Extraction method of L-histidine

Abstract

The invention provides an extraction method of L-histidine. The extraction method comprises the following steps: S1) carrying out filtration and first decoloration on an L-histidine fermentation liquid to obtain a first liquid; s2) performing first crystallization treatment on the first liquid to obtain a first crystallization product and first crystallization mother liquor; s3) performing continuous chromatographic purification on the first crystallization mother liquor to obtain second liquid; and S4) mixing the first crystallization product with a second liquid, and carrying out second crystallization treatment to obtain the L-histidine. The method can solve the problem that an extraction method of L-histidine in the prior art is difficult to carry out industrial enlarged production, and is suitable for the field of extraction of L-histidine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of L-histidine extraction, in particular, to a method for extracting L-histidine. BACKGROUND

[0002] L-histidine is a semi-essential amino acid, which is particularly important for the growth of infants and animals. It can be used as a biochemical reagent and a medicament, and can also be used in the preparation of drugs for treating heart disease, anemia, rheumatoid arthritis, etc. The main applications are: preparation of synthetic myopeptide, amino acid infusion, protection of amino acids, preparation of nutritional additives and preparation of feed additives, etc. The methods for producing histidine mainly include protein hydrolysis, chemical synthesis and microbial fermentation.

[0003] 1) Protein hydrolysis method

[0004] The protein hydrolysis method is the most traditional method for producing histidine, which mainly extracts L-histidine from pig blood, cow blood and soybean powder. The operation method is as follows: mix blood powder, industrial hydrochloric acid and distilled water, oil bath reflux for 24 h, hydrolyze at -4℃ for 2 d, and remove impurities by filtration. The filtrate is adjusted to pH 2.5 with hydrochloric acid, and then column chromatography is performed, and distilled water is used for washing until pH = 5-6. This method mainly depends on the availability of natural resources, but the yield is low and it is difficult to meet the demand for histidine; and due to the prohibition of animal-derived feed in ruminant feed, the development of this process has been limited.

[0005] 2) Chemical synthesis method

[0006] The chemical synthesis method has the advantage of being able to prepare amino acids of various types. The disadvantages are long cycle, low yield, and complicated steps. Moreover, the product obtained by chemical synthesis is a DL racemate, and the racemic mixture is a "non-natural" compound, which does not meet the standards of the Food and Drug Administration and is difficult for consumers to accept. Finally, in order to obtain L or D amino acids, optical resolution is required, which is a costly method.

[0007] 3) Biological fermentation method

[0008] At present, the mainstream method for producing L-histidine is microbial fermentation. This method uses microorganisms to synthesize target products through metabolic pathway transformation under suitable conditions. L-histidine prepared by microbial fermentation and extraction and purification has controllable risk of virus introduction and controllable quality, and is suitable for the preparation of pharmaceutical raw materials. However, due to the low content of L-histidine in the fermentation broth and the presence of other amino acid impurities, separation and purification treatment is required.

[0009] In the prior art, the fermentation liquor is filtered through a ceramic membrane and then subjected to continuous chromatographic resin separation. In the ceramic membrane treatment process, in order to ensure the yield, after the impurities are intercepted, additional water is added for dialysis, and then the ceramic clear liquid is directly subjected to chromatographic purification to obtain a crude crystal with a high crystallization rate, so as to improve the yield and purity of the product. However, the volume of the ceramic clear liquid obtained by this method is 2-3 times larger than that of the initial fermentation liquor, which leads to a too large volume for subsequent treatment, a large amount of resin, a too large amount of water for eluting the resin, an increased subsequent concentration power loss, a high cost, and is not conducive to industrial production. Therefore, it is necessary to develop a process for extracting L-histidine from fermentation liquor, which can reduce energy consumption under the premise of ensuring the recovery rate and is suitable for industrial scale-up production. SUMMARY

[0010] The main purpose of the present application is to provide an extraction method of L-histidine to solve the problem that the extraction method in the prior art is difficult to be industrialized and scaled up.

[0011] In order to achieve the above-mentioned purpose, according to a first aspect of the present application, an extraction method of L-histidine is provided, which comprises: S1) filtering and first decolorizing L-histidine fermentation liquor to obtain a first liquid; S2) subjecting the first liquid to first crystallization treatment to obtain a first crystallization product and a first crystallization mother liquor; S3) subjecting the first crystallization mother liquor to continuous chromatographic purification to obtain a second liquid; and S4) mixing the first crystallization product and the second liquid to perform second crystallization treatment to obtain L-histidine.

[0012] Further, S1) comprises: S1-1) filtering the L-histidine fermentation liquor to obtain a first filtrate; S1-2) filtering the first filtrate to obtain a second filtrate; S1-3) mixing the second filtrate with a first decolorizing agent to perform first decolorization to obtain a first decolorized liquid; and S1-4) performing first concentration on the first decolorized liquid to obtain the first liquid; the content of L-histidine in the first liquid is 200-300 g / L; preferably, the vacuum degree of the first concentration is -0.085 to -0.095 MPa; and preferably, the temperature of the first concentration is 30-70℃.

[0013] Further, in S1-1), the first filtration comprises ceramic membrane filtration; preferably, the content of L-histidine in the first filtrate is 20-35 g / L; and preferably, the volume ratio of the first filtrate to the L-histidine fermentation liquor is 1.1-2:1.

