Preparation method and device of L-citrulline

Abstract

The present disclosure relates to a method for preparing L-citrulline, comprising: a step 1 of preparing a fermentation broth containing L-citrulline; a step 2 of performing a continuous chromatography process on the fermentation broth to obtain a purified citrulline process liquid; and a step 3 of crystallizing and separating L-citrulline in the purified citrulline process liquid. According to the present disclosure, high-purity L-citrulline crystals can be obtained in high yield from a fermentation broth.

Description

Technical Field

[0001] This disclosure relates to a method and apparatus for preparing high-purity L-citrulline crystals in high yield. Background Technology

[0002] Food-grade citrulline is typically manufactured through synthesis or fermentation. When producing citrulline via fermentation, removing impurities such as fermentation byproducts and crystals is essential to obtaining high-purity citrulline. Obtaining high-purity citrulline through fermentation requires multiple purification processes; however, the increased number of purification steps leads to a decrease in citrulline recovery yield.

[0003] The main methods for obtaining high-purity citrulline from fermentation broth are purification methods such as crystallization or resin column adsorption.

[0004] For example, CN 105039216 B discloses a method for obtaining citrulline by converting citrulline from arginine fermentation broth, passing the obtained citrulline through a cation exchange chromatography resin, and crystallizing citrulline by adding an alcohol. This process uses a chromatography resin but also an organic solvent, alcohol, and therefore involves the use of chemical substances.

[0005] In the case of previously developed existing technologies, chemicals must be used in both the resin column process and the crystallization step, which is disadvantageous from an ESG perspective. Furthermore, such methods cannot produce citrulline crystals with a purity of 98.5% (the specification required for food-grade applications). Moreover, with each additional process step, the amount of citrulline obtained decreases relative to the amount of citrulline in the initial fermentation broth, and without sufficient impurity removal in each step, a product with a purity of 98.5% or higher cannot be obtained, thus failing to meet the specifications required for food use.

[0006] Therefore, there is a need to develop a process that can produce food-grade, high-purity L-citrulline in high yield without using chemicals such as organic solvents.

[0007] Existing technical documents

[0008] Patent documents

[0009] (Patent Document 1) CN 105039216 B Summary of the Invention

[0010] Technical issues

[0011] One object of this disclosure is to provide a technique for purifying high-purity L-citrulline without using organic solvents in the process of producing L-citrulline using fermentation.

[0012] Another object of this disclosure is to provide a technique for obtaining high-purity L-citrulline in high yield while reducing the number of steps in the purification process.

[0013] Technical solution

[0014] To achieve the above objectives, this disclosure provides a technique for purifying high-purity L-citrulline without using organic solvents in the production of L-citrulline using a fermentation process.

[0015] Furthermore, this disclosure provides a technique for obtaining high-purity L-citrulline in high yield while reducing the number of steps in the purification process.

[0016] Beneficial effects

[0017] The method for preparing L-citrulline according to embodiments of this disclosure allows for the removal of impurities from the fermentation broth using a continuous chromatography process without the use of chemical solvents, thus being environmentally friendly. Furthermore, after impurity removal, the L-citrulline crystals can be easily separated from the process liquid by concentrating and cooling crystallization to change the crystal form of L-citrulline. The L-citrulline crystals separated by this method have high purity and low moisture content, thus reducing the energy required for drying. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating a method for preparing L-citrulline according to the present disclosure.

[0019] Figure 2 This is a flowchart illustrating a method for preparing L-citrulline according to one embodiment of the present disclosure.

[0020] Figure 3 This is a block diagram illustrating a continuous chromatography unit in an L-citrulline preparation system according to one embodiment of the present disclosure.

[0021] Figure 4 This is a diagram showing the separation pattern of citrulline and impurities using a chromatographic resin column.

[0022] Figure 5 These are images analyzing various crystal forms of L-citrulline. Detailed Implementation

[0023] This disclosure will now be described in detail. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited by the specific descriptions below.

