Method for separating and extracting L-histidine from fermentation liquor

Abstract

The invention relates to the technical field of biological engineering, in particular to a method for separating and extracting L-histidine from fermentation broth, which comprises the following steps: adjusting the acid of feed liquid, filtering by a ceramic membrane, decolorizing by a carbon column, adjusting the alkali of the feed liquid, removing impurities by a negative column, desalting by nanofiltration, performing column chromatography by a positive column, concentrating and crystallizing to obtain the L-histidine with the purity of more than 99%. Wherein the pH value of the feed liquid after acid adjustment is 4 + / -1; the pore diameter of the ceramic membrane is 15-100 nm; the pH value of the reduced feed liquid is 9 + / -1; and a nanofiltration membrane with the molecular weight of 90-150 Daltons is used for nanofiltration desalination. The extraction method provided by the invention overcomes the problems of complex process route, high energy consumption and the like in the existing production process, and has the advantages of energy conservation and environmental protection, the product purity can reach 99% or above, and the yield can reach 85% or above.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a method for separating and extracting L-histidine from fermentation liquid. Background Art

[0002] Histidine (α-aminoβ-imidazole propionic acid) has a molecular formula of C6H9N3O2 and a molecular weight of 155.15. It is a white crystal or crystalline powder, odorless, slightly bitter, soluble in water, extremely difficult to dissolve in ethanol, and insoluble in ether. Histidine is an essential amino acid for humans. It is found in bananas, grapes, meat, poultry, milk and dairy products. Histidine is also found in green vegetables, but the content is relatively small. Histidine is the basic unit of protein and one of the 21 amino acids that make up human protein. Histidine has a wide range of uses. In medicine, it is the main component of amino acid infusion and comprehensive amino acid preparations. It is used to treat gastric ulcers, anemia, angina pectoris, aortitis, heart failure and other cardiovascular diseases; it can be used as a nutritional enhancer; it is also used in biochemical research and food additives. There are many methods for producing histidine, such as extracting it from pig blood and cow blood; it can also be extracted from the hydrolyzate of defatted soybeans; the industrial production methods include: one is the direct fermentation method, using glucose as the carbon source, and using induced drug-resistant strains of Brevibacterium flavum, Corynebacterium glutamicum, and Seymourne mucosa to produce it; the other is the protein hydrolysis method, using pig blood powder or pig hair or hoof as raw materials, hydrolyzing it with acid, and then separating and refining it.

[0003] CN103360319A discloses a process for extracting and purifying histidine, which specifically includes: firstly, the amino acid mother liquor containing histidine is concentrated and filtered, stored, adsorbed by cation exchange resin, washed and dehydrated, histidine liquid is collected, low pressure permeation is performed, pH value is adjusted, crude product is concentrated, crude product is centrifuged and dried, crude product is dissolved and decolorized, plate and frame filtration is performed, refined and concentrated, refined product is centrifuged and dried, and a series of processes are performed to produce histidine. Although the purity of the finished product of this method is relatively high, the operation process is complicated, multiple concentrations and centrifugations are required before and after, which is time-consuming and labor-intensive, and the mass production cost is relatively high.

[0004] CN114940664A discloses a method for separating and extracting histidine from fermentation broth, which requires two decolorizations after filtration with a ceramic membrane, exchange with a cationic resin, one concentration and redissolution, and one concentration and crystallization to prepare the finished histidine product. The process is complex and the production cost is high.

[0005] Therefore, the present invention aims to provide a method for extracting histidine which has simple process, high purity of finished product, low cost and can be applied to large-scale production. Summary of the invention

[0006] The present invention provides a method for separating and extracting L-histidine from a fermentation broth. The method has high equipment utilization, simple process, a finished product purity greater than 99%, low cost, and can quantitatively produce L-histidine in a more environmentally friendly manner.

[0007] Specifically, the technical solution provided by the present invention is as follows: In a first aspect, the present invention provides a method for separating and extracting L-histidine from a fermentation broth, wherein the method sequentially carries out the steps of acidification of the slurry, ceramic membrane filtration, carbon column decolorization, alkali adjustment of the slurry, anion column impurity removal, nanofiltration desalination, cation column chromatography, concentration, and crystallization to obtain L-histidine with a purity of more than 99%; wherein the pH value of the slurry after acidification is 4±1; the pore size of the ceramic membrane is 15-100nm; the pH value of the slurry after reduction is 9±1; and the nanofiltration desalination uses a nanofiltration membrane with a molecular weight of 90-150 Daltons.

