high energy consumption. The use of activated carbon for decolorization causes difficulty in recycling and regeneration of the activated carbon, resulting in high material cost. In the crystallization process, organic reagents are used for solvent crystallization, on the one hand, the recycling of the organic solvent is difficult, and there is a certain loss, on the other hand, 10%-20% of the gamma-aminobutyric acid remains in the crystallization mother liquor, which affects the recovery rate. In addition, in the process of preparing the gamma-aminobutyric acid fermentation liquor by the fermentation process, sulfate ions are introduced in the culture medium or the feeding process, but the related technology does not test the sulfate residue in the gamma-aminobutyric acid product. If the gamma-aminobutyric acid product with sulfate residue is applied to food additives or health products, it is easy to cause gastrointestinal disorders and diarrhea and other problems.

[0038] The embodiment of the present application provides a separation and purification method for removing sulfate in a gamma-aminobutyric acid fermentation liquor, comprising:

[0039] Providing a gamma-aminobutyric acid fermentation liquor;

[0040] The gamma-aminobutyric acid fermentation liquor is subjected to solid-liquid separation and first concentration to obtain a first concentrated liquor;

[0041] The first concentrated liquor is subjected to chromatographic separation to obtain a separated liquor;

[0042] The separated liquor is subjected to anion exchange resin purification to obtain a purified liquor;

[0043] The purified liquor is subjected to second concentration and drying to obtain a gamma-aminobutyric acid product.

[0044] The separation and purification method for removing sulfate in the gamma-aminobutyric acid fermentation liquor provided by the embodiment of the present application can remove impurities in the fermentation liquor through solid-liquid separation, and after concentration, decolorization and impurity removal are performed through chromatographic separation, and then the conductivity is reduced through anion exchange resin, and after re-concentration and drying, a gamma-aminobutyric acid product can be obtained. In the separation and purification method for removing sulfate in the gamma-aminobutyric acid fermentation liquor provided by the embodiment of the present application, through chromatographic separation and anion exchange resin purification, the sulfate residue in the gamma-aminobutyric acid product can be reduced, the purity of the gamma-aminobutyric acid product is improved, and the separation and purification process is simple, and is suitable for industrialized continuous batch production application.

[0045] Specifically, the separation and purification method for removing sulfate from the fermentation broth of gamma-aminobutyric acid provided by the embodiments of the present application can produce a gamma-aminobutyric acid product with high purity, the content of gamma-aminobutyric acid can reach more than 99%, and the yield of gamma-aminobutyric acid can reach more than 90%. The chromatographic separation and anion exchange resin purification can reduce the sulfate residue in the gamma-aminobutyric acid product, so that the mass percentage of sulfate ions in the gamma-aminobutyric acid product is less than 0.03%.

[0046] In some embodiments, the first concentrated liquid is subjected to chromatographic separation, comprising:

[0047] The first concentrated liquid is loaded into the chromatographic separation column, and then 1-3 times the volume of the chromatographic separation column of pure water is used for elution, and the eluate with a conductivity of less than 1.0 mS / cm is collected.

[0048] In some embodiments, the first concentrated liquid is subjected to chromatographic separation, comprising:

[0049] The chromatographic separation is a separation method based on the difference in partition coefficient between different substances in the stationary phase and the mobile phase, and has the characteristics of high efficiency and rapidity. In the chromatographic separation process, by controlling the loading amount and elution conditions, the precise separation and purification of gamma-aminobutyric acid can be realized.

[0050] In the present embodiment, the volume ratio of the first concentrated liquid to the filler in the chromatographic separation column is 10%-20% each time, which can avoid overloading of the chromatographic separation column filler, reduce the risk of chromatographic separation column blockage, and help prolong the service life of the chromatographic separation column. The use of 1-3 times the volume of the chromatographic separation column of pure water for elution can ensure that the gamma-aminobutyric acid in the chromatographic separation column can be fully eluted, improve the purity of gamma-aminobutyric acid in the separation liquid, and at the same time reduce the process difficulty and cost of subsequent concentration and other treatments.