[0014] Further, in S1-2), preferably, the second filtration comprises nanofiltration; preferably, the size of the filtration membrane for nanofiltration is 500-2000 da; preferably, the temperature of the second filtration is 1-35℃; and preferably, the pressure of the second filtration is 1-2 MPa.

[0015] Further, the temperature of the first decoloring in S1-3) is 50-70℃, and the time of the first decoloring is 30-60min; preferably, the first decoloring agent comprises activated carbon; preferably, the addition amount of the first decoloring agent is 0.5%-1.0% of the mass volume of the second filtrate.

[0016] Further, in S2), the temperature of the first crystallization treatment is 5-10℃, and the time is 2-5h; preferably, after the first crystallization treatment, centrifugation is performed to obtain the first crystallization product and the first crystallization mother liquor.

[0017] Further, in S3), the first crystallization mother liquor is subjected to continuous chromatography purification using a chromatography resin and a purification agent; wherein the chromatography resin comprises zgspc106-Na, LX-38S or LX-40S, and more preferably zgspc106-Na; preferably, the purification agent is water.

[0018] Further, the volume ratio of the purification agent to the first crystallization mother liquor is 1.1-1.3:1.

[0019] Further, in S3), after the continuous chromatography purification, the chromatography resin is eluted to obtain the second liquid and the purification residue; the volume ratio of the second liquid to the purification residue is 0.9-1.0:1.

[0020] Further, S4) comprises: S4-1) mixing the first crystallization product, the second liquid and water to obtain a third liquid with the content of L-histidine being 40-50g / L; S4-2) mixing the third liquid with a second decoloring agent to perform a second decoloring treatment to obtain a second decoloring liquid; S4-3) performing a second concentration and a second crystallization treatment on the second decoloring liquid to obtain L-histidine; preferably, the second decoloring agent comprises activated carbon; preferably, the addition amount of the second decoloring agent is 0.5%-1.0% of the mass volume of the third liquid; preferably, the time of the second decoloring is 30-60min, and the temperature is 50-70℃; preferably, the vacuum degree of the second concentration is -0.085--0.095MPa; preferably, the temperature of the second concentration is 50-70℃; preferably, the temperature of the second crystallization treatment is 5-10℃, and the time is 2-5h.

[0021] The technical scheme of the present application is used to filter and first decolorize the L-histidine fermentation liquor to obtain a first liquid from which most of the fermentation liquor bacteria and impurities are removed, and then directly use the first liquid for crystallization treatment to obtain a first crystallization product containing L-histidine crude product and a first crystallization mother liquor. The first crystallization mother liquor is subjected to continuous chromatography purification to obtain a chromatography clear liquid, i.e. a second liquid. In this process, since the filtration treatment and the decolorization treatment are performed, and most of the bacterial impurities and macromolecular impurities are removed after the crystallization, the amount of resin consumed after the chromatography purification is completed is small, and the amount of water used for subsequent elution is also reduced. Then, the second liquid is used as a dissolving agent of the first crystallization product, mixed with the first crystallization product, and subjected to final crystallization treatment (second crystallization treatment), thereby improving the recovery rate of L-histidine. DETAILED DESCRIPTION

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0023] As mentioned in the background, in the prior art, after removing the bacteria by ceramic membrane filtration, chromatography purification is usually performed on the L-histidine in the fermentation liquor. However, due to the special properties of the fermentation liquor, water dialysis is performed to increase the volume to 2-3 times during the membrane filtration, which makes the subsequent chromatography purification treatment large, consumes a large amount of resin, and consumes a large amount of eluent during elution. This extraction method has high energy consumption and high cost, and is therefore not suitable for industrialized scale production. Therefore, the inventors attempt to develop an L-histidine extraction method more suitable for industrialized scale production, which reduces the consumption during chromatography purification of L-histidine in the fermentation liquor, and thus propose a series of protection schemes in the present application.

[0024] In the first typical embodiment of the present application, an L-histidine extraction method is provided, which comprises: S1) filtering and first decolorizing the L-histidine fermentation liquor to obtain a first liquid; S2) performing first crystallization treatment on the first liquid to obtain a first crystallization product and a first crystallization mother liquor; S3) performing continuous chromatography purification on the first crystallization mother liquor to obtain a second liquid; and S4) mixing the first crystallization product and the second liquid to perform second crystallization treatment to obtain L-histidine.

[0025] L-histidine fermentation broth contains a large amount of organic pigments, other amino acid impurities, and some inorganic salt impurities, requiring purification to obtain the final product. The high content of solids such as raw materials, cell bodies, and impurities in the fermentation broth necessitates additional water dialysis after filtration to remove impurities, in order to ensure crystallization quality and yield, current techniques require this to avoid raw material loss. However, the volume of the initial filtrate obtained through this method is 2-3 times that of the initial fermentation broth, and the volume of the chromatographically purified supernatant is approximately 3-4 times that of the initial fermentation broth. This large processing volume leads to higher resin and elution water consumption, increasing the energy loss for subsequent concentration and resulting in higher costs. Furthermore, recrystallization is still required to obtain the product, making the operation complex and unsuitable for industrial production.

