Method for separating and purifying beta-alanine from fermentation liquor

Abstract

The invention relates to the technical field of purification of beta-alanine, and discloses a method for separating and purifying beta-alanine from fermentation liquor. The invention provides a method for separating and purifying beta-alanine from fermentation liquor by combining flocculation and resin to overcome the defects that an existing process for separating and purifying beta-alanine from fermentation liquor is tedious in route, high in cost and technology, large in energy consumption and the like. Impurity substances can be effectively removed through the steps of flocculation impurity removal, protein removal, resin adsorption decoloration, resin adsorption elution refining and crystallization extraction of beta-alanine, and the obtained product is high in purity. Meanwhile, the method is simple to operate, mild in process condition and low in cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of purification of beta-alanine, and in particular to a method for separating and purifying beta-alanine from fermentation broth. BACKGROUND

[0002] Alanine is a non-polar alpha-amino acid with the chemical formula C3H7NO2, and is one of the simplest amino acids in protein composition. Alanine can be divided into alpha type and beta type due to the difference in the amino position. Alanine is an alpha-amino acid, while beta-alanine is a beta-amino acid, which is the only beta-type amino acid existing in nature and has important physiological functions in the metabolism of animals, plants and microorganisms.

[0003] Beta-alanine, also known as 3-aminopropanoic acid, is an important precursor for the synthesis of pantothenic acid in organisms. Although beta-alanine does not participate in the synthesis of proteins, as one of the most promising three-carbon chemical products in the world, beta-alanine and its derivatives are widely used in medicine, environment, food, feed and chemical industry, and are a potential functional amino acid with important value.

[0004] Chemical synthesis is the main method for mass production of beta-alanine in most factories at present, but its reaction conditions are harsh (high temperature, high pressure, strong acid and strong base), and it is inevitable to produce by-products, which is not environmentally friendly. Nowadays, microbial fermentation method has attracted widespread attention due to its genetic modification potential and green and sustainable advantages, and has gradually become the mainstream method for producing beta-alanine. This method has the advantages of green environmental protection and low cost. However, microbial fermentation broth is a complex multiphase system containing cells, metabolites, unused culture medium and other solid and colloidal substances dispersed therein, which have compressibility and density close to liquid, and belong to non-Newtonian liquid due to high viscosity, making it difficult to separate solids from the culture broth. Therefore, subsequent biochemical separation engineering means is needed to extract the required product from the fermentation broth.

[0005] Downstream separation and purification technology is the core link connecting upstream biosynthesis and terminal product application in the production of beta-alanine, and directly affects the product purity, production cost, environmental performance and market competitiveness. At present, there is still a blank in the technology for separating and purifying beta-alanine from fermentation broth, and the process route still has problems such as complexity, high energy consumption and high cost. Therefore, a process route for separating beta-alanine from fermentation broth needs to be developed to realize further industrial application. SUMMARY

[0006] In order to solve the technical problem of separating and purifying beta-alanine from fermentation broth, the present application provides a method for separating and purifying beta-alanine from fermentation broth.

[0007] The specific technical scheme of the present application is: The present application provides a method for separating and purifying beta-alanine from fermentation liquor, which comprises the following steps: Step S1, adding a flocculating agent to the fermentation liquor containing beta-alanine for flocculation treatment, solid-liquid separation, and collecting the liquid; Step S2, acidifying the liquid obtained in step S1, centrifuging, and collecting the supernatant; Step S3, performing chromatographic adsorption decolorization on the supernatant obtained in step S2, wherein the adsorption decolorization is performed by a chromatographic column, and the filling resin of the chromatographic column is DA201-C; Step S4, performing chromatographic adsorption elution on the decolorized supernatant to obtain a refined liquid, wherein the adsorption elution is performed by a chromatographic column, and the filling resin of the chromatographic column is a cation exchange resin; Step S5, taking the refined liquid, and performing crystallization by combining evaporation concentration and cooling to obtain beta-alanine fine product.