[0051] Illustratively, the volume ratio of the first concentrated liquid to the filler in the chromatographic separation column can be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% each time. When eluting, the volume ratio of the pure water used to the chromatographic separation column can be 1, 1.5, 2, 2.5 or 3. The eluate with a conductivity of less 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.

[0052] In some embodiments, the chromatographic separation column is provided with a first filler, and the first filler comprises at least one of LX1850NH filler and LX1880NH filler.

[0053] The LX1850NH filler and the LX1880NH filler have specific pore sizes and surface properties, are beneficial to the effective distribution of γ-aminobutyric acid between the stationary phase and the mobile phase, have strong selectivity for γ-aminobutyric acid, reduce the interference of other impurity molecules, and thus realize efficient separation and improve the separation purity. Moreover, the LX1850NH filler and the LX1880NH filler both have high chemical stability and mechanical strength, can prolong the service life of the chromatographic separation column, and reduce the cost.

[0054] In some embodiments, the conductivity of the purified liquid is 10 μS / cm - 50 μS / cm.

[0055] The anion exchange resin is a resin material with specific ion exchange function. When the separation liquid is purified by the anion exchange resin, the anion exchange resin can selectively adsorb anions in the separation liquid, so as to realize the separation of γ-aminobutyric acid and other impurities. After the separation liquid is purified by the anion exchange resin, the conductivity of the obtained purified liquid is 10 μS / cm - 50 μS / cm, further reducing the ion content, improving the purity of γ-aminobutyric acid in the purified liquid, reducing the interference of other impurities, and also reducing the difficulty and cost of subsequent processing.

[0056] In some embodiments, the separation liquid is purified by the anion exchange resin, including:

[0057] The separation liquid is passed through the anion exchange resin column, and the first permeate is collected. Then, 1-3 times the volume of the anion exchange resin column of pure water is used for elution to obtain a first eluate, and the first permeate and the first eluate are combined.

[0058] During the process of passing the separation liquid through the anion exchange resin column, the anion exchange resin selectively adsorbs anions, inorganic salts, organic small molecules and other components in the separation liquid, and most of the γ-aminobutyric acid permeates with the separation liquid to obtain the first permeate. A small amount of γ-aminobutyric acid remaining in the anion exchange resin column can be eluted by 1-3 times the volume of the anion exchange resin column of pure water to obtain the first eluate. By combining the first permeate and the first eluate, the content of γ-aminobutyric acid can be improved, and the loss of γ-aminobutyric acid can be reduced.

[0059] By using pure water as the elution solvent in the chromatographic separation and the anion exchange resin purification process, the use of hydrochloric acid, ethanol and other reagents can be reduced, and the cost of consumables can be reduced.

[0060] In some embodiments, the second filler is arranged in the anion exchange resin column, and the second filler includes at least one of a D315 filler and a D311 filler.

[0061] The D315 filler and the D311 filler have high adsorption capacity for anions, can effectively adsorb and separate anions and other impurities in the separation liquid, improve the purification efficiency, and have high selectivity, which helps to improve the purity of the γ-aminobutyric acid in the purified liquid.

[0062] In some embodiments, the γ-aminobutyric acid fermentation liquor includes a corynebacterium glutamicum fermentation liquor. Corynebacterium glutamicum itself lacks a glutamate decarboxylase system, and through genetic modification, corynebacterium glutamicum can obtain the ability to convert glutamic acid to γ-aminobutyric acid. Corynebacterium glutamicum is a safe strain, which guarantees the safety of fermentation of γ-aminobutyric acid. Corynebacterium glutamicum has a significant advantage in synthesizing glutamic acid, which is the main precursor of γ-aminobutyric acid, and can efficiently utilize substrates to synthesize glutamic acid, which is then converted to γ-aminobutyric acid. In addition, corynebacterium glutamicum has strong adaptability to fermentation processes, and can improve the yield of γ-aminobutyric acid by optimizing fermentation conditions. Moreover, the fermentation process of corynebacterium glutamicum is relatively stable, which is conducive to industrial production.