[0026] In this application, after filtering and decolorizing the L-histidine fermentation broth, a first crystallization is performed before continuous chromatographic purification to obtain a first crystalline product and a first crystallization mother liquor. At this point, the first crystalline product is the crude L-histidine crystal, which already contains most of the L-histidine component. The first crystallization mother liquor containing a small amount of L-histidine is continuously pumped into the continuous chromatographic purification system of this application for continuous chromatographic purification, reducing the throughput of continuous chromatographic purification, resin usage, and subsequent eluent usage. After continuous chromatographic purification, a second liquid is obtained. The first crystalline product and the second liquid are then mixed, with the second liquid used as a solvent for the first crystalline product, for a second crystallization treatment, thus obtaining a purified L-histidine product with increased yield. Therefore, compared with existing technologies, the L-histidine extraction method in this application reduces energy consumption, improves the purification effect of the mother liquor and the recovery rate of the final product, and also reduces wastewater discharge, making it more suitable for industrial-scale production.

[0027] In a preferred embodiment, S1) includes: S1-1) filtering the L-histidine fermentation broth first to obtain a first filtrate; S1-2) filtering the first filtrate second to obtain a second filtrate; S1-3) mixing the second filtrate with a first decolorizing agent for a first decolorization, followed by a third filtration to obtain a first decolorized liquid; S1-4) concentrating the first decolorized liquid first to obtain a first liquid; the L-histidine content in the first liquid is 200-300 g / L, including but not limited to... 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 g / L; preferably, the vacuum degree of the first concentration is -0.095 to -0.085 MPa, including but not limited to -0.095, -0.093, -0.09 or -0.085 MPa; preferably, the temperature of the first concentration is 30 to 70°C, including but not limited to 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C or 70°C.

[0028] This application, after filtering and top-water dialysis of the L-histidine fermentation broth, does not directly perform chromatographic purification. Instead, it further concentrates the filtrate to obtain a first liquid for subsequent crystallization. The L-histidine fermentation broth undergoes two filtration processes: the first filtration is preferably ceramic membrane filtration, and the second filtration is preferably nanofiltration membrane filtration. After the first and second filtrations, the product concentration in the fermentation broth is further concentrated to obtain a second filtrate. To improve product yield, the second filtrate still needs to be top-water dialysis. However, if the filtrate is directly subjected to chromatographic processing as in existing technologies, the volume of the filtrate after top-water dialysis is also large, leading to increased water and resin consumption during chromatographic processing. This application further concentrates the second filtrate to obtain the first liquid, and then chromatographically purifies the crystallization mother liquor obtained from crystallization. The volume of this crystallization mother liquor is much smaller than that of the second filtrate, significantly reducing the water and resin consumption in subsequent chromatographic processing, lowering the overall extraction cost, and maintaining a high yield and purity of the final product. Compared with existing technologies, this is more suitable for industrial-scale production.

[0029] This application further controls the concentration of L-histidine in the first liquid to be maintained at 200-300 g / L by controlling the vacuum degree and temperature of the first concentration within the aforementioned range, facilitating the subsequent first crystallization process. If the concentration of L-histidine in the first liquid is too high, the crystals will be too viscous during the crystallization process, making centrifugation difficult and affecting product quality and subsequent industrial production operations. However, if the concentration of the first solute is slightly lower, although the crystallization difficulty is reduced, the final product content in the crystals will be low, and the final mother liquor volume will be large, increasing the pressure of chromatographic processing. Therefore, controlling the concentration of the first liquid within the aforementioned range is beneficial to crystal formation, improves crystallization efficiency, and also helps to increase the yield of L-histidine in the final product.

[0030] In a preferred embodiment, in S1-1), the first filtration includes ceramic membrane filtration; preferably, the L-histidine content in the first filtrate is 20 g / L to 35 g / L, including but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 27.6, 28, 29, 30, 31, 32, 33, 34 or 35 g / L; preferably, the volume ratio of the first filtrate to the L-histidine fermentation broth is 1.1 to 2:1, including but not limited to 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1.

[0031] After the first filtration, this application obtains a first filtrate with a volume ranging from 1.1 to 2 times the original fermentation broth volume. This first filtrate allows for the preliminary filtration and separation of the bacterial cells and some proteins. Compared with the filtrate obtained by preliminary ceramic membrane filtration of L-histidine fermentation broth in the prior art, the volume is reduced, further reducing the amount of resin used, the workload of elution, and the amount of liquid consumed in chromatographic purification. This reduces the energy consumption and cost of subsequent purification steps. Furthermore, by controlling the concentration of L-histidine in the first filtrate to be between 20 g / L and 35 g / L, bacterial cells and some protein impurities are filtered out, ensuring the effectiveness of the first filtration and facilitating the subsequent first crystallization process.

[0032] In a preferred embodiment, in S1-2), preferably, the second filtration includes nanofiltration; preferably, the nanofiltration membrane size is 500-2000 da, including but not limited to 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 da; preferably, the temperature of the second filtration is 1-35°C, including but not limited to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C. The temperature is ℃, 25℃, 30℃ or 35℃; preferably, the pressure of the second filtration is 1 to 2 MPa, including but not limited to 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 MPa; preferably, the acid content of the second filtrate is 1 to 6 g / L, including but not limited to 1, 2, 3, 4, 5 or 6 g / L; preferably, the concentration of L-histidine in the second filtrate is 20 to 30 g / L, including but not limited to 20, 21, 22, 23, 24, 25, 26, 27, 27.6, 28, 29 or 30 g / L.

[0033] In the second filtration, the molecular weight cutoff of the nanofiltration system is controlled to be 500–2000 da, preferably 800–1500 da, and more preferably 1000–1200 da. The pressure and temperature range of the nanofiltration system are also controlled to improve filtration efficiency and ensure the effectiveness of the second filtration. The second filtration further separates and filters most of the large molecular impurities, proteins, and organic pigments present in the first filtrate.