[0024] This disclosure relates to a process for obtaining L-citrulline from fermentation broth using continuous chromatography. According to this disclosure, using continuous chromatography simplifies the purification process, thereby enabling the high-yield acquisition of L-citrulline. Furthermore, by avoiding the use of organic solvents in the purification process involving continuous chromatography, high-purity L-citrulline can be obtained.

[0025] Figure 1 This is a flowchart illustrating a method for preparing L-citrulline according to the present disclosure.

[0026] refer to Figure 1 The method for preparing L-citrulline according to this disclosure includes: step 1 (S100) of preparing a fermentation broth containing L-citrulline; step 2 (S200) of subjecting the fermentation broth to continuous chromatography to obtain a purified citrulline process liquid; and step 3 (S300) of crystallizing and separating L-citrulline from the purified citrulline process liquid. Each step is described in detail below.

[0027] Step 1 (S100) is the step of preparing a fermentation broth containing L-citrulline. As used herein, L-citrulline, represented by the chemical formula H2NC(O)NH(CH2)3CH(NH2)CO2H, is known in IUPAC nomenclature as 2-amino-5-(carbamoylamino)valerate. L-citrulline is considered a major metabolite in the urea cycle.

[0028] As used herein, the term "fermentation product" can refer to a product formed by the enzymatic or metabolic breakdown of organic matter using microorganisms. For example, a fermentation product can comprise the culture itself obtained by culturing microorganisms in a culture medium, or a concentrate, dried product, or lyophilized product of a culture obtained after removing the microbial strain. Furthermore, in this case, the fermentation broth can contain the entire fermentation product containing L-citrulline, or it can be a fermentation product containing L-citrulline in which impurities have been removed.

[0029] The "microorganism producing L-citrulline" or "microorganism producing L-citrulline or a desired product" used in step 1 (S100) includes wild-type microorganisms and naturally or artificially genetically modified microorganisms. It may be a genetically modified microorganism to produce the desired polypeptide or L-citrulline, in which a specific mechanism is weakened or enhanced due to factors such as the insertion of a foreign gene or the enhancement or inactivation of endogenous gene activity.

[0030] The microorganisms for producing L-citrulline disclosed herein may be naturally capable of producing L-citrulline, or may be microorganisms in which the L-citrulline production capacity has been conferred by a parent strain lacking L-citrulline production capacity, but are not limited thereto. Specifically, in this disclosure, the microorganisms that produce L-citrulline or the desired product, or microorganisms exhibiting the ability to produce L-citrulline or the desired product, may be microorganisms in which some genes in the biosynthetic pathway of the desired protein or the desired product are enhanced or weakened, or microorganisms in which some genes in the degradation pathway of the desired protein or the desired product are enhanced or weakened. "Enhancing" or "increasing" the L-citrulline production capacity of the microorganisms of this disclosure means that the L-citrulline production capacity of the microorganisms of this disclosure is improved compared to the fermentation product production capacity of microorganisms, parent strains, or unmodified microorganisms different from those of this disclosure. For example, compared to the L-citrulline production rate of another microorganism, the microorganism of this disclosure may have an increased L-citrulline production capacity of about 1% or more, 10% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 1100% or more, 1200% or more, or 1300% or more, and the microorganism of this disclosure may have an increased L-citrulline production capacity of about 1.01 times or more, 2 times or more, 5 times or more, 10 times or more, 11 times or more, 12 times or more, or 13 times or more, but is not limited thereto. The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., which includes all values ​​equal to or approximately the range of values ​​following the term "about," but is not limited thereto.

[0031] The microorganisms used in step 1 (S100) can be at least one microorganism selected from the group consisting of yeast Candida famata, Ascomycetes Eremothecium ashbyii and Ashbya gossypii, and bacteria Bacillus subtilis and Corynebacterium sp.

[0032] When the microorganism used in step 1 (S100) is an undetermined species of Corynebacterium, the microorganism may specifically be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, or Corynebacterium testis. Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, or more specifically, Corynebacterium glutamicum, but not limited thereto.