[0063] For example, the corynebacterium glutamicum can be corynebacterium glutamicum FF10 independently developed by the company (Sunrise Biotechnology Co., Ltd.). For materials related to corynebacterium glutamicum FF10, please refer to the patent with publication number CN114752544A.

[0064] In some embodiments, the γ-aminobutyric acid fermentation liquor is subjected to solid-liquid separation and first concentration, including:

[0065] The ceramic membrane is used to filter the γ-aminobutyric acid fermentation liquor, the second permeate is collected, and then the concentrated phase is washed with pure water, and the water washing liquid is collected. The second permeate and the water washing liquid are combined to obtain a filtration liquid.

[0066] The filtration liquid is concentrated to a light transmittance of 50%-70% to obtain a first concentrated liquid.

[0067] The ceramic membrane has a fine pore structure, which can effectively separate small molecular impurities, soluble proteins, colloids, polysaccharides and small particle insoluble substances in the gamma-aminobutyric acid fermentation liquor. The filtered filtrate has high light transmittance, which can reduce the interference of impurities and is conducive to the subsequent process, thereby improving the production efficiency. The separation and filtration process of the ceramic membrane can be carried out at room temperature without the need for high-temperature heating and chemical additives, thereby reducing the loss of effective components and saving energy consumption. By performing pure water top washing of the concentrated phase, a small amount of gamma-aminobutyric acid adsorbed in the pores of the ceramic membrane can be washed to form a water washing liquid, thereby improving the yield of gamma-aminobutyric acid. During the concentration process, the water and other soluble impurities in the filtrate can be removed, thereby improving the purity of gamma-aminobutyric acid. By controlling the light transmittance of the concentrated filtrate to be 50%-70%, the concentration degree can be controlled within a certain range. The volume of the first concentrated liquid after concentration is reduced, thereby reducing the workload of subsequent processing, and the high-concentration gamma-aminobutyric acid is conducive to the extraction and purification of subsequent products.

[0068] In some embodiments, the membrane pore size of the ceramic membrane is 50nm-200nm. When the membrane pore size of the ceramic membrane is 50nm-200nm, bacteria and other impurities in the gamma-aminobutyric acid fermentation liquor can be accurately intercepted, thereby ensuring the filtration effect and reducing the influence of impurities.

[0069] For example, the membrane pore size of the ceramic membrane can be 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm or 200nm.

[0070] In some embodiments, the purified liquid is subjected to second concentration and drying, comprising:

[0071] The purified liquid is concentrated to an optical density of 10%-25% to obtain a second concentrated liquid;

[0072] The second concentrated liquid is subjected to spray drying to obtain a gamma-aminobutyric acid product.

[0073] By concentrating the purified liquid and controlling the light transmittance of the concentrated liquid to be 10%-25%, the water and other soluble impurities in the purified liquid can be removed, thereby improving the concentration and purity of gamma-aminobutyric acid, providing convenience for subsequent processes, reducing material loss in the subsequent processing process, and improving the overall production efficiency. Spray drying can disperse the second concentrated liquid into small droplets through an atomizer, and the droplets are fully contacted with hot air to achieve rapid drying. The efficiency of spray drying is high, and the spray drying process is stable and easy to realize automatic control, thereby shortening the preparation period. The gamma-aminobutyric acid product prepared by spray drying has good biological activity due to the short heating time, high purity, and uniform morphology.

[0074] In some embodiments, the second concentrated solution is subjected to spray drying, including:

[0075] The second concentrated solution is dried by a spray dryer, wherein the inlet air temperature of the spray dryer is set to 150-180℃, the outlet air temperature is set to 100-110℃, the feed rotation speed is set to 25-40rpm, and the fan frequency is set to 30-50Hz.