[0034] In a preferred embodiment, the temperature of the first decolorization in S1-3) is 50-70°C, including but not limited to 50, 55, 60, 65 or 70°C; the time of the first decolorization is 30-60 min, including but not limited to 30, 35, 40, 45, 50, 55 or 60 min; preferably, the first decolorizing agent includes activated carbon; preferably, the amount of the first decolorizing agent added is 0.5%-1.0% (w / v) of the mass volume of the second filtrate, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%; preferably, the concentration of L-histidine in the first decolorizing solution is 20-30 g / L, including but not limited to 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 g / L.

[0035] This application improves the efficiency and effectiveness of decolorization by controlling the conditions of decolorization treatment, including temperature, time, and the amount of decolorizing agent added, to decolorize the second filtrate. This removes residual pigments, metabolic products of fermentation cells, and impurities from the second filtrate, increases the purity of the product, and obtains a first decolorized solution with an L-histidine concentration of 20-30 g / L, which facilitates subsequent separation and purification.

[0036] In a preferred embodiment, in S2), the temperature of the first crystallization treatment is 5-10°C, including but not limited to 5°C, 6°C, 7°C, 8°C, 9°C or 10°C, and the time is 2-5 hours, including but not limited to 2, 3, 4 or 5 hours; preferably, after the first crystallization treatment is completed, centrifugation is performed to obtain the first crystallization product and the first crystallization mother liquor.

[0037] After filtration, decolorization, and concentration processes (S1-1) to (S1-4), the obtained first liquid is directly subjected to a first crystallization process. By controlling the low-temperature range and processing time of the first crystallization process, most of the L-histidine in the first liquid can be crystallized to obtain the first crystalline product, namely crude L-histidine crystals. The remaining L-histidine and some impurities in the first crystallization mother liquor that did not precipitate during the first crystallization process are subjected to subsequent chromatographic purification. Chromatographic purification of the first crystallization mother liquor reduces the processing volume and energy consumption of continuous chromatographic purification. Compared with existing technologies, water consumption and resin consumption are significantly reduced, lowering the cost of the chromatographic purification process and further reducing the overall extraction process cost.

[0038] After removing residual impurities such as pigments, salts, and miscellaneous amino acids from the first crystallization mother liquor by chromatographic purification, a second liquid with high product purity is obtained. In this application, the second liquid is used as a solvent to dissolve the first crystallized product. The mixture of the two liquids is then subjected to a second crystallization process, further enriching the product and achieving product yield and purity comparable to existing technologies. The technical solution of this application reduces processing costs while achieving product yield and purity comparable to existing technologies, making it more suitable for industrial-scale production compared to existing technologies.

[0039] In a preferred embodiment, in S3), the first crystallization mother liquor is continuously purified by chromatography using a chromatographic resin and a purifying agent; wherein the chromatographic resin includes zgspc106-Na, LX-38S or LX-40S, more preferably zgspc106-Na.

[0040] In a preferred embodiment, the volume ratio of the purifying agent to the first crystallization mother liquor is 1.1-1.3:1, including but not limited to 1.1:1, 1.15:1, 1.2:1, 1.25:1 or 1.3:1; preferably, the purifying agent is water.

[0041] In a preferred embodiment, in S3), after continuous chromatographic purification is completed, the chromatographic resin is eluted to obtain a second liquid and a purification residue; the volume ratio of the second liquid to the purification residue is 0.9-1.0:1, including but not limited to 0.9:1, 0.91:1, 0.92:1, 0.93:1, 0.94:1, 0.95:1, 0.96:1, 0.97:1, 0.98:1, 0.99:1 or 1:1.

[0042] The chromatographic residue contains most of the pigments, salts, and residual amino acids eluted from the first crystallization mother liquor, while the chromatographic clear liquid (second liquid) contains most of the L-histidine eluted from the first crystallization mother liquor. The term "continuous operation" refers to the continuous pumping and collection of the target product from the crude mother liquor, achieving continuous operation, improving separation efficiency, and ultimately obtaining the corresponding chromatographic purification residue and the qualified chromatographic clear liquid (second liquid). In the embodiments of this application, the second liquid obtained after chromatographic purification, compared with the first crystallization mother liquor, has a salt removal rate ≥90%, a product yield ≥95%, and a chromatographic purity increased from the initial 50% to over 92%, proving that most of the chromatographic impurities in the first crystallization mother liquor of this application can be removed in this step.

[0043] Compared with existing technologies, the volume ratio between the purifying agent and the first crystallization mother liquor added in this application is relatively small. Within this addition range, most of the impurities such as pigments, salts, and miscellaneous amino acids in the first crystallization mother liquor can be separated from the target product. Moreover, the volume ratio between the second liquid obtained after elution and the purified residue is smaller than that of existing technologies, resulting in less elution liquid consumption, less purification material consumption, lower cost, reduced wastewater discharge, and environmental friendliness, which is conducive to its promotion in industrial-scale production.