[0033] Step 1 (S100) may further include culturing "microorganisms that produce L-citrulline". The cultivation of said microorganisms may be carried out in suitable culture media and under suitable culture conditions known in the art. Those skilled in the art can readily adapt and use such a culture method according to the selected strain. Specifically, said culture may be batch culture, continuous culture, and / or fed-batch culture, but is not limited thereto. As used herein, the term "culture medium" refers to a substance in which nutrients required for the cultivation of microorganisms are mixed as a major component, and said culture medium provides nutrients, growth factors, etc., including water essential for survival and growth. Specifically, any culture medium and culture conditions may be used as the culture medium and culture conditions for culturing the microorganisms of this disclosure, as long as the culture medium is used for the general cultivation of microorganisms, without particular limitation. The microorganisms of this disclosure may be cultured under aerobic conditions in a universal culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0034] The fermentation broth prepared in step 1 (S100) is then introduced into a continuous chromatography process.

[0035] In step 2 (S200), the fermentation broth is subjected to continuous chromatography to obtain purified citrulline process liquid.

[0036] "Continuous chromatography" refers to a process that evolves traditional intermittent chromatography into a continuous process. Specifically, in a chromatographic apparatus, a solid and liquid phase can be continuously supplied, and as the solid and liquid phases move in opposite directions, countercurrent contact occurs, thereby enabling more efficient separation of substances. As used herein, continuous chromatography can be understood as encompassing both true moving bed (TMB) chromatography and simulated moving bed (SMB) chromatography. Furthermore, since TMB and SMB chromatography share the same principles, those skilled in the art can appropriately select and use them by considering productivity and other factors.

[0037] Continuous chromatography may include multiple resin columns, and a strongly acidic cation exchange resin may be provided as the stationary phase within the resin columns. The functional groups of the strongly acidic cation exchange resin may be sulfate groups, but are not limited thereto. Furthermore, the base material of the strongly acidic cation exchange resin used herein is not limited, as long as it is capable of adsorbing onto the strongly acidic functional groups. For example, styrene-divinylbenzene copolymers may be used, but are not limited thereto. As a specific example, the strongly acidic cation exchange resin may be a sulfonated styrene-divinylbenzene copolymer, but is not limited thereto.

[0038] Compared to single-column chromatography using only a single resin column, continuous chromatography exhibits superior resolution between L-citrulline and impurities. Therefore, it significantly reduces the co-eluting fraction of L-citrulline and impurities, and allows for the acquisition of high-purity L-citrulline from the majority of the eluted fraction. This increases the purity and recovery rate of L-citrulline. In contrast, single-column chromatography results in relatively low resolution, leading to a co-eluting fraction of L-citrulline and impurities. To obtain high-purity L-citrulline, it can only be obtained from the fraction where impurities are not eluted, not from the co-eluting fraction. Consequently, a large amount of L-citrulline is discarded along with the impurities, resulting in a decreased recovery rate.

[0039] In step 2 (S200), when the fermentation broth passes through a continuous chromatography resin column, in addition to L-citrulline, other substances that may be present in the fermentation broth, such as ions, organic acids, other amino acids, and residual sugars, can also be removed.

[0040] Specifically, step 2 (S200) may further include step 2-1 of introducing the fermentation broth into multiple resin columns to separate L-citrulline from impurities, and step 2-2 of introducing water into the resin columns that have retained L-citrulline to obtain a purified citrulline process liquid containing L-citrulline and water.

[0041] Continuous chromatography may include multiple resin columns and provide a strongly acidic cation exchange resin in the resin columns. In step 2-1, the fermentation broth can pass through the aforementioned resin columns and contact with the strongly acidic cation exchange resin provided therein, thereby allowing impurities in the fermentation broth to be adsorbed and removed by the strongly acidic cation exchange resin.

[0042] Next, water, a non-organic solvent, can be introduced into the resin column that retains L-citrulline to obtain a purified citrulline process liquid containing L-citrulline and water. The purified citrulline process liquid can refer to a solution from which impurities (such as ions, organic acids, other amino acids, residual sugars, etc.) initially present in the fermentation broth have been removed as described above, and in which the concentration of L-citrulline has increased.