[0008] Preferably, the pore size of the ceramic membrane is 15-50 nm, the working pressure is 0.2-0.3 MPa, and the working temperature is 40-60°C.

[0009] Preferably, the molecular weight of the nanofiltration membrane is 90-100 Daltons, the working pressure is 2.9-3.1 MPa, and the working temperature is 30-45°C.

[0010] Preferably, the carbon column filler is activated carbon particles.

[0011] Preferably, the anion column is an anion resin selected from one of D730, D315 or J18; preferably D730.

[0012] Preferably, the cation column is a strong acid cationic resin selected from one of D75, 650L, and 1850L; preferably D75.

[0013] Preferably, the material after column chromatography is concentrated under reduced pressure to a content of 30-40 wt%, and crystallized and separated to obtain L-histidine pure wet crystals and L-histidine crystallization mother liquor.

[0014] Preferably, the pure L-histidine wet crystals are dried and then ground to obtain pure L-histidine powder with a purity greater than 99%; the L-histidine crystallization mother liquor repeats the steps of anion column impurity removal, nanofiltration desalting, cation column chromatography, concentration, and crystallization.

[0015] Preferably, the drying method is vacuum heating, the drying temperature is 60±10° C., and the drying time is 4±1 h.

[0016] In a second aspect, the present invention provides the use of high-purity L-histidine as a biopharmaceutical raw material or a cosmetic raw material, wherein the high-purity L-histidine is prepared by the method for separating and extracting L-histidine from a fermentation broth.

[0017] Beneficial effects: The present invention provides a method for separating and extracting L-histidine from a fermentation broth, wherein the method sequentially undergoes the steps of acidification of the feed liquid, ceramic membrane filtration, carbon column decolorization, alkali adjustment of the feed liquid, anion column impurity removal, nanofiltration desalination, cation column chromatography, concentration, and crystallization to obtain L-histidine with a purity of more than 99%; wherein the pH value of the feed liquid after acidification is 4±1; the pore size of the ceramic membrane is 15-100nm; the pH value of the feed liquid after reduction is 9±1; and nanofiltration desalination uses a nanofiltration membrane with a molecular weight of 90-150 Daltons. The extraction method of the present invention overcomes the problems of complex process routes and high energy consumption in the existing production process, has the advantages of energy saving and environmental protection, and the product purity can reach more than 99%, and the yield can reach more than 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be described below.

[0019] Figure 1 The process flow chart of extracting L-histidine provided in the embodiment of the present invention. DETAILED DESCRIPTION

[0020] The present invention provides a method for separating and extracting L-histidine from a fermentation broth, the method comprising the following steps: adjusting the pH of the fermentation broth with a strong acid, allowing the broth to settle, and then entering an inorganic membrane separation system to remove most of the proteins, bacteria, pigments and other macromolecular substances in the fermentation broth. Decolorizing the inorganic membrane filtrate with a carbon column, adjusting the pH with a strong base, and performing ion exchange with a strong base anion resin to perform salt release. Desalting the released liquid with nanofiltration and concentrating the feed liquid at the same time. Mixing the nanofiltration concentrate and water washing with a strong acid cationic resin for chromatography, washing with water to obtain a high-purity L-histidine solution, and then performing reduced pressure concentration, crystallization, drying and grinding to obtain a finished L-histidine product.

[0021] In some embodiments of the present invention, the method for separating and extracting L-histidine from the fermentation broth specifically comprises the following steps: (1) Use strong acid to adjust the pH of the fermentation broth to 3-5.

[0022] (2) Use a ceramic membrane with a pore size of 15-100 nm to perform preliminary filtration on the fermentation broth to remove most of the proteins, bacteria and other macromolecular impurities in the fermentation broth.

[0023] (3) Decolorizing the ceramic membrane filtrate by using activated carbon powder, decolorizing resin or carbon column, preferably carbon column, wherein the carbon column filler is activated carbon particles. After carbon column decolorization, a decolorized liquid is obtained.