[0044] In a preferred embodiment, S4) includes: S4-1) mixing the first crystalline product, the second liquid, and water to obtain a third liquid with an L-histidine content of 40-50 g / L; S4-2) subjecting the third liquid to a second decolorization treatment with a second decolorizing agent to obtain a second decolorized solution; S4-3) subjecting the second decolorized solution to a second concentration and second crystallization treatment to obtain L-histidine; preferably, the second decolorizing agent includes activated carbon; preferably, the amount of the second decolorizing agent added is 0.5% to 1.0% (w / v) of the mass volume of the third liquid, including but not limited to 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1%; preferably, the second decolorization time is 30-60 min, including but not limited to 30 min. The second concentration is performed for 40, 50, or 60 minutes at a temperature of 50–70°C, including but not limited to 50, 60, or 70°C. Preferably, the vacuum degree of the second concentration is -0.085 to -0.095 MPa, including but not limited to -0.095, -0.093, -0.09, or -0.085 MPa. Preferably, the temperature of the second concentration is 50–70°C, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, or 70°C. Preferably, the temperature of the second crystallization treatment is 5–10°C, including but not limited to 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C, and the time is 2–5 hours, including but not limited to 2, 3, 4, or 5 hours.

[0045] This application achieves optimal extraction of L-histidine by extensively controlling the processing conditions of filtration, decolorization, and the first crystallization treatment prior to continuous chromatographic purification. This results in a system where each step works in synergy to obtain a relatively high yield of the first crystalline product, namely crude L-histidine crystals. Retaining these crude crystals allows them to be used in the chromatographic supernatant (second liquid) obtained after subsequent chromatographic purification, further improving the final product yield. The first crystallization mother liquor is then introduced into the chromatogram, reducing the pressure and energy consumption of subsequent purification processes. Furthermore, it allows for the separation of residual L-histidine from the mother liquor. Using the chromatographic supernatant (second liquid) as a solvent for the first crystallization product, the second crystallization treatment is carried out. Controlling the temperature and time of the second crystallization treatment, along with further decolorization and the acquisition of the purified product, further improves the product yield.

[0046] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0047] Example 1

[0048] (1): Take 10L of histidine fermentation broth (concentration 50.1g / L), filter it through a ceramic membrane (first filtration) to remove the bacterial cells and obtain the first filtrate.

[0049] After the fermentation broth was concentrated to nearly 5 times its original volume by ceramic membrane filtration, water was added in several batches for dialysis, with 2L of water added each time. A total of 4 dialysis cycles were performed, and the clear liquid from the ceramic membrane, i.e., the first filtrate, was collected, with a concentration of 31g / L. The volume ratio of the first filtrate to the L-histidine fermentation broth was 1.6:1.

[0050] (2): Pour the first filtrate into a 1000 da nanofiltration membrane system, control the temperature ≤35℃ and the pressure ≤2Mpa, and perform nanofiltration (second filtration).

[0051] After the first filtrate was concentrated by nanofiltration membrane filtration to nearly 10 times, water was added and the concentrated liquid was dialyzed to control the L-histidine content (i.e. the concentration mentioned above) to within 6 g / L, and nanofiltration clear liquid, i.e. the second filtrate, was obtained. 18 L of the second filtrate (concentration 27 g / L) was collected.

[0052] (3): Add 0.5% (w / v) of powdered activated carbon to the obtained second filtrate for decolorization. The decolorization temperature is controlled at 60℃. Stir and decolorize for 30 minutes. After filtration (conventional filtration methods are sufficient), the decolorized liquid is obtained, namely 18L of the first decolorized liquid (concentration of about 27g / L).

[0053] (4): The first decolorized liquid obtained is concentrated under vacuum at 60°C to 200 g / L (vacuum degree controlled at ≤-0.085 MPa), and the concentration temperature is controlled at 60°C to obtain the first liquid.

[0054] (5): The first liquid was subjected to the first crystallization treatment, stirred and cooled to 5°C, and crystallized for 2 hours. The resulting crystallized liquid was centrifuged to obtain the first crystallized product (316g in total), with a water content of about 30%, and 2.05L of the first crystallization mother liquor (concentration 83g / L).

[0055] (6): The first crystallization mother liquor was pumped into a continuous chromatography system using ZGSPC106-Na resin. Pure water was used for elution, with the ratio of pure water to the first crystallization mother liquor controlled at 1.1:1. The chromatographic supernatant, i.e., 2.04 L of the second liquid, was collected. The ratio of the chromatographic supernatant to the chromatographic purification residue was 0.9:1. The desalination rate in the second liquid was 93%, and the product yield was 95.5%.

[0056] (7): The second liquid was used to dissolve the first crystallized product and diluted with pure water to a concentration of 50 g / L to obtain the third liquid. 0.5% (w / v) of powdered activated carbon was added to the third liquid for decolorization. The decolorization temperature was controlled at 60°C, and the mixture was stirred for 30 min. After filtration (using conventional filtration methods), 9.5 L of the second decolorized liquid (concentration 50 g / L) was obtained.

[0057] (8): The second decolorizing solution was concentrated under vacuum at 60°C to 200 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature was controlled at 60°C.

[0058] (9): Stir the concentrated liquid obtained in step (8) and cool it down to 5°C, and grow crystals for 2 hours. Centrifuge the resulting crystal liquid to obtain wet product (382g in total). After drying at 60°C, obtain 380g of finished product with a moisture content of 0.18%, a purity of 99.2%, and a white color.

[0059] Example 2

[0060] (1): Take 10L of histidine fermentation broth (concentration 45g / L), filter it through a ceramic membrane (first filtration) to remove the bacterial cells and obtain Taoqing liquid.

[0061] After the fermentation broth was concentrated by filtration through a ceramic membrane to nearly 5 times its original volume, water was added in several stages for dialysis, with 2L of water added each time. A total of 4 dialysis cycles were performed, and the clear liquid from the ceramic membrane, i.e., the first filtrate, was collected in 16L with a concentration of 27.6g / L. The volume ratio of the first filtrate to the L-histidine fermentation broth was 1.6:1.