[0043] In step 2 (S200), as described above, L-citrulline is not adsorbed onto the resin, while impurities are adsorbed onto the resin and thus removed. Therefore, no separate eluent or regenerator is needed to separate L-citrulline from the resin, making the process environmentally friendly. Furthermore, since continuous chromatography does not require an adsorption / elution process, the hourly productivity is higher, and the amount of process water can be reduced compared to ion exchange methods. In conventional techniques, to purify fermentation broth containing L-citrulline, L-citrulline is adsorbed onto an ion exchange resin and then separated using solvents such as ammonia or alcohol. From an ESG perspective, these techniques are disadvantageous because they use chemicals.

[0044] Then, the purified citrulline process liquid obtained in step 2 (S200) is introduced into the crystallization and separation process.

[0045] Step 3 (S300) Crystallization, thereby separating L-citrulline from the purified citrulline process liquid. As used herein, “crystallization” refers to the phenomenon of a liquid or amorphous solid forming crystals, and may be accompanied by two phenomena: the formation and growth of crystal nuclei.

[0046] In step 3 (S300), L-citrulline crystals are obtained by morphological transformation of the concentrated and purified citrulline process liquid and L-citrulline crystals. These crystals allow for easy separation of the mother liquor and contain a small amount of water. Further details regarding step 3 (S300) will be described below.

[0047] According to one embodiment of this disclosure, a continuous chromatography and crystallization process is used to prepare L-citrulline products from fermentation broth. The use of continuous chromatography allows for the efficient removal of impurities from the fermentation broth without requiring complex multi-step processes and without the use of chemical solvents, thus making the process environmentally friendly. Furthermore, the crystallization process can obtain L-citrulline crystals by concentrating and purifying the citrulline process liquid and transforming the crystal form of the L-citrulline crystals, which allow for easy separation from the mother liquor and contain a small amount of water. According to this disclosure, high-purity L-citrulline crystals can be obtained in high yield in an environmentally friendly manner.

[0048] The method for preparing L-citrulline according to this disclosure has been described above. The detailed process for preparing L-citrulline according to embodiments of this disclosure will be further described below.

[0049] Figure 2 This is a flowchart illustrating a method for preparing L-citrulline according to one embodiment of the present disclosure.

[0050] exist Figure 2 In the following description, explanations of processes identical to those described above will be omitted to avoid redundancy.

[0051] refer to Figure 2 Step 1 (S100) for preparing the fermentation broth containing L-citrulline includes preparing the fermentation solution and adjusting the pH. In the pH adjustment step, the pH of the prepared fermentation broth can be adjusted to a range of 2 to 7. Typically, the pH of the fermentation broth can be 6 to 7, and an acidic solution such as HCl or H2SO4 can be added to adjust the pH. However, any substance used to adjust the pH of the fermentation broth can be appropriately selected, as long as it does not affect L-citrulline. Adjusting the pH of the fermentation broth to the above range enables a highly efficient cell separation process through enhanced cell aggregation, thereby improving the effectiveness of the subsequent decolorization process.

[0052] After step 1 (S100), an additional process for removing cells from the fermentation broth can be performed. For example, before or after step 2 (S200) and / or step 3 (S300), the fermentation broth can be filtered through a membrane that does not allow cells to pass through, thereby removing cells from the fermentation broth.

[0053] Furthermore, after step 1 (S100), a decolorization process can be performed. The decolorization process aims to remove residual colorant from the fermentation broth and can be carried out by adsorption decolorization using activated carbon or clay (activated clay, acidic clay) as adsorbents. However, the above description is exemplary, and the method for performing the decolorization process can be appropriately selected by considering the properties of the fermentation broth containing L-citrulline, the characteristics of subsequent processes, etc.

[0054] The accompanying diagram illustrates that pH adjustment, cell filtration, and decolorization are performed sequentially after the fermentation broth is prepared. However, the order of these processes can be altered as needed. For example, pH can be adjusted after cell filtration and decolorization, or decolorization can be performed before cell filtration.