[0076] By setting the inlet air temperature of the spray dryer to a range of 150-180℃, sufficient heat can be provided by the hot air to quickly evaporate and dry the second concentrated solution, shortening the drying time. Controlling the outlet air temperature between 100-110℃ helps to ensure that the γ-aminobutyric acid product reaches an appropriate degree of drying, while avoiding overheating that can cause quality degradation, ensuring the stability and consistency of the γ-aminobutyric acid product. Suitable feed rotation speed and fan frequency can ensure the continuity and stability of spray drying, improving the uniform distribution and sufficient drying of the second concentrated solution in the drying chamber.

[0077] For example, the inlet air temperature of the spray dryer can be set to 150℃, 160℃, 170℃ or 180℃, the outlet air temperature can be set to 100℃, 105℃ or 110℃, the feed rotation speed can be set to 25rpm, 30rpm, 35rpm or 40rpm, and the fan frequency can be set to 30Hz, 35Hz, 40Hz, 45Hz or 50Hz.

[0078] The embodiments of the present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples are not specified, which are generally according to the conditions recommended by the manufacturer.

[0079] In the examples, the γ-aminobutyric acid fermentation broth is Corynebacterium glutamicum FF10 fermentation broth, which is derived from Sunwise Biotech Co., Ltd. (For more information about Corynebacterium glutamicum FF10, please refer to Chinese Patent CN114752544B). The process of Corynebacterium glutamicum FF10 fermentation broth is shown as follows:

[0080] Corynebacterium glutamicum FF10 was cultured in BHIS medium at 30℃ for 24 hours to obtain a seed solution; 500 mL of fermentation medium was added to a 1L fermenter, and the seed solution was inoculated into the fermenter at a rate of 10%; the fermentation temperature was 30℃, the dissolved oxygen was 30%, and the pH was adjusted to 7.0±0.5 using ammonia water to produce glutamic acid; as the fermentation proceeded, the pH was controlled at 5.5 after 76 hours.

[0081] The BHIS medium formula is as follows: 0.74 g of BHI (brain heart infusion broth), 3.64 g of sorbitol dissolved in 40 ml of water, and filtered. The fermentation medium formula is as follows: glucose 100 g / L, ammonium sulfate 12 g / L, magnesium sulfate 0.87 g / L, corn syrup 3 ml / L, phosphoric acid 0.4 ml / L, potassium chloride 0.53 g / L, ferrous sulfate 120 mg / L, manganese sulfate 120 mg / L, nicotinamide 42 mg / L, calcium pantothenate 6.3 mg / L, vitamin B1 6.3 mg / L, and biotin 0.5 mg / L.

[0082] Example 1

[0083] (1) After the Corynebacterium glutamicum FF10 fermentation broth is inactivated, it is discharged, 10 L of the fermentation broth is filtered by a ceramic membrane with a pore size of 50 nm, and pure water is used for top washing. The permeate and the water washing liquid are collected and concentrated by reduced pressure distillation. The refractive index of the concentrated liquid is 57%, and the first concentrated liquid is obtained.

[0084] (2) The first concentrated liquid is loaded by a simulated moving bed. The chromatographic resin filler is LX1850NH. The single loading is 15% of the chromatographic separation column volume. The eluate with a conductivity lower than 400 μS / cm is collected after pure water is washed for 2.2 chromatographic separation column volumes, and the separation liquid is obtained.

[0085] (3) The loading amount is calculated according to the treatment of 135 g of γ-aminobutyric acid per liter of anion exchange resin D315 for low conductivity treatment. After the water is washed for 2 anion exchange resin column volumes, the loading effluent and the water washing effluent are combined, and the purified liquid with a conductivity of 17 μS / cm is obtained.

[0086] (4) The purified liquid is concentrated to a γ-aminobutyric acid concentration of about 130 g / L, and the second concentrated liquid is obtained.

[0087] (5) The second concentrated liquid is subjected to spray drying. The spray drying parameters are as follows: the inlet air temperature is 180°C, the outlet air temperature is 100°C, the feeding speed is 25 rpm, the fan frequency is 35 Hz, and the γ-aminobutyric acid product is collected.