[0008] In view of the defects in the prior art process for purifying beta-alanine from fermentation liquor, more specifically, in view of the complicated route, high cost and energy consumption, the present application provides a method for separating and purifying beta-alanine from fermentation liquor by combining flocculation and resin, which comprises the steps of flocculation impurity removal, protein removal, resin adsorption decolorization, resin adsorption elution, and crystallization extraction of beta-alanine, which can effectively remove impurities and obtain a product with high purity. At the same time, the method is simple to operate, has mild process conditions, and is low in cost.

[0009] As a preferred method of the above method, in step S5, the crystallization method is: evaporating and concentrating the refined liquid under the conditions of a temperature of 40-60 DEG C and a vacuum degree of-0.1-0.1 MPa, concentrating to a liquid surface to appear a crystal film, then transferring to 0-20 DEG C for cooling crystallization, filtering, drying, and obtaining beta-alanine crystals.

[0010] The present application evaporates and concentrates to appear a crystal film, which is used as a crystal seed, so that a large amount of beta-alanine crystals can be crystallized in the cooling crystallization process, thereby realizing the extraction of pure beta-alanine.

[0011] As a preferred method of the above method, in step S3, the sample flow rate of the adsorption decolorization is 1-6 BV / h.

[0012] Step S3 is an adsorption and decolorization step for the chromatography resin. In this step, DA201-C is used as the packing resin for adsorption and decolorization. DA201-C is particularly suitable for removing pigment substances from a fermentation liquor containing β-alanine, and is therefore selected. Through experiments, it is verified that the DA201-C macroporous adsorption resin selected as the indicator of decolorization rate and yield can effectively remove pigment substances from the acidified liquor, and has the best purification effect; other macroporous adsorbents, including D101, H103 and AB-8, have general impurity removal effects. Further, the sample flow rate of the acidified liquor in step S3 is optimized through dynamic adsorption, and is 1-6 BV / h, and more preferably 2 BV / h.

[0013] As a preferred method described above, in step S4, the cation exchange resin is selected from HD-8, LX-160, D314, D301, D001, LXT-104 and LX-6703.

[0014] Step S4 is a refining step of adsorption and elution of the chromatography resin. The reason for performing this step is that the decolorized liquor after step S3 has a very high salt content, and the conductivity can be as high as 110 ms / cm. The present inventors have found in experiments that if these salts are not removed completely, it will greatly hinder the subsequent crystallization and purification process, and it is difficult to obtain pure β-alanine by crystallization.

[0015] For the removal of salt from the decolorized liquor, it was initially intended to use a desalination resin or electro-osmosis method to remove the high salt content in the fermentation liquor, but the experiment was not satisfactory. First, the nodes of salt and product β-alanine flow are very similar, and second, when β-alanine is deprotonated, it exists in an ionic state, which requires additional steps or the addition of solvents to restore the non-ionic state of β-alanine. Ultimately, the present inventors selected a cation exchange resin as a packing agent for adsorption and elution to remove salt. It is found that this method can obtain high-purity β-alanine liquid with salt removed. The principle of adsorption and elution for salt removal is that the cation exchange resin adsorbs β-alanine onto the resin, and then the salt is eluted first, and then β-alanine is eluted from the cation exchange resin, thereby obtaining high-purity β-alanine liquid. Through verification, the cation exchange resin is selected from HD-8, LX-160, D314, D301, D001, LXT-104 and LX-6703, and more preferably LXT-104.

[0016] As a preferred method described above, in step S4, the sample flow rate of the decolorized liquor is 0.8-3 BV / h, and more preferably the sample flow rate is 1 BV / h.

[0017] When the decoloring solution is loaded into the cation exchange resin column, the loading flow rate of the decoloring solution should be controlled within a proper range to ensure that the positively charged β-alanine can be effectively combined with the ion exchange resin column after ion exchange, preventing the β-alanine from flowing out and reducing the yield.