[0055] Next, after step 2 (S200) of performing continuous chromatography on the fermentation broth to obtain purified citrulline process liquid, a filtration and decolorization process can be performed additionally. The filtration and decolorization process further removes any impurities that may remain in the purified citrulline process liquid. The filtration and decolorization process can be performed in a conventional manner similar to step 1 (S100) described above, and can be omitted if necessary to simplify the process.

[0056] Subsequently, in step 3, crystallization is performed to separate and purify L-citrulline from the citrulline process liquid. Step 3 (S300) may specifically include a concentration and crystallization step, a crystal separation step, and a drying step.

[0057] Step 3, the concentration and crystallization step (S300), may further include concentrating the purified citrulline process liquid prior to L-citrulline crystallization. The purified citrulline process liquid can be concentrated in a conventional concentrator (e.g., a forced circulation concentrator, a thin-film concentrator, or a rotary concentrator) selected by those skilled in the art. The concentration of L-citrulline in the concentrated process liquid can be from about 100 g / L to less than about 800 g / L, from about 200 g / L to less than about 700 g / L, from about 300 g / L to less than about 600 g / L, from about 100 g / L to less than about 500 g / L, from about 200 g / L to less than about 500 g / L, from about 300 g / L to less than about 500 g / L, and more specifically, from about 400 g / L to less than about 500 g / L, but is not limited thereto. By increasing the concentration of the purified citrulline process liquid, the recovery rate of citrulline can be improved. However, if the concentration of L-citrulline in the concentrated process liquid exceeds approximately 500 g / L, excessive crystals will form, making stirring difficult. Therefore, the concentration process can be implemented such that the concentration of L-citrulline in the concentrated process liquid is less than approximately 500 g / L.

[0058] The concentration process described in step 3 (S300) can be carried out in a temperature range of approximately 50°C to approximately 90°C. In some cases, the concentration process can be carried out in a temperature range of approximately 50°C to approximately 80°C, approximately 50°C to approximately 70°C, approximately 60°C to approximately 80°C, approximately 60°C to approximately 70°C, or approximately 70°C to approximately 80°C. In the concentration process, L-citrulline in the purified citrulline process liquid can form L-citrulline crystals. The L-citrulline crystals formed in the concentration process may be amorphous. Because amorphous crystals are difficult to separate from the mother liquor of the process liquid, it is difficult to obtain clean crystals. Therefore, step 3 may also include, after concentrating the purified citrulline process liquid, converting the amorphous crystalline form of the L-citrulline crystals into an amorphous form by a cooling process. When the concentration process is carried out in the above temperature range, the crystalline form of the L-citrulline crystals may change. Specifically, by carrying out the concentration process in the above temperature range and then performing a cooling crystallization process, L-citrulline crystals can be formed in a form that is easily separated from the mother liquor. The L-citrulline crystals formed after the cooling crystallization process can be small, rectangular anhydrous crystals.

[0059] The cooling crystallization process can be carried out by cooling the concentrated process liquid containing L-citrulline crystals to a temperature ranging from approximately 5°C to approximately 45°C. In some cases, the cooling temperature can be approximately 5°C to approximately 40°C, approximately 10°C to approximately 35°C, approximately 15°C to approximately 30°C, approximately 20°C to approximately 25°C, approximately 5°C to approximately 35°C, or approximately 10°C to approximately 30°C. In the cooling crystallization process, the temperature of the process liquid can be gradually reduced so that all amorphous L-citrulline crystals can be converted into anhydrous crystalline form. For example, the cooling rate can be approximately 3°C / h to approximately 7°C / h. The cooling rate can be determined by taking into account process factors such as the temperature of the concentrated process liquid and the amount of L-citrulline.

[0060] The anhydrous L-citrulline crystals produced after the cooling crystallization process have a relatively uniform shape and are easily separated from the mother liquor of the process liquid. Therefore, the separated L-citrulline crystals may have a relatively low moisture content compared to amorphous crystals. Low moisture content L-citrulline crystals ensure stable quality because there are no dehydration or washing issues during crystal separation. Furthermore, using a dryer eliminates risks associated with transfer and flow and reduces the amount generated during the drying process, thereby minimizing losses. Additionally, the reduced moisture content improves the economics of the process by decreasing the amount of steam used in the dryer.