[0088] Example 2

[0089] (1) After the Corynebacterium glutamicum FF10 fermentation broth is inactivated, it is discharged, 10 L of the fermentation broth is filtered by a ceramic membrane with a pore size of 50 nm, and pure water is used for top washing. The permeate and the water washing liquid are collected and concentrated by reduced pressure distillation. The refractive index of the concentrated liquid is 54%, and the first concentrated liquid is obtained.

[0090] (2) The first concentrated solution is loaded by simulated moving bed, the chromatographic resin filler is LX1850NH, the single loading is 14% of the chromatographic separation column volume, the pure water is washed for 2 chromatographic separation column volumes, the eluent with conductivity lower than 350 μS / cm is collected, and the separation solution is obtained;

[0091] (3) The loading amount is calculated according to the treatment of 140 g of γ-aminobutyric acid per liter of anion exchange resin D315 to carry out the low conductivity treatment, the outflow of loading and water washing is combined after the water washing for 2 anion exchange resin column volumes, and the purified solution with conductivity of 13 μS / cm is obtained;

[0092] (4) The purified solution is concentrated to the γ-aminobutyric acid concentration of about 150 g / L, and the second concentrated solution is obtained;

[0093] (5) The second concentrated solution is subjected to spray drying, the spray drying parameters are as follows: the inlet air temperature is 175 ℃, the outlet air temperature is 105 ℃, the feeding speed is 25 rpm, the fan frequency is 30 Hz, and the γ-aminobutyric acid product is collected.

[0094] Example 3

[0095] (1) The Corynebacterium glutamicum FF10 fermentation liquor is inactivated and then discharged, 10 L of the fermentation liquor is filtered by a ceramic membrane with a membrane pore size of 50 nm, the permeate and the water washing liquid are collected and concentrated by vacuum distillation, the concentrated solution has a refractive index of 70%, and the first concentrated solution is obtained;

[0096] (2) The first concentrated solution is loaded by simulated moving bed, the chromatographic resin filler is LX1880NH, the single loading is 10% of the chromatographic separation column volume, the pure water is washed for 1 chromatographic separation column volume, the eluent with conductivity lower than 1.0 mS / cm is collected, and the separation solution is obtained;

[0097] (3) The loading amount is calculated according to the treatment of 200 g of γ-aminobutyric acid per liter of anion exchange resin D311 to carry out the low conductivity treatment, the outflow of loading and water washing is combined after the water washing for 3 anion exchange resin column volumes, and the purified solution with conductivity of 10 μS / cm is obtained;

[0098] (4) The purified solution is concentrated to the γ-aminobutyric acid concentration of about 150 g / L, and the second concentrated solution is obtained;

[0099] (5) The second concentrated solution is subjected to spray drying, the spray drying parameters are as follows: the inlet air temperature is 150 ℃, the outlet air temperature is 100 ℃, the feeding speed is 40 rpm, the fan frequency is 50 Hz, and the γ-aminobutyric acid product is collected.

[0100] Example 4

[0101] (1) The fermentation broth of Corynebacterium glutamicum FF10 was inactivated and then discharged. 10 L of the fermentation broth was filtered by a ceramic membrane with a pore size of 50 nm, and pure water was used for top washing. The permeate and the water washing liquid were collected and concentrated by vacuum distillation. The refractive index of the concentrated liquid was 50%, and a first concentrated liquid was obtained;

[0102] (2) The first concentrated liquid was loaded by a simulated moving bed. The chromatographic resin filler was LX1880NH. The single loading was 20% of the chromatographic separation column volume. Pure water was used for washing for 3 chromatographic separation column volumes. The eluate with a conductivity lower than 0.1 mS / cm was collected, and a separated liquid was obtained;

[0103] (3) The loading amount was calculated according to the treatment of 100 g of γ-aminobutyric acid per liter of anion exchange resin D311, and the reduced conductivity treatment was performed. After water washing for 1 anion exchange resin column volume, the loading effluent and the water washing effluent were combined, and a purified liquid with a conductivity of 50 μS / cm was obtained;

[0104] (4) The purified liquid was concentrated to a γ-aminobutyric acid concentration of about 150 g / L, and a second concentrated liquid was obtained;

[0105] (5) The second concentrated liquid was subjected to spray drying. The spray drying parameters were as follows: the inlet air temperature was 180 ℃, the outlet air temperature was 110 ℃, the feeding speed was 25 rpm, and the fan frequency was 30 Hz. The γ-aminobutyric acid product was collected.