[0018] As a preferred method, in step S4, the eluent for adsorption elution is ammonia water. The elution concentration of the ammonia water is preferably 1%-6% (volume ratio), and the elution flow rate of the ammonia water is preferably 0.5 BV / h-3.0 BV / h, and more preferably 1.0 BV / h.

[0019] When the cation exchange resin column is eluted after loading, the type and concentration of the eluent should be considered to ensure that the β-alanine combined with the ion exchange resin column is completely eluted as much as possible, thereby improving the elution yield. The eluent is preferably ammonia water.

[0020] As a preferred method, in step S1, the flocculant is a chitosan-sodium alginate composite flocculant.

[0021] Step S1 is a flocculation step. The chitosan-sodium alginate composite flocculant is used, which has a better flocculation effect than polymeric ferrous sulfate, polyacrylamide, sodium alginate, or chitosan.

[0022] The principle is that sodium alginate has good thickening properties, and the aqueous solution has high viscosity. It can form a thermally irreversible gel or a divalent ion in a complex system under the action of divalent metal ions, thereby enhancing the flocculation effect. This is suitable for the high salt content in the β-alanine fermentation broth, and is suitable for the flocculation treatment of the β-alanine fermentation broth. However, the viscosity of the flocculation liquid after adding sodium alginate is high, which inhibits the further uniform diffusion of sodium alginate in the system. However, when the chitosan-sodium alginate composite flocculant is used as a composite flocculant, chitosan has good diffusion in the fermentation broth and can penetrate the viscous environment of the bacterial aggregates, so that sodium alginate and chitosan can better play a flocculation role. Therefore, the flocculation effect of sodium alginate or chitosan as a flocculant is better than that of the chitosan-sodium alginate composite flocculant.

[0023] The reason for the high salt content in the β-alanine fermentation broth is that inorganic salts need to be added as nutrient components for the growth and production of the strain during fermentation, so the fermentation broth has high salt content.

[0024] As a preferred method, the mass ratio of chitosan to sodium alginate in the chitosan-sodium alginate composite flocculant is (1-7):1.

[0025] As a preferred method, in step S1, the method for solid-liquid separation is microfiltration, plate and frame filtration, or centrifugation.

[0026] As the above method preferably, in step S2, the pH of the acidification is 2-3.

[0027] Based on the above method, the present application also provides a high-purity beta-alanine product.

[0028] Compared with the prior art, the present application has the following technical effects: (1) In view of the defects of the prior art, such as complicated process route, high cost and energy consumption, the present application provides a method for separating and purifying beta-alanine from fermentation broth by combining flocculation and resin. The steps of flocculation, protein removal, resin adsorption, decolorization, resin adsorption, elution and purification, and crystallization extraction of beta-alanine can effectively remove impurities, and the obtained product has high purity.

[0029] (2) More specifically, the present application can induce the formation of a large amount of beta-alanine crystals during cooling crystallization at 0-20℃ by evaporating and concentrating to form a crystal film as a seed, thereby realizing the extraction of pure beta-alanine. Without the initiation of the seed, it is difficult to form beta-alanine crystals during cooling crystallization.

[0030] (3) More specifically, the present application also provides a macroporous adsorbent DA201-C as an index of decolorization rate and yield, which can effectively remove pigment substances in the acidified liquid and has the best purification effect. Other macroporous adsorbents, including D101, H103 and AB-8, generally have poor impurity removal effect.

[0031] (4) More specifically, the present application also provides a chitosan-sodium alginate composite flocculant, which is particularly suitable for the flocculation treatment of beta-alanine fermentation broth. The flocculant can simultaneously play the roles of sodium alginate and chitosan in the flocculation treatment of beta-alanine fermentation broth, and can achieve a flocculation effect that cannot be achieved by adding single sodium alginate or chitosan, thereby achieving a 1+1>2 impurity removal effect.