[0061] The separation of L-citrulline crystals and mother liquor can be performed using a liquid-solid separator, such as a vacuum membrane filtration unit, a pressurized membrane filtration unit, or a centrifuge, but is not limited thereto. The remaining mother liquor after obtaining L-citrulline crystals can be reintroduced into step 1 (S100) or step 2 (S200) to improve the final purification recovery rate of L-citrulline.

[0062] Next, the obtained L-citrulline crystals can be washed and dried to prepare the final product, L-citrulline crystals. Washing and drying can be performed using any commonly used method.

[0063] As described above, the L-citrulline preparation process according to the embodiments of this disclosure allows for the conversion of L-citrulline crystals into their crystalline form during the concentration and crystallization process, thereby enabling easy separation from the mother liquor and obtaining L-citrulline crystals with low moisture content. Therefore, by using the above preparation method, high-purity L-citrulline crystals can be obtained, and the energy required for the process can also be reduced.

[0064] Mode of implementing the present invention

[0065] This disclosure will be described in detail by way of examples. However, these examples are for illustrative purposes only, and the scope of the invention is not limited to these examples. Furthermore, any technical descriptions lacking in this disclosure can be fully understood and readily practiced by those skilled in the art or related fields.

[0066] The preparation process and system for L-citrulline according to embodiments of the present disclosure are described below. The advantageous effects involved in this disclosure will be described below with reference to experimental results from examples and comparative examples.

[0067] Overview of Experimental Methods

[0068] The analysis of the experimental results described in this disclosure was carried out in the following manner.

[0069] (1) HPLC method for quantitative analysis of L-citrulline

[0070] In this disclosure, the HPLC analytical conditions used to analyze the purity and concentration of L-citrulline are as follows:

[0071] Equipment: HPLC 1260 Infinity system (Agilent Technology Inc.)

[0072] Column: HP C18 (150 mm × 3.9 mm; 5 μm)

[0073] Mobile phase: 25 mM KH2PO4, 12.2 mM OSA aqueous solution

[0074] Flow rate: 1 mL / min

[0075] Temperature: 40℃

[0076] Detection: UV at 210 nm

[0077] Injection volume: 5 μL

[0078] (2) Method for measuring the purity of L-citrulline crystals

[0079] In this disclosure, the quality of L-citrulline crystals is evaluated based on the purity of L-citrulline, and the method follows this order:

[0080] (a) L-citrulline crystals were prepared by placing L-citrulline crystals in a vacuum dryer containing silica gel and keeping them at a vacuum level of 20 mmHg or lower for 24 hours to remove residual moisture and cooling the L-citrulline crystals to room temperature.

[0081] (b) Weigh 0.5000 g of L-citrulline crystals cooled to room temperature, place the crystals in a 1 L volumetric flask, and dilute with triple-distilled water to prepare a sample with a concentration of 0.5000 g / L.

[0082] (c) First, process the L-citrulline standard crystals (Sigma, ≥ 99.0%) in the same manner as in step (a). Then, weigh 0.5000 g of crystals, place them in a 1 L volumetric flask, and dilute with triple-distilled water to prepare a sample with a concentration of 0.5000 g / L. Confirm the purity of the standard product according to the analytical certificate provided by the standard reagent manufacturer, and calculate the concentration of L-citrulline in the sample ([converted L-citrulline concentration] = 0.5000 g / L × [purity of the standard]); and

[0083] (d) The purity of the L-citrulline crystals used in step (a) was determined by analyzing the sample prepared in step (b) with HPLC and using the sample prepared in step (c) as an external standard.

[0084] (3) Method for analyzing L-citrulline content based on solids in fermentation broth or eluent obtained from chromatographic processes.