[0106] Comparative Example 1

[0107] (1) The fermentation broth of Corynebacterium glutamicum FF10 was inactivated and then discharged. 10 L of the fermentation broth was filtered by a ceramic membrane with a pore size of 50 nm, and pure water was used for top washing. The permeate and the water washing liquid were collected and concentrated by vacuum distillation. The refractive index of the concentrated liquid was 50%, and a first concentrated liquid was obtained;

[0108] (2) The first concentrated liquid was loaded by a simulated moving bed. The single loading was 20% of the chromatographic separation column volume. Pure water was used for washing for 3 chromatographic separation column volumes. The eluate with a conductivity lower than 0.1 mS / cm was collected, and a separated liquid was obtained;

[0109] (3) The first concentrated liquid was loaded by a simulated moving bed. The single loading was 20% of the chromatographic separation column volume. Pure water was used for washing for 3 chromatographic separation column volumes. The eluate with a conductivity lower than 0.1 mS / cm was collected, and a separated liquid was obtained;

[0110] (4) The second concentrated liquid was subjected to spray drying. The spray drying parameters were as follows: the inlet air temperature was 180 ℃, the outlet air temperature was 102 ℃, the feeding speed was 30 rpm, and the fan frequency was 37 HZ. The γ-aminobutyric acid product was collected.

[0111] Comparative Example 2

[0112] In the present comparative example, the Corynebacterium glutamicum FF10 fermentation broth was inactivated and then discharged. 10 L of the fermentation broth was subjected to high-speed centrifugation at a centrifugal force of 9500 xg. The supernatant after centrifugation was heated to 80°C and then clarified by filtration through a 0.45 μm filter membrane. The collected filtrate was adjusted to pH 5.0 with 2M acetic acid, and activated carbon powder was added at an addition amount of 2%. The mixture was stirred and heated to 60°C for 30 min. The mixture was first filtered through double filter paper and then precisely filtered through a 0.45 μm filter membrane. The filtrate was concentrated by vacuum rotary evaporation. The water bath heating temperature was set to 55°C. When a large amount of crystals precipitated, the concentrated material was added with 95% ethanol at a volume of 3 times the weight of the concentrated material. The mixture was cooled to 4°C, and then stirring was stopped and the mixture was incubated for 12 h. The filtered cake was washed with 95% ethanol at a ratio of 3 mL / g of crystals. The mixture was heated to 40°C and stirred for 1 h, and then filtered. The wet crystals after filtration were dried in an oven at 80°C for 12 h to constant weight. After cooling, the finished product of γ-aminobutyric acid was obtained.

[0113] The content of γ-aminobutyric acid and the content of sulfate salt in the γ-aminobutyric acid product in Test Examples 1-2 and Comparative Example 1 were tested.

[0114] The test method for the content of γ-aminobutyric acid refers 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.

[0115] The test method for the content of sulfate salt (calculated as SO4) is as follows:

[0116] 1. Reagents and materials

[0117] 1) Potassium sulfate standard solution: 0.1 mg / mL.

[0118] 2) Hydrochloric acid solution: 10%.

[0119] 3) Barium chloride solution: 1 mol / L.

[0120] 2. Analysis steps

[0121] 1) Weigh 0.70 g of the sample and dissolve in water to 40 mL (if the solution is alkaline, add hydrochloric acid dropwise to make it neutral; if the solution is not clear, filter). Place in a 50 mL Nessler colorimetric tube, add 2 mL of hydrochloric acid solution, and shake well to obtain the sample solution;

[0122] 2) Take 2.1 mL of the potassium sulfate standard solution and place it in another 50 mL Nessler colorimetric tube. Dilute with water to 40 mL, add 2 mL of hydrochloric acid solution, and shake well to obtain the standard control solution;

[0123] 3) In the sample solution and the standard control solution, respectively, add barium chloride solution 5 mL, dilute with water to 50 mL, shake well, stand for 10 min, and place on a black background, observe and compare from the top of the colorimetric tube to the bottom.