[0024] (4) The decolorized solution is adjusted to pH 8-10 with a strong base and then pumped into a strong base anion resin D730 for decolorization. A 1-10% (preferably 2%) sodium chloride solution is used as a decolorizing agent to obtain a decolorized solution.

[0025] (5) The strong base anion resin ionization solution is desalted by nanofiltration using a nanofiltration membrane with a molecular weight of 90-150 Daltons, and the solution is concentrated while removing excess salt.

[0026] (6) The nanofiltration concentrate is collected and pumped into a strong acid cationic resin D75 for chromatography transformation, and then flushed with water to obtain a pure L-histidine solution.

[0027] (7) The pure L-histidine solution obtained by chromatography is concentrated under reduced pressure to a content of 30-40 wt % L-histidine, and then crystallized to obtain pure L-histidine wet crystals and L-histidine crystallization mother liquor.

[0028] (8) The pure L-histidine wet crystals are vacuum-heated and dried, and then ground to obtain pure L-histidine powder with a purity greater than 99%.

[0029] Furthermore, the strong acid in step (1) is concentrated hydrochloric acid or concentrated sulfuric acid, preferably concentrated hydrochloric acid. The pH of the fermentation liquid is preferably adjusted to pH=4.

[0030] Furthermore, the ceramic membrane described in step (2) is preferably 15 nm, its working pressure is preferably 0.20-0.30 MPa, preferably 0.25 MPa, and its working temperature is preferably 40-60°C, more preferably 40-55°C.

[0031] Furthermore, the activated carbon particle column mentioned in step (3) is preferably regenerable activated carbon, and the regeneration method is preferably to introduce steam into the carbon column.

[0032] Furthermore, the strong base in step (4) is sodium hydroxide, and the pH of the decolorizing solution is preferably adjusted to pH=9.

[0033] Furthermore, the anion resin described in step (4) preferably includes D730, D315, and J18, and D730 is more preferably used as the ionization resin of the L-histidine decolorizing solution.

[0034] Furthermore, the molecular weight of the nanofiltration membrane in step (5) is preferably 90 Daltons, its working pressure is preferably 3 MPa, and its temperature is preferably 30-45°C.

[0035] Furthermore, the strong acid cationic resin described in step (6) preferably includes strong acid cationic resins such as D75, 650L, 1850L, etc., and D75 strong acid cationic resin is more preferably used as the chromatography resin for the nanofiltration concentrate.

[0036] Furthermore, the crystallization method described in step (7) is preferably reduced pressure concentration, with high vacuum degree, low concentrated liquid temperature and high concentrated liquid concentration. When the feed liquid concentration reaches a certain level, it can be taken out and directly separated, and the separation method can be selected from centrifugal separation or suction filtration separation. Centrifugal separation is preferred.

[0037] Furthermore, the L-histidine crystallization mother liquor obtained in step 7 is recycled to step (4) to achieve the purpose of mother liquor recovery, thereby improving the overall yield.

[0038] Furthermore, the drying method in step (8) can be freeze drying or vacuum heating drying, preferably vacuum heating drying, and the drying method is 60° C. for 4 hours.