[0032] (5) The method of the present application is simple to operate, has mild process conditions and low cost. DETAILED DESCRIPTION

[0033] The present application will be further described below with reference to the examples. Those skilled in the art will be able to implement the present application based on these descriptions. In addition, the examples of the present application involved in the following descriptions are generally only a part of the examples of the present application, but not all the examples. Therefore, based on the examples in the present application, all other examples obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present application.

[0034] As a total embodiment, a method for separating and purifying beta-alanine from fermentation liquor is provided, which comprises the following steps: Step S1, adding a flocculating agent to the fermentation liquor containing beta-alanine for flocculation treatment, solid-liquid separation, and collecting the liquid; Step S2, acidifying the liquid obtained in step S1, centrifuging, and collecting the supernatant; Step S3, performing chromatographic adsorption decolorization on the supernatant obtained in step S2, wherein the adsorption decolorization is performed by a chromatographic column, and the filling resin of the chromatographic column is DA201-C; Step S4, performing chromatographic adsorption elution on the decolorized supernatant to obtain a refined liquid, wherein the adsorption elution is performed by a chromatographic column, and the filling resin of the chromatographic column is a cation exchange resin; Step S5, taking the refined liquid, and performing crystallization by combining evaporation concentration and cooling to obtain beta-alanine fine product.

[0035] In view of the defects of the existing process for purifying beta-alanine from fermentation liquor, such as complicated route, high cost and technology, and large energy consumption, the above method combines flocculation and resin to effectively remove impurities and obtain a product with high purity by the steps of flocculation impurity removal, protein removal, resin adsorption decolorization, resin adsorption elution, and crystallization extraction of beta-alanine.

[0036] As a preferred embodiment of the above method, in step S5, the crystallization method is: evaporating and concentrating the refined liquid under the conditions of a temperature of 40-60°C and a vacuum degree of -0.1-0.1 MPa, concentrating to a liquid surface with a crystal film, then transferring to 0-20°C for cooling crystallization, filtering, drying, and obtaining beta-alanine crystals.

[0037] In step S5, the crystal film is used as a crystal seed by evaporation and concentration, so that a large amount of beta-alanine crystals can be precipitated during the cooling crystallization process, thereby realizing the extraction of pure beta-alanine.

[0038] As a preferred embodiment of the above method, in step S3, the loading flow rate of the adsorption decolorization is 1-6 BV / h.

[0039] Step S3 is an adsorption and decolorization step for the chromatography resin. In this step, DA201-C is used as the packing resin for adsorption and decolorization. DA201-C is particularly suitable for removing pigment substances from a fermentation liquor containing β-alanine, and is therefore selected. Through experiments, it is verified that the DA201-C macroporous adsorbent selected as the indicator of decolorization rate and yield can effectively remove pigment substances from the acidified liquor, and has the best purification effect; other macroporous adsorbents, including D101, H103 and AB-8, have general impurity removal effects. Further, the sample flow rate of the acidified liquor in step S3 is optimized through dynamic adsorption, and is 1-6 BV / h, and more preferably 2 BV / h.

[0040] As a preferred method described above, in step S4, the cation exchange resin is selected from HD-8, LX-160, D314, D301, D001, LXT-104 and LX-6703.

[0041] Step S4 is a refining step of adsorption and elution of the chromatography resin. The reason for performing this step is that the decolorized liquor after step S3 has a very high salt content, and the conductivity can be as high as 110 ms / cm. The present inventors have found in experiments that if these salts are not removed, it will greatly hinder the subsequent crystallization and purification process, and it is difficult to obtain pure β-alanine by crystallization.