[0124] If the turbidity of the sample solution is not higher than that of the standard control solution, the sulfate content is ≤0.03%.

[0125] The test results are shown in Table 1. Figures 1-2

[0126] Table 1 Comparison table of test results of γ-aminobutyric acid products in different examples and comparative examples

[0127]

[0128] Among them, Figure 1 is the detection spectrum of the γ-aminobutyric acid product in Example 1, Figure 2 is a comparison chart of sulfate content results of the γ-aminobutyric acid products prepared 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 indicates that the sulfate content is ≤0.03%. From Table 1 and Figures 1-2 It can be seen from Table 1 that the γ-aminobutyric acid product prepared in the examples of the present application has a higher content of γ-aminobutyric acid, higher purity, and the sulfate content can be controlled below 0.3%, which ensures the quality of the γ-aminobutyric acid product and has a high yield.

[0129] The above describes the examples of the present application in detail, and the principles and implementation modes of the present application are described by applying specific examples. The above example descriptions are only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation mode and application range will be changed, and the above description should not be understood as a limitation of the present application.​

Claims

1. A method for separating and purifying sulfate from γ-aminobutyric acid fermentation broth, characterized in that, include: Provide γ-aminobutyric acid fermentation broth; The γ-aminobutyric acid fermentation broth was subjected to solid-liquid separation and a first concentration to obtain a first concentrated liquid; The first concentrate was subjected to chromatographic separation to obtain the separated solution; The separated solution was purified by anion exchange resin to obtain a purified solution; The purified solution was further concentrated and dried to obtain γ-aminobutyric acid product; The step of separating the first concentrate by chromatography includes: The first concentrated solution is loaded onto a chromatographic column, and then eluted with 1-3 times the volume of pure water of the chromatographic column, and the eluent with a conductivity of less than 1.0 mS / cm is collected. The anion exchange resin column is provided with a second packing material, which is at least one of D315 packing material and D311 packing material. The γ-aminobutyric acid fermentation broth is a Corynebacterium glutamicum fermentation broth.

2. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 1, characterized in that, For each sample loading, the ratio of the volume of the first concentrate to the volume of the chromatographic 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 packing material, which is at least one of LX1850NH packing material and LX1880NH packing material.

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 solution 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 purification of the separated solution using anion exchange resin includes: The separated liquid is passed through an anion exchange resin column, the first permeate is collected, and then eluted with 1-3 times the volume of the anion exchange resin column of pure water to obtain the first eluent. The first permeate and the first eluent are then combined.

6. 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, including: The γ-aminobutyric acid fermentation broth was filtered using a ceramic membrane, and the second permeate was collected. Then, the concentrated phase was washed with pure water, and the washing liquid was collected. The second permeate and the washing liquid were combined to obtain the filtrate. The filtrate is concentrated to a refractive index of 50%-70% to obtain the first concentrate.

7. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 6, characterized in that, The ceramic membrane has a pore size of 50 nm, 100 nm, or 200 nm.

8. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to any one of claims 1-7, characterized in that, The second concentration and drying of the purified solution includes: The purified solution was concentrated to a refractive index of 10%-25% to obtain a second concentrated solution; The second concentrate was spray-dried to obtain the γ-aminobutyric acid product.

9. The separation and purification method for removing sulfate from γ-aminobutyric acid fermentation broth according to claim 8, characterized in that, The step of spray drying the second concentrate includes: The second concentrate is dried using a spray dryer, wherein the inlet air temperature of the spray dryer is set to 150℃-180℃, the outlet air temperature is set to 100℃-110℃, the feed speed is set to 25rpm-40rpm, and the fan frequency is set to 30Hz-50Hz.

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