[0039] In summary, the present invention provides a method for separating and extracting L-histidine from a fermentation broth, the method comprising: adjusting the pH of the fermentation broth to 4 using a strong acid, and removing impurities such as bacteria, most proteins, and pigments in the fermentation broth using an inorganic membrane separation system to achieve a preliminary purification effect. The feed liquid after inorganic membrane filtration is pumped into a decolorization column filled with activated carbon particles to obtain a decolorized liquid. The decolorized liquid is adjusted to pH=9 using a strong base and then separated using a strong base anion resin. A certain concentration of salt solution is used as a release agent to obtain a release liquid. The salt in the release liquid is removed by nanofiltration membrane filtration, and the release liquid is concentrated at the same time. The concentrated solution after nanofiltration is pumped into a strong acid cation resin column for chromatography, and water is washed to obtain a pure L-histidine solution, and the cation column water wash liquid is concentrated under reduced pressure to an L-histidine concentration of 30%-40%, crystallized and separated, and dried to obtain L-histidine with a purity of more than 99%. Compared with the prior art, the present invention provides a method of using a ceramic membrane to remove most of the impurities and then using a carbon column for decolorization, which has a high yield, good decolorization effect, and can be reused, which is green and environmentally friendly. The anionic resin column removes most impurities such as pigments and proteins. Nanofiltration desalination and concentration improves the purity of the solution while concentrating the solution, achieving the purpose of saving energy while removing impurities and reducing costs. Cationic resin chromatography eliminates the use of a release solution, and transforms L-histidine under the condition of using only deionized water to obtain pure L-histidine. It reduces the generation of sewage, protects the environment and reduces costs. The concentration of L-histidine in the concentrate is increased by vacuum concentration, and the vacuum degree of vacuum concentration is also high. At the end of concentration, the temperature of the concentrate is room temperature. At this time, due to the low solubility of L-histidine, centrifugal separation can be directly performed. This method optimizes the crystallization process. The complex cooling crystallization is removed, and time and energy consumption can be saved under the condition of similar yields. Centrifugal separation is preferably used for crystallization separation. It is found that L-histidine is easy to agglomerate during suction filtration, and it is not easy to separate by suction filtration, and the moisture content of the obtained L-histidine wet crystals is large. Vacuum heating drying is selected during crystallization drying. The L-histidine finished product obtained by freeze drying has high moisture content and is easy to harden. Vacuum heating and drying can avoid high moisture content in the finished product and prevent the finished product from becoming hardened. After grinding, L-histidine with a purity greater than 99% is obtained.

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some specific implementations" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0044] In the following examples, the purity of the L-histidine product was determined using a Spursil high performance liquid chromatograph, with a C18 column, a column temperature of 35° C., a mobile phase of 10% acetonitrile, a flow rate of 0.5 mL / min, and an ultraviolet detection wavelength of 248 nm.

[0045] Example 1 This embodiment provides a method for separating and extracting L-histidine from a fermentation broth. The process flow is as follows: Figure 1 As shown, the specific steps are as follows: a. Take 30L of fermentation liquid with L-histidine concentration of 70g / L, detect its pH=6.89, use concentrated hydrochloric acid to adjust the fermentation liquid to pH=4 while stirring, stir evenly and prepare for filtration.

[0046] b. Pump the fermentation liquid with pH=4 obtained in step a into the inorganic membrane separation system. The inorganic membrane is a 15nm ceramic membrane for filtration. The working pressure is 0.25MPa and the working temperature is 45°C. When the solid content of the clear liquid side discharge of the filtrate is lower than 5g / L, stop filtration.

[0047] c. Pump the filtrate collected in step b directly into the decolorization carbon column filled with activated carbon particles, collect the decolorized liquid after the activated carbon particles, use pure water to top wash the carbon column, and stop top washing when the solid content of the solution flowing out of the carbon column is less than 10g / L. The light transmittance of the decolorized liquid is 99.5%, and steam is injected into the carbon column to regenerate the activated carbon.

[0048] d. The decolorizing solution collected in step c is adjusted to pH=9 using sodium hydroxide.

[0049] e. Pump the decolorized solution with pH=9 obtained in step d into the strong base anion resin D730. After L-histidine is completely adsorbed, flush with water until the conductivity of the resin column effluent is lower than 200μs / cm. At this time, a 2% sodium chloride solution is used as a release agent to release the adsorbed L-histidine at a flow rate of 1BV / h. At the end of the release, the conductivity of the ion exchange effluent is 20ms / cm, and the collection of the release solution is stopped at this time.

[0050] f. The release solution obtained in step e is pumped into a nanofiltration membrane device, and a 90-Dalton nanofiltration membrane is used to desalt and remove impurities from the release solution. The pressure of the device is 3 MPa and the working temperature is 40° C. The feed liquid is concentrated while desalting. The nanofiltration concentrate is obtained by dialyzing with 50 wt% of the feed liquid with deionized water.

[0051] g. Pump the nanofiltration concentrate obtained in step f into the strong acid cationic resin D75 for chromatography. After the concentrate is completely loaded onto the column, it is rinsed with deionized water to obtain a high-purity L-histidine chromatographic solution.