[0042] For the removal of salt from the decolorized liquor, it was initially intended to use a desalination resin or electro-osmosis method to remove the high salt content in the fermentation liquor, but the experiment was not satisfactory. First, the nodes of salt and product β-alanine flow are very similar, and second, when β-alanine is deprotonated, it exists in an ionic state, which requires additional steps or the addition of solvents to restore the non-ionic state of β-alanine. Ultimately, the present inventors selected a cation exchange resin as a packing agent for adsorption and elution to remove salt. It is found that this method can obtain high-purity β-alanine liquid with salt removed. The principle of adsorption and elution for salt removal is that the cation exchange resin adsorbs β-alanine onto the resin, and then the salt is eluted first, and then β-alanine is eluted from the cation exchange resin, thereby obtaining high-purity β-alanine liquid. Through verification, the cation exchange resin is selected from HD-8, LX-160, D314, D301, D001, LXT-104 and LX-6703, and more preferably LXT-104.

[0043] As a preferred method described above, in step S4, the sample flow rate of the decolorized liquor is 0.8-3 BV / h, and more preferably the sample flow rate is 1 BV / h.

[0044] When the decoloring solution is loaded into the cation exchange resin column, the loading flow rate of the decoloring solution should be controlled within a proper range to ensure that the positively charged β-alanine can be effectively combined to the ion exchange resin column after ion exchange, and prevent the β-alanine from flowing out to reduce the yield.

[0045] As the preferred method, in step S4, the eluent for the adsorption elution is ammonia water. The elution concentration of the ammonia water is preferably 1%-6% (volume ratio), and the elution flow rate of the ammonia water is preferably 0.5 BV / h-3.0 BV / h, and more preferably 1.0 BV / h.

[0046] When the cation exchange resin column is eluted after loading, the type and concentration of the eluent should be considered to ensure that the β-alanine combined to the ion exchange resin column is as completely eluted as possible to improve the elution yield. The eluent is preferably ammonia water.

[0047] As the preferred method, in step S1, the flocculant is a chitosan-sodium alginate composite flocculant.

[0048] Step S1 is a flocculation step. The chitosan-sodium alginate composite flocculant has a better flocculation effect than polymeric ferrous sulfate, polyacrylamide, sodium alginate, or chitosan.

[0049] The principle is that sodium alginate has good thickening properties, and the aqueous solution has high viscosity. It can form a thermally irreversible gel or a divalent ion in a complex system under the action of divalent metal ions, enhancing the flocculation effect, which is suitable for the flocculation treatment of β-alanine fermentation broth. However, the flocculation liquid after adding sodium alginate has high viscosity, which inhibits the further uniform diffusion of sodium alginate in the system. However, when the chitosan-sodium alginate composite flocculant is used as a composite flocculant, chitosan has good diffusion in the fermentation broth and can penetrate the viscous environment of bacterial aggregation, so that sodium alginate and chitosan can better play a flocculation role. Therefore, the flocculation effect of sodium alginate or chitosan as a flocculant is better than that of the chitosan-sodium alginate composite flocculant.

[0050] The reason for the high salt content in the β-alanine fermentation broth is that inorganic salts need to be added as nutrient ingredients for strain growth and production during fermentation, so the fermentation broth has high salt content.

[0051] As the preferred method, the mass ratio of chitosan to sodium alginate in the chitosan-sodium alginate composite flocculant is (1-7):1.

[0052] As the preferred method, in step S1, the method of solid-liquid separation is microfiltration, plate and frame filtration or centrifugation. The rotation speed and time of centrifugation are preferably 6000-10000 rpm and 8-15 min, respectively, and more preferably 8000-10000 rpm and 10-12 min, respectively.

[0053] As the preferred method, in step S2, the pH of acidification is 2-3.