[0062] (2): Pour the first filtrate into a 1000 da nanofiltration membrane system, control the temperature ≤35℃ and the pressure ≤2Mpa, and perform nanofiltration (second filtration).

[0063] After concentrating the first filtrate to nearly 10 times its original volume using nanofiltration, water was added and the concentrated liquid was dialyzed. The L-histidine content (i.e., the concentration mentioned above) was controlled to be below 6 g / L to obtain the nanofiltration solution, i.e., the second filtrate. 18 L of the second filtrate (concentration 24 g / L) was collected.

[0064] (3): Add 0.5% (w / v) of powdered activated carbon to the obtained second filtrate for decolorization. The decolorization temperature is controlled at 60℃. Stir and decolorize for 30 minutes. After filtration (conventional filtration methods are sufficient), the decolorized liquid is obtained, namely 18L of the first decolorized liquid (concentration 23.8g / L).

[0065] (4): The first decolorized liquid obtained is concentrated under vacuum at 60°C to 250 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature is controlled at 60°C to obtain the first liquid.

[0066] (5): The first liquid was subjected to the first crystallization treatment, stirred and cooled to 10°C, and crystallized for 2 hours. The resulting crystallized liquid was centrifuged to obtain the first crystallized product (about 308g in total), with a water content of about 30%, and 1.5L of the first crystallization mother liquor (concentration 82g / L).

[0067] (6): The first crystallization mother liquor was pumped into a continuous chromatography system. The resin type was ZGSPC106-Na. Pure water was used for elution. The ratio of pure water to the first crystallization mother liquor was controlled at 1.1:1. The chromatographic clear liquid, i.e., 1.58L of the second liquid, was collected. The ratio of the chromatographic clear liquid to the chromatographic purification residue was 1:1. The desalination rate in the second liquid was 91%, and the product yield was 97.2%.

[0068] (7): The second liquid was used to dissolve the first crystallized product and diluted with pure water to a concentration of 45 g / L to obtain the third liquid. 0.5% (w / v) of powdered activated carbon was added to the third liquid for decolorization. The decolorization temperature was controlled at 60°C and the mixture was stirred for 30 min. After filtration (conventional filtration methods are sufficient), 9.4 L of the second decolorized liquid (concentration 45 g / L) was obtained.

[0069] (8): The second decolorizing solution was concentrated under vacuum at 60°C to 250 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature was controlled at 60°C.

[0070] (9): Stir the concentrated liquid obtained in step (8) and cool it down to 10°C. Then, grow crystals for 2 hours. Centrifuge the resulting crystal liquid to obtain a wet product (336g in total). After drying at 60°C, obtain a finished product of 337g with a moisture content of 0.25%, a purity of 99.5%, and a white color.

[0071] Example 3

[0072] Steps (1) to (2) in this embodiment are the same as in embodiment 2, except that:

[0073] (3): Add 0.5% (w / v) of powdered activated carbon to the obtained second filtrate for decolorization. The decolorization temperature is controlled at 60℃. Stir and decolorize for 30 minutes. After filtration (conventional filtration methods are sufficient), the decolorized liquid is obtained, namely 18L of the first decolorized liquid (concentration 23.8g / L).

[0074] (4): The first decolorized liquid obtained is concentrated under vacuum at 60°C to 300 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature is controlled at 60°C to obtain the first liquid.

[0075] (5): The first liquid was subjected to the first crystallization treatment, stirred and cooled to 10°C, and crystallized for 2 hours. The resulting crystallized liquid was centrifuged to obtain the first crystallized product (about 328g in total), with a water content of about 30% and 1.3L of the first crystallization mother liquor (concentration 85g / L).

[0076] (6): The first crystallization mother liquor was pumped into a continuous chromatography system. The resin type was ZGSPC106-Na. Pure water was used for elution. The ratio of pure water to the first crystallization mother liquor was controlled at 1.3:1. The chromatographic clear liquid, i.e., 1.5L of the second liquid, was collected. The ratio of the chromatographic clear liquid to the chromatographic purification residue was 1:1. The desalination rate in the second liquid was 92%, and the product yield was 96.5%.

[0077] (7): The second liquid was used to dissolve the first crystallized product and diluted with pure water to a concentration of 45 g / L to obtain the third liquid. 0.5% (w / v) of powdered activated carbon was added to the third liquid for decolorization. The decolorization temperature was controlled at 60°C, and the mixture was stirred for 30 min. After filtration (using conventional filtration methods), 9.5 L of the second decolorized liquid (concentration 45 g / L) was obtained.

[0078] (8): The second decolorizing solution was concentrated under vacuum at 60°C to 300 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature was controlled at 60°C.

[0079] (9): Stir the concentrated liquid obtained in step (8) and cool it down to 10°C. Then, grow crystals for 2 hours. Centrifuge the resulting crystal liquid to obtain a wet product (352g in total). After drying at 60°C, obtain a finished product of 353g with a moisture content of 0.2%, a purity of 99.2%, and a white color.