[0052] h. Pump the high-purity L-histidine chromatographic solution obtained in step g into a vacuum concentration device, and stop concentrating the chromatographic solution when the L-histidine content is 35wt%. After collecting the concentrated solution, directly separate it, and use a centrifuge to centrifuge to obtain pure L-histidine wet crystals.

[0053] i. The pure L-histidine wet crystals obtained in step h were dried by vacuum heating at 60° C. for 4 h, and then ground to obtain 1905.6 g of pure L-histidine with a total yield of 90.74%.

[0054] The pure L-histidine obtained in step i was diluted with deionized water and measured using a Spursil high performance liquid chromatograph, and the purity of the L-histidine was found to be 99.85%.

[0055] Example 2 This embodiment provides a method for separating and extracting L-histidine from a fermentation broth. The process flow is as follows: Figure 1 As shown, the specific steps are as follows: a. Take 30L of fermentation liquid with an L-histidine concentration of 60g / L, detect its pH = 6.84, use concentrated hydrochloric acid to adjust the fermentation liquid to pH = 4 while stirring, stir evenly and prepare for filtration.

[0056] b. Pump the fermentation liquid with pH=4 obtained in step a into an inorganic membrane separation system. A 50nm ceramic membrane is selected for filtration. The working pressure is 0.2MPa and the working temperature is 45°C. When the solid content of the clear liquid side discharge of the filtrate is lower than 5g / L, stop filtration.

[0057] c. Pump the filtrate collected in step b directly into a decolorizing carbon column filled with activated carbon particles, collect the decolorized liquid after the activated carbon particles, use pure water to top wash the carbon column, and stop top washing when the solid content of the solution flowing out of the carbon column is less than 10g / L. The light transmittance of the decolorized liquid is 99.5%. Inject steam into the carbon column to regenerate the activated carbon.

[0058] d. The decolorizing solution collected in step c is adjusted to pH=9 using sodium hydroxide.

[0059] e. Pump the decolorized solution with pH=9 obtained in step d into the strong base anion resin D730. After L-histidine is completely adsorbed, flush with water until the conductivity of the resin column effluent is lower than 200μs / cm. At this time, a 2% sodium chloride solution is used as a release agent to release the adsorbed L-histidine at a flow rate of 1BV / h. At the end of the release, the conductivity of the ion exchange effluent is 20ms / cm, and the collection of the release solution is stopped at this time.

[0060] f. The release solution obtained in step e is pumped into a nanofiltration membrane device, and a 150 Dalton nanofiltration membrane is used to desalt and remove impurities from the release solution. The pressure of the device is 3 MPa and the working temperature is 40° C. The feed liquid is concentrated while desalting. The nanofiltration concentrate is obtained by dialyzing with 50 wt% deionized water.

[0061] g. Pump the nanofiltration concentrate obtained in step f into the strong acid cationic resin D75 for chromatography. After the concentrate is completely loaded onto the column, it is rinsed with deionized water to obtain a high-purity L-histidine chromatographic solution.

[0062] h. Pump the high-purity L-histidine chromatographic solution obtained in step g into a vacuum concentration device, and stop concentrating the chromatographic solution when the L-histidine content is 35wt%. After collecting the concentrated solution, directly separate it, and use a centrifuge to centrifuge to obtain pure L-histidine wet crystals.

[0063] i. The pure L-histidine wet crystals obtained in step h were dried by vacuum heating at 60° C. for 4 h, and then ground to obtain 1593.6 g of pure L-histidine with a total yield of 88.53%.

[0064] The pure L-histidine obtained in step i was diluted with deionized water and measured using a Spursil high performance liquid chromatograph, and the purity of the L-histidine was found to be 99.03%.

[0065] Example 3 This embodiment provides a method for separating and extracting L-histidine from a fermentation broth. The process flow is as follows: Figure 1 As shown, the specific steps are as follows: a. Take 30L of fermentation liquid with L-histidine concentration of 65g / L, detect its pH=6.82, use concentrated hydrochloric acid to adjust the fermentation liquid to pH=4 while stirring, stir evenly and prepare for filtration.

[0066] b. Pump the fermentation liquid with pH=4 obtained in step a into the inorganic membrane separation system. The inorganic membrane is a 15nm ceramic membrane for filtration. The working pressure is 0.25MPa and the working temperature is 45°C. When the solid content of the clear liquid side discharge of the filtrate is lower than 5g / L, stop filtration.