[0054] The β-alanine fermentation broth can be understood as a fermentation broth containing the target product β-alanine obtained in the fermentation process of a strain in bioengineering. In the embodiments of the present application, the preparation method of the β-alanine fermentation broth is as follows: in the presence of carbon and nitrogen sources, a strain engineering strain producing β-alanine is used to ferment to prepare a fermentation broth containing β-alanine, which comprises the following steps: inoculating the engineering strain on a 50 mg / L kanamycin-resistant LB plate and culturing overnight at 37°C, picking a single colony to a 50 mg / L kanamycin-resistant LB test tube and culturing overnight at 37°C and 150 rpm to prepare a seed liquid; inoculating the seed liquid into a 50 mg / L kanamycin-containing LB medium at a volume concentration of 5%, and culturing overnight at 37°C and 150 rpm to obtain a secondary seed liquid; inoculating the secondary seed liquid into a fermentation tank containing a 50 mg / L kanamycin-containing fermentation medium at a volume concentration of 15%, and adding IPTG at a final concentration of 0.1 mM, and then fermenting and culturing under the conditions of 30°C, 500 rpm and aeration rate of 0.5 V / V / min; when the pH value is higher than 6.80, automatically starting the feeding and adding a 50 mg / L kanamycin-containing IPTG feeding medium at a final concentration of 0.2 mM until the pH value is lower than 6.80; then stopping the feeding and culturing for 106 h to obtain a fermentation broth containing β-alanine.

[0055] Example 1 Step (1): 1 L of β-alanine fermentation broth was prepared, and the content of β-alanine in the fermentation broth was determined by high performance liquid chromatography to be 115 g / L. A chitosan-sodium alginate flocculant complex was added at a mass percentage of 1% to the fermentation broth for flocculation treatment, and a flocculated fermentation broth was obtained. The preparation method of the chitosan-sodium alginate flocculant complex is as follows: a 0.5 g / L sodium alginate aqueous solution was slowly added dropwise into a chitosan acetic acid solution with a concentration of 1.0 g / L to obtain a chitosan-sodium alginate flocculant complex, and the mass ratio of chitosan to sodium alginate in the complex was 5:1.

[0056] Step (2): The fermentation broth of step (1) was centrifuged at 10000 rpm for 10 min, and the fermentation supernatant was collected. The removal rate of the bacterial cells was determined to be 98.5%.

[0057] Step (3), the supernatant of step (2) is added to hydrochloric acid to adjust the pH to 3, and steam heating is performed at 70°C for 15 min to make the protein coagulate, and the protein is removed by centrifugation to obtain a supernatant, and the protein removal rate in the supernatant is determined to be 96.7% or more, and the yield of β-alanine after the pretreatment process of the above three steps is 91.8%.

[0058] Step (4), the decolorized resin after treatment is loaded by wet method, and the pretreatment method of DA201-C macroporous adsorption resin is as follows: a proper amount of resin is weighed and placed in a 250 mL conical flask, 95% ethanol is added, and the conical flask is placed in a 30°C shaking bed, and the ethanol is replaced every 1 h, and the washing is performed 2 to 3 times, then pure water is used for washing, and the washing is performed until there is no ethanol smell, then the water is removed by filtration and used for standby. The acidified supernatant of step (3) is loaded onto a DA201-C macroporous adsorption resin chromatographic column at a loading flow rate of 2.0 BV / h to perform decolorization, and after the loading is completed, the effluent is collected and the yield of β-alanine and the decolorization rate are detected, and in this process, the yield of β-alanine is 91.9%, and the decolorization rate is 93.7%.

[0059] Step (5), the pretreated LXT-104 cation exchange resin is loaded into a chromatographic column, and the LXT-104 resin is a macroporous strong acid cation exchange resin in Na type. The pretreatment method is as follows: after washing with deionized water until the immersion washing water is colorless, the resin is loaded by wet method to make it fully swell to avoid breakage; after loading, 4% HCl is passed through the resin at a flow rate of 1.5 BV / h, and the column volume is 5.0 BV, to remove inorganic impurities such as iron and calcium in the resin; then deionized water is used for washing until the pH of the effluent is between 5.8 and 7.0, and then 4% HCl is used to flow through the resin at a flow rate of 1.5 BV / h, and the column volume is 5.0 BV, to remove organic matter and pigment; deionized water is used for washing until the pH of the effluent is between 7.0 and 9.2; 5% NaCl is used again to flow through the resin at a flow rate of 1.5 BV / h, and the column volume is 6.0 BV, to convert the resin to sodium type, and the sodium chloride solution needs to be excessive to ensure complete conversion, otherwise the residual H type resin will affect the exchange efficiency of cations in subsequent use; finally, deionized water is used to continuously wash the resin until the effluent pH is neutral, i.e. 6-7, at which time there is no residual electrolyte on the surface of the resin, and the pretreatment is completed.