[0080] Example 4

[0081] Steps (1) to (5) in this embodiment are the same as in embodiment 2, except that:

[0082] (6): The first crystallization mother liquor was pumped into the continuous chromatography system. The resin type was LX-40S. Pure water was used for elution. The ratio of pure water to the first crystallization mother liquor was controlled at 1.3:1. The chromatographic clear liquid, i.e., the second liquid, was collected in 1.7L. The ratio of the chromatographic clear liquid to the chromatographic purification residue was 0.9:1. The desalination rate in the second liquid was 90%, and the product yield was 95.6%.

[0083] (7): The second liquid was used to dissolve the first crystallized product and diluted with pure water to a concentration of 45 g / L to obtain the third liquid. 0.5% (w / v) of powdered activated carbon was added to the third liquid for decolorization. The decolorization temperature was controlled at 60°C, and the mixture was stirred for 30 min. After filtration (using conventional filtration methods), 9.5 L of the second decolorized liquid (concentration 45 g / L) was obtained.

[0084] (8): The second decolorizing solution was concentrated under vacuum at 60°C to 250 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature was controlled at 60°C.

[0085] (9): Stir the concentrated liquid obtained in step (8) and cool it down to 10°C. Then, grow crystals for 2 hours. Centrifuge the resulting crystal liquid to obtain a wet product (332g in total). After drying at 60°C, obtain a finished product of 330g with a moisture content of 0.22%, a purity of 99.3%, and a white color.

[0086] Comparative Example 1

[0087] The main difference from Example 1 is that the first crystallized product is not incorporated into the chromatographic clear liquid (second liquid), and the chromatographic clear liquid is decolorized, concentrated, and crystallized separately.

[0088] (1): Take 10L of histidine fermentation broth (concentration 45g / L), filter it through a ceramic membrane (first filtration) to remove the bacteria, and obtain the ceramic clear liquid. After the ceramic membrane is concentrated nearly 5 times, water is added in several times for dialysis, with each addition of 2L of water. Dialysis is performed 4 times in total, and the ceramic membrane clear liquid, i.e., the first filtrate, is collected in 16L with a concentration of 27g / L.

[0089] (2): Pour the first filtrate into a 1000da nanofiltration membrane system, control the temperature ≤35℃ and the pressure ≤2Mpa, and perform nanofiltration (second filtration). After the nanofiltration is concentrated by nearly 10 times, add dialysis water and dialyze until the concentrated solution contains less than 6g / L of acid. Collect the nanofiltration solution, which is the second filtrate. Collect 18L of the second filtrate (concentration 23.4g / L).

[0090] (3): Add 0.5% (w / v) of powdered activated carbon to the obtained second filtrate for decolorization. The decolorization temperature is controlled at 60℃. Stir and decolorize for 30 minutes. After filtration (conventional filtration methods are sufficient), the decolorized liquid is obtained, namely 18L of the first decolorized liquid (concentration 23.2g / L).

[0091] (4): The first decolorized liquid obtained is concentrated under vacuum at 60°C to 200 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature is controlled at 60°C to obtain the first liquid.

[0092] (5): The first liquid was subjected to the first crystallization treatment, stirred and cooled to 10°C, and crystallized for 5 hours. The resulting crystallized liquid was centrifuged to obtain the first crystallized product (about 270g in total), with a water content of about 30% and 1.7L of the first crystallization mother liquor (concentration 86g / L).

[0093] (6): The first crystallization mother liquor was pumped into the continuous chromatography system and eluted with pure water. The ratio of pure water to the first crystallization mother liquor was controlled at 1.3:1. The chromatographic clear liquid, i.e., the second liquid, was collected in 1.96L. The ratio of the chromatographic clear liquid to the chromatographic purification residue was 1:1. The desalination rate in the second liquid was 93.2%, and the product yield was 95.5%.

[0094] (7): Add 1% (w / v) of powdered activated carbon to the second liquid for decolorization. The decolorization temperature is controlled at 60℃. Stir and decolorize for 30 min. After filtration, obtain 2L of the second decolorized liquid (concentration 70g / L).

[0095] (8): The second decolorizing solution was concentrated under vacuum at 60°C to 200 g / L (vacuum degree ≤ -0.085 MPa), and the concentration temperature was controlled at 60°C.

[0096] (9): Stir the concentrated liquid obtained in step (8) and cool it down to 10°C. Then, grow crystals for 2 hours. Centrifuge the resulting crystal liquid to obtain wet product (105g in total). After drying at 60°C, obtain 105g of finished product with a moisture content of 0.28%, a purity of 98.5%, and a color close to white.

[0097] Comparative Example 2

[0098] The first crystalline product obtained in Example 1 was used for processing. The difference from Example 1 is that the first crystalline product in this comparative example was subjected to decolorization, concentration and crystallization treatment alone, without being dissolved by a second liquid.

[0099] (1): The first crystallized product was dissolved in pure water to make its concentration 50 g / L. 0.5% (w / v) of powdered activated carbon was added for decolorization. The decolorization temperature was controlled at 60℃. The decolorization was stirred for 30 min. After filtration, about 5.4 L of decolorized liquid (concentration 50 g / L) was obtained.

[0100] (2): The obtained decolorized clear liquid was concentrated under vacuum at 60℃ to 200g / L (vacuum degree ≤ -0.085MPa), and the concentration temperature was controlled at 60℃.

[0101] (3): The concentrated liquid was stirred and cooled to 10°C and crystallized for 2 hours. The resulting crystallized liquid was centrifuged to obtain a wet product (216g in total). After drying at 60°C, 213g of the finished product was obtained with a moisture content of 0.15%, a purity of 99.9%, and a color close to white.