[0067] c. Pump the filtrate collected in step b directly into the decolorization carbon column filled with activated carbon particles, collect the decolorized liquid after the activated carbon particles, use pure water to top wash the carbon column, and stop top washing when the solid content of the solution flowing out of the carbon column is less than 10g / L. The light transmittance of the decolorized liquid is 99.5%, and steam is injected into the carbon column to regenerate the activated carbon.

[0068] d. The decolorizing solution collected in step c is adjusted to pH=9 using sodium hydroxide.

[0069] e. Pump the decolorized solution with pH=9 obtained in step d into the strong base anion resin D315. After L-histidine is completely adsorbed, flush with water until the conductivity of the resin column effluent is lower than 200μs / cm. At this time, a 2% sodium chloride solution is used as a release agent to release the adsorbed L-histidine at a flow rate of 1BV / h. At the end of the release, the conductivity of the ion exchange effluent is 20ms / cm, and the collection of the release solution is stopped at this time.

[0070] f. The release solution obtained in step e is pumped into a nanofiltration membrane device, and a 90-Dalton nanofiltration membrane is used to desalt and remove impurities from the release solution. The pressure of the device is 3 MPa and the working temperature is 40° C. The feed liquid is concentrated while desalting. The nanofiltration concentrate is obtained by dialyzing with 50 wt% of the feed liquid with deionized water.

[0071] g. Pump the nanofiltration concentrate obtained in step f into 650 L of strong acid cationic resin for chromatography. After the concentrate is completely loaded onto the column, it is rinsed with deionized water to obtain a high-purity L-histidine chromatographic solution.

[0072] h. Pump the high-purity L-histidine chromatographic solution obtained in step g into a vacuum concentration device, and stop concentrating the chromatographic solution when the L-histidine content is 35wt%. After collecting the concentrated solution, directly separate it, and use a centrifuge to centrifuge to obtain pure L-histidine wet crystals.

[0073] i. The pure L-histidine wet crystals obtained in step h were dried by vacuum heating at 60° C. for 4 h, and then ground to obtain 1694.5 g of pure L-histidine with a total yield of 86.9%.

[0074] The pure L-histidine obtained in step i was diluted with deionized water and measured using a Spursil high performance liquid chromatograph, and the purity of the L-histidine was found to be 98.72%.

[0075] Comparative Example 1 This comparative example provides a method for separating and extracting L-histidine from a fermentation broth, and the specific steps are as follows: a. Take 30L of fermentation liquid with L-histidine concentration of 62g / L, detect its pH=6.93, use concentrated hydrochloric acid to adjust the fermentation liquid to pH=4 while stirring, stir evenly and prepare for filtration.

[0076] b. Pump the fermentation liquid with pH=4 obtained in step a into the inorganic membrane separation system. The inorganic membrane is a 15nm ceramic membrane for filtration. The working pressure is 0.25MPa and the working temperature is 45°C. When the solid content of the clear liquid side discharge of the filtrate is lower than 5g / L, stop filtration.

[0077] c. Pump the filtrate collected in step b directly into a decolorizing carbon column filled with activated carbon particles, collect the decolorized liquid after the activated carbon particles, use pure water to top wash the carbon column, and stop top washing when the solid content of the solution flowing out of the carbon column is less than 10g / L. The light transmittance of the decolorized liquid is 99.5%. Inject steam into the carbon column to regenerate the activated carbon.

[0078] d. The decolorizing solution collected in step c is adjusted to pH=9 using sodium hydroxide.

[0079] e. Pump the decolorized solution with pH=9 obtained in step d into the strong base anion resin D730. After L-histidine is completely adsorbed, flush with water until the conductivity of the resin column effluent is lower than 200μs / cm. At this time, a 2% sodium chloride solution is used as a release agent to release the adsorbed L-histidine at a flow rate of 1BV / h. At the end of the release, the conductivity of the ion exchange effluent is 20ms / cm, and the collection of the release solution is stopped at this time.

[0080] f. Pump the release solution obtained in step e into the strong acid cationic resin D75 for chromatography. After the concentrated solution is completely loaded onto the column, it is rinsed with deionized water to obtain a high-purity L-histidine chromatographic solution.