[0060] After pretreatment, the decolorized liquid of step (4) is loaded onto a LXT-104 cation exchange resin chromatographic column at a loading flow rate of 1.0 BV / h, and after the loading is completed, deionized water is used for washing at a flow rate of 2.0 BV / h to remove impurities, and then 4% ammonia water (4% is a volume ratio) is used for elution at an elution flow rate of 1.0 BV / h, and the eluate is collected and the yield of β-alanine is detected by high performance liquid chromatography, and in this process, the yield of β-alanine is 89.3%.

[0061] Step (6): The eluent was concentrated by rotary evaporator, the temperature was 40℃ water bath, the vacuum degree was -0.1 MPa, and the concentration was performed until the liquid surface appeared a crystal film, at this time the concentration of β-alanine reached 400 g / L. Then it was transferred to 4℃ for 10 hours, and the crystal was obtained by cooling crystallization, filtration and drying. The purity of β-alanine was detected by high performance liquid chromatography, and the results showed that the purity of β-alanine was 90.1%, and the yield of crystallization process was 83.7%.

[0062] Example 2 The difference between this example and example 1 is that the flocculating agent in step (1) is replaced by polymeric ferrous sulfate, polyacrylamide, sodium alginate and chitosan respectively to perform 4 groups of experiments. The other steps are the same as example 1. This example is based on the purification process of example 1, and the single adjustment of certain process parameters is performed to determine the optimal selection of certain process parameters.

[0063] Through this example, it is found that when different flocculants are used for flocculation treatment, the removal rate of bacterial cells in step (2) is shown in Table 1.

[0064] flocculants removal rate of bacteria body / % chitosan-sodium alginate complex 98.5 polyferric sulfate 87.2 polyacrylamide 83.5 sodium alginate 90.8 chitosan 87.6

[0065] As shown in Table 1, compared with polymeric ferrous sulfate, polyacrylamide, sodium alginate or chitosan, the flocculation treatment step using chitosan-sodium alginate composite flocculant has better flocculation effect, and the chitosan-sodium alginate composite flocculant is especially suitable for the flocculation treatment of β-alanine fermentation broth. The flocculant can simultaneously play the role of sodium alginate and chitosan in the flocculation treatment of β-alanine fermentation broth, and produce a flocculation effect that cannot be achieved when adding single sodium alginate and chitosan, and produce a 1+1>2 impurity removal effect.

[0066] Although the flocculation effect does not directly affect the purity of the β-alanine crystal product in the later stage, if the flocculation effect is poor, it will lead to an increase in the cost of passing through the chromatography column and damage to the chromatography column. The optimization and improvement of the flocculant still have important significance.

[0067] Example 3 The difference between this example and example 1 is that the macroporous adsorption resin DA201-C in step (4) is replaced by macroporous adsorption resins D101, H103 and AB-8 respectively to perform 3 groups of experiments. The other steps are the same as example 1. This example is based on the purification process of example 1, and the single adjustment of certain process parameters is performed to determine the optimal selection of certain process parameters.

[0068] Through this example, it is found that when different macroporous adsorption resins are used for decolorization of fermentation broth, the yield and decolorization rate of β-alanine in step (4) and the purity of β-alanine in step (6) are shown in Table 2.