[0085] In this disclosure, the quality of fermentation broth or eluent obtained from a chromatographic process is evaluated based on the content of L-citrulline in the solid obtained by removing water from the solution, and the method follows this order:

[0086] (a) After deodorizing about 5 grams of sea sand (10 to 20 mesh; Daejung Chemicals & Metals Co., Ltd.), place it in a ceramic container, put the container in a forced convection oven at 105°C for 3 hours to remove residual moisture, and then put it in a vacuum desiccator containing silica gel for 1 hour to cool to room temperature.

[0087] (b) The solution to be analyzed is added to the container from step (a), and the mass of the added solution is quantified by measuring the weight difference before and after the addition.

[0088] (c) Place the container from step (b) in a forced convection oven at 105°C for 3 hours to remove residual moisture, then place it in a vacuum desiccator containing silica gel for 1 hour to cool to room temperature. Quantify the amount of water removed based on the weight difference and calculate the content based on the mass of solids in the solution to be tested ([solid mass content] = ([solution mass] – [mass of water removed]) / [solution mass]).

[0089] (d) Measure the density of the solution to be analyzed using a hydrometer;

[0090] (e) The concentration of L-citrulline in the analyte solution was measured using HPLC; and

[0091] (f) The content of L-citrulline in the solid obtained by removing water from the solution is calculated using the content, density and L-citrulline concentration based on the mass of solids in the solution ([content of L-citrulline in the solid obtained by removing water from the solution] = [concentration of L-citrulline] / [density of solution] / [mass of solids in the solution]).

[0092] Experimental Example 1. Separation process of L-citrulline from fermentation broth

[0093] In Experimental Example 1, during the process of separating L-citrulline from fermentation broth, the results obtained using continuous chromatography according to the embodiments of this disclosure (Example 1) were compared with the results obtained using cation exchange resin according to conventional techniques (Comparative Example 1) or using single-column chromatography resin (Comparative Example 2).

[0094] Preparation example. Preparation of fermentation broth containing L-citrulline.

[0095] A fermentation broth containing L-citrulline was obtained by culturing L-citrulline-producing microorganisms in a fermentation medium. The prepared L-citrulline fermentation broth had a pH of 7.0 and an L-citrulline concentration of 130 g / L. The solid obtained by removing water from the L-citrulline fermentation broth contained 70% L-citrulline. The pH of the fermentation broth was adjusted to 4.0 using 98% sulfuric acid. Cell separation of the fermentation broth was performed using a ceramic membrane filtration system from TAMI Co., employing a membrane with a pore size of 0.14 μm to obtain the filtrate. After cell separation, the L-citrulline fermentation broth was prepared as the feed solution for the following SMB chromatography.

[0096] Example 1. Separation of citrulline using continuous chromatography

[0097] To obtain the eluent for separating L-citrulline from the fermentation broth containing L-citrulline prepared in the preparation example, an SMB chromatography apparatus (NOVASEP) was used, which uses a resin (TRILITE MCK32L) with strongly acidic functional groups as the stationary phase. A schematic diagram of the SMB chromatography apparatus is shown below. Figure 3 As shown.

[0098] like Figure 3 As shown, the SMB chromatography apparatus consists of a total of eight resin columns. Each column has a volume of 7.0 L, and the resin is packed to 95% of the column volume. The SMB feed solution is injected into the fifth resin column at a flow rate of 4.5 L / hr. The mobile phase (water) is injected into the first resin column at a flow rate of 18.2 L / hr. The SMB product process liquid (elution) is discharged from the first resin column at a flow rate of 18.2 L / hr. The SMB process wastewater is discharged from the seventh resin column at a flow rate of 4.5 L / hr. The impurities migrating into the process wastewater are mainly ions, organic acids, residual sugars, and non-citrulline amino acids. These resin columns are operated in a circulatory manner, moving from the first resin column to the eighth resin column every 25 minutes.