[0102] The finished product quality, yield, moisture content, and purity data of the above embodiments and comparative examples are shown in Table 1.

[0103] Table 1

[0104] mass yield moisture purity example 1 380g 75.85％ 0.18％ 99.2％ example 2 337g 74.88％ 0.25％ 99.5％ example 3 353g 78.44％ 0.20％ 99.2％ example 4 330g 73.33％ 0.22％ 99.3％ comparative example 1 105g 20.95％ 0.28％ 98.5％ comparative example 2 213g 42％ 0.15％ 99.9％

[0105] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: In the technical solution of this application, the fermentation broth of L-histidine is subjected to ceramic filtration, nanofiltration, and decolorization to remove most of the bacterial cells and macromolecules in the fermentation broth. Furthermore, the number of dialysis cycles and the amount of water used are reduced in the two filtration processes, resulting in a smaller volume of the first liquid obtained after filtration. The first liquid is then directly subjected to a first crystallization process to obtain the first crystalline product, namely crude L-histidine, and the first crystallization mother liquor. The first crystallization mother liquor is then purified by chromatography, which reduces the energy consumption of chromatographic purification. Subsequently, the second liquid obtained after chromatography is used as a solvent for the first crystalline product in a second crystallization process, improving the product recovery rate. The L-histidine extraction method of this application reduces the processing energy consumption in the extraction steps without affecting the recovery rate, resulting in lower costs and less actual consumption. Compared with existing technologies, it is more environmentally friendly and more suitable for industrial-scale production.

[0106] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for extracting L-histidine, characterized in that, The extraction method includes: S1) The L-histidine fermentation broth is filtered and first decolorized to obtain the first liquid; S2) Perform a first crystallization treatment on the first liquid to obtain a first crystallization product and a first crystallization mother liquor; S3) The first crystallization mother liquor is purified by continuous chromatography to obtain the second liquid; S4) The first crystalline product and the second liquid are mixed and subjected to a second crystallization process to obtain the L-histidine.

2. The extraction method according to claim 1, characterized in that, S1) includes: S1-1) After the L-histidine fermentation broth is subjected to a first filtration, a first filtrate is obtained; S1-2) After the first filtrate is filtered a second time, a second filtrate is obtained; S1-3) The second filtrate is mixed with the first decolorizing agent, and after the first decolorization, the first decolorized solution is obtained; S1-4) The first decolorizing solution is concentrated to obtain the first liquid; The content of L-histidine in the first liquid is 200-300 g / L; Preferably, the vacuum degree of the first concentration is -0.085 to -0.095 MPa; Preferably, the temperature of the first concentration is 30–70°C.

3. The extraction method according to claim 2, characterized in that, In S1-1), the first filter includes a ceramic membrane filter; Preferably, the L-histidine content in the first filtrate is 20 g / L to 35 g / L; Preferably, the volume ratio of the first filtrate to the L-histidine fermentation broth is 1.1 to 2:

1.

4. The extraction method according to claim 2, characterized in that, In S1-2), preferably, the second filtration includes nanofiltration; Preferably, the nanofiltration membrane size is 500–2000 da; Preferably, the temperature of the second filter is 1–35°C; Preferably, the pressure of the second filter is 1 to 2 MPa.

5. The extraction method according to claim 2, characterized in that, In S1-3), the temperature of the first decolorization is 50-70°C, and the time of the first decolorization is 30-60 min. Preferably, the first decolorizing agent comprises activated carbon; Preferably, the amount of the first decolorizing agent added is 0.5% to 1.0% of the mass volume of the second filtrate.

6. The extraction method according to claim 1, characterized in that, In S2), the temperature of the first crystallization treatment is 5-10℃ and the time is 2-5h. Preferably, after the first crystallization treatment is completed, centrifugation is performed to obtain the first crystallized product and the first crystallization mother liquor.

7. The extraction method according to claim 1, characterized in that, In step S3), the first crystallization mother liquor is subjected to continuous chromatographic purification using chromatographic resin and purifying agent. The chromatographic resin includes zgspc106-Na, LX-38S or LX-40S, more preferably zgspc106-Na; Preferably, the purifying agent is water.

8. The extraction method according to claim 7, characterized in that, The volume ratio of the purifying agent to the first crystallization mother liquor is 1.1-1.3:

1.

9. The extraction method according to claim 7, characterized in that, In step S3), after the continuous chromatographic purification is completed, the chromatographic resin is eluted to obtain the second liquid and the purified residue. The volume ratio of the second liquid to the purified residue is 0.9-1.0:

1.

10. The extraction method according to claim 1, characterized in that, S4) includes: S4-1) The first crystalline product, the second liquid, and water are mixed to obtain a third liquid with an L-histidine content of 40-50 g / L; S4-2) The third liquid is mixed with the second decolorizing agent to perform a second decolorization treatment, thereby obtaining a second decolorized liquid; S4-3) The second decolorizing solution is subjected to a second concentration and a second crystallization treatment to obtain the L-histidine; Preferably, the second decolorizing agent comprises activated carbon; Preferably, the amount of the second decolorizing agent added is 0.5% to 1.0% of the mass volume of the third liquid; Preferably, the second decolorization time is 30-60 minutes and the temperature is 50-70°C; Preferably, the vacuum degree of the second concentration is -0.085 to -0.095 MPa; Preferably, the temperature of the second concentration is 50–70°C; Preferably, the temperature of the second crystallization treatment is 5-10℃, and the time is 2-5 hours.