[0069] decoloring resin decolorization rate / % yield / % purity of final product / % da201-c 93.7 91.9 90.1 D101 87.6 92.0 87.4 H103 82.1 91.4 84.9 ab-8 87.8 91.7 88.6

[0070] From Table 2, it can be seen that the DA201-C macroporous adsorbent can effectively remove the pigment material in the acidizing fluid, and has the best purification effect; and other macroporous adsorbents, including D101, H103 and AB-8, have general impurity removal effect. At the same time, from Table 2, it can be seen that if the pigment cannot be effectively removed, the purity of the final crystalline product will be affected.

[0071] Example 3 The difference between this example and Example 1 is only that step (6) does not produce a crystal film. The other steps are the same as those of Example 1. Step (6) of this example is as follows: The eluent is evaporated and concentrated by a rotary evaporator with a water bath temperature of 40℃ and a vacuum degree of 0.1MPa. The system is observed at all times to prevent the formation of a crystal film (if a crystal film appears, the experiment is stopped and redone). When the volume is concentrated to 1 / 5, the concentration is stopped, and the concentration of β-alanine in the system is 389g / L. After the concentration is stopped, the system is transferred to 4℃ for 10 hours of cooling crystallization. After filtration and drying of the crystals, β-alanine is obtained. The purity of β-alanine is 89.8% as detected by high performance liquid chromatography, and the crystallization process has a yield of 70.1%.

[0072] Therefore, by evaporating and concentrating to form a crystal film, which is used as a crystal seed, a large amount of β-alanine crystals can be precipitated during the cooling crystallization process, which is beneficial to the extraction of pure β-alanine and improves the high yield of pure β-alanine.

[0073] The raw materials and equipment used in the present application are conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are conventional methods in the art unless otherwise specified.

[0074] The above description is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change and equivalent transformation of the above embodiment based on the technical essence of the present application are still within the protection scope of the technical solution of the present application.

Claims

1. A method for separating and purifying β-alanine from a fermentation broth, characterized in that: The following steps are involved: Step S1, adding a flocculant to the fermentation broth containing β-alanine to perform flocculation treatment, separate the solid and liquid, and collect the liquid; Step S2, acidifying the liquid obtained in step S1, centrifuging, and collecting the supernatant; Step S3, performing chromatographic adsorption decolorization on the supernatant obtained in step S2, wherein the adsorption decolorization is performed using a chromatography column, and the filling resin of the chromatography column is DA201-C; Step S4, performing chromatographic adsorption elution on the decolorized supernatant to obtain a refined solution, wherein the adsorption elution is performed through a chromatography column, and the filling resin of the chromatography column is a cation exchange resin; Step S5: taking the refined liquid and crystallizing it by combining evaporation concentration and cooling to obtain the refined β-alanine.

2. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S5, the crystallization method is: evaporating and concentrating the refined liquid at a temperature of 40°C to 60°C and a vacuum degree of -0.1 MPa to 0.1 MPa until a crystal film appears on the surface of the liquid, then transferring it to 0-20°C for cooling and crystallization, filtering, and drying to obtain β-alanine crystals.

3. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S3, the sample loading flow rate of the adsorption decolorization is 1-6 BV / h.

4. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S4, the cation exchange resin is selected from HD-8, LX-160, D314, D301, D001, LXT-104, and LX-6703.

5. A method for separating and purifying β-alanine from a fermentation broth according to claim 1 or 4, characterized in that: In step S4, the eluent for the adsorption elution is aqueous ammonia.

6. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S1, the flocculant is a chitosan-sodium alginate composite flocculant.

7. The method for separating and purifying β-alanine from a fermentation broth according to claim 4, wherein: The mass ratio of chitosan to sodium alginate in the chitosan-sodium alginate composite flocculant is (1-7):

1.

8. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S1, the solid-liquid separation method is microfiltration, plate and frame filtration or centrifugation.

9. The method for separating and purifying β-alanine from a fermentation broth according to claim 1, wherein: In step S2, the acidified pH is 2-3.

10. The β-alanine product obtained by separating according to the method according to any one of claims 1 to 9.