[0081] g. Pump the high-purity L-histidine chromatographic solution obtained in step f into a vacuum concentration device, and stop concentrating the chromatographic solution when the L-histidine content is 35wt%. After collecting the concentrated solution, directly separate it and use a centrifuge to centrifuge to obtain pure L-histidine wet crystals.

[0082] h. The pure L-histidine wet crystals obtained in step g were dried by vacuum heating at 60° C. for 4 h, and then ground to obtain 1458.7 g of pure L-histidine with a total yield of 78.42%.

[0083] The pure L-histidine obtained in step h was diluted with deionized water and measured using a Spursil high performance liquid chromatograph, and the purity of the L-histidine was found to be 96.62%.

[0084] Comparative Example 2 This comparative example provides a method for separating and extracting L-histidine from a fermentation broth. The difference between this method and Example 1 is that in step c, the decolorization method is changed from decolorization by an activated carbon particle column to decolorization by a decolorizing resin. The light transmittance of the decolorized liquid is 94%. As a result, the yield of L-histidine is 78.13% and the purity is 97.45%.

[0085] Comparative Example 3 This comparative example provides a method for separating and extracting L-histidine from a fermentation broth, which differs from Example 1 in that in step i, vacuum heating drying is replaced by vacuum freeze drying.

[0086] Results showed that the yield of L-histidine was 79.56% and the purity was 95.32%. Compared with the finished product dried by vacuum heating, the freeze-dried product is easy to harden and difficult to dry, and the high water content of the finished product affects its purity.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating and extracting L-histidine from a fermentation broth, characterized in that: The process is sequentially followed by acidification of the feed solution, ceramic membrane filtration, carbon column decolorization, base adjustment of the feed solution, anion column impurity removal, nanofiltration desalination, cation column chromatography, concentration, and crystallization to obtain L-histidine with a purity of more than 99%. The pH value of the feed solution after acidification is 4±1. The pore size of the ceramic membrane is 15-100 nm. The pH value of the feed solution after reduction is 9±1. Nanofiltration desalination uses a nanofiltration membrane with a molecular weight of 90-150 Daltons.

2. The method for separating and extracting L-histidine from a fermentation broth according to claim 1, characterized in that: The pore size of the ceramic membrane is 15-50nm, the working pressure is 0.2-0.3MPa, and the working temperature is 40-60℃.

3. The method for separating and extracting L-histidine from a fermentation broth according to claim 1 or 2, characterized in that: The molecular weight of the nanofiltration membrane is 90-100 Daltons, the working pressure is 2.9-3.1MPa, and the working temperature is 30-45°C.

4. The method for separating and extracting L-histidine from a fermentation broth according to any one of claims 1 to 3, characterized in that: The carbon column is filled with activated carbon particles.

5. The method for separating and extracting L-histidine from a fermentation broth according to any one of claims 1 to 4, characterized in that: The anion column is an anion resin selected from one of D730, D315 or J18, preferably D730.

6. The method for separating and extracting L-histidine from a fermentation broth according to any one of claims 1 to 5, characterized in that: The positive column is a strong acid cationic resin selected from one of D75, 650L, and 1850L; preferably D75.

7. The method for separating and extracting L-histidine from a fermentation broth according to any one of claims 1 to 6, characterized in that: The material after column chromatography is concentrated under reduced pressure to a content of 30-40 wt%, and then crystallized and separated to obtain L-histidine pure wet crystals and L-histidine crystal mother liquor.

8. The method for separating and extracting L-histidine from a fermentation broth according to claim 7, characterized in that: The pure L-histidine wet crystals are dried and then ground to obtain pure L-histidine powder with a purity greater than 99%; the L-histidine crystal mother liquor repeats the steps of anion column impurity removal, nanofiltration desalting, cation column chromatography, concentration, and crystallization.

9. The method for separating and extracting L-histidine from a fermentation broth according to claim 8, characterized in that: The drying method is vacuum heating, the drying temperature is 60±10℃, and the drying time is 4±1h.

10. Application of high-purity L-histidine as a biopharmaceutical raw material or a cosmetic raw material, characterized in that: The high-purity L-histidine is prepared by the method for separating and extracting L-histidine from fermentation broth according to any one of claims 1 to 9.

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