[0099] TRILITE MCK32L resin, a styrene-divinylbenzene copolymer resin with sulfate groups (a strong acid) as functional groups, was loaded onto a resin column of a chromatography apparatus. 0.1 kL or more of SMB chromatographic feed solution was injected, and the apparatus was operated to obtain the SMB product process liquid (elution). The pH of the fermentation broth was adjusted to different values ​​in the range of 2 to 7 using 98% sulfuric acid and 50% NaOH, and the recovery rate of the continuous chromatography process and the purity (%) of L-citrulline in the eluent obtained from the continuous chromatography process were measured. The recovery rate of the continuous chromatography process was calculated as the recovery rate of L-citrulline in the eluent and compared with the fermentation broth introduced into the continuous chromatography apparatus. The results are shown in Table 1 below.

[0100] [Table 1]

[0101]

[0102] The yields of the continuous chromatography process were found to be 80% or more in the pH range of 2.0 to 7.0, 85% or more in the pH range of 3.0 to 6.5, and 90% in the pH range of 4.0 to 5.5. The purity of L-citrulline in the eluent obtained from the continuous chromatography process was found to be 85% or higher in the pH range of 2.0 to 6.5, and 90% or higher in the pH range of 3.5 to 5.5.

[0103] Comparative Example 1. Separation of citrulline using cation exchange resin

[0104] Prepare the fermentation broth as described in the preparation example, and adjust the pH of the filtrate to the range of 0 to 2 using 98% sulfuric acid or 35% hydrochloric acid. Regenerate the cation exchange resin using 35% hydrochloric acid (using TRILITE SCR-B), and use its H... + The citrulline cell filtrate was then introduced into a cation exchange resin, and water was added so that only citrulline in the filtrate was adsorbed onto the resin, while remaining impurities were removed. To further adsorb citrulline onto the resin, it was treated with 35% hydrochloric acid to achieve the desired H2O concentration. + The citrulline is adsorbed onto the resin and eluted, resulting in a citrulline process liquid with impurities removed. The purity of the citrulline solution thus obtained was approximately 90% according to analysis. In Comparative Example 1, when using a cation exchange resin, major impurities such as ions, other amino acids, and residual sugars can be removed from the fermentation broth; however, organic acids are difficult to remove because they are cations at pH 2 or lower.

[0105] Comparative Example 2. Separation of citrulline using single-column chromatography resin

[0106] The filtrate prepared as the fermentation broth in the preparation example was passed through a single-column chromatographic resin column. TRILITE MCK32L resin, the same resin used in continuous chromatography, was used; the column volume was 520 mL, and over 95% of the column was packed with resin. The process liquid was introduced at a flow rate of 0.5 L / hr, and the mobile phase (water) was introduced at a flow rate of 1.5 L / hr. Citrulline, ions, organic acids, residual sugars, and other impurities in the citrulline fermentation broth were separated by chromatographic resin, size exclusion, ion adsorption, and van der Waals adsorption, respectively. Anions were separated and eluted first, followed by residual sugars, organic acids, and cations. Citrulline was eluted last. The pH of the fermentation broth was adjusted to different values ​​in the range of 2 to 7 using 98% sulfuric acid and 50% NaOH, and the recovery rate of the single-column chromatography process and the purity (%) of L-citrulline in the eluent obtained from the single-column chromatography process were measured according to the pH value of the fermentation broth. The recovery rate of the single-column chromatography process was calculated as the recovery rate of L-citrulline from ions, residual sugars, organic acids, and other impurities compared to the fermentation broth introduced into the single-column chromatography apparatus. The results are shown in Table 2 below.

[0107] [Table 2]

[0108]

[0109] The purity of L-citrulline in the eluent obtained from single-column chromatography (the method of Comparative Example 2) was found to be 87% or higher in the pH range of 2.0 to 7.0, and 90% or higher in the pH range of 3.5 to 5.5. However, as... Figure 4 As shown, under the conditions of single-column chromatography, the peaks of impurities and citrulline significantly overlapped, and it was confirmed that the recovery rate of citrulline decreased significantly to 10% or more in the pH range of 2.0 to 7.0, and significantly decreased to about 13% in the pH range of 4.0 to 6.0. Therefore, the comparison between Example 1 and Comparative Examples 1 and 2 confirms that using continuous chromatography to purify the fermentation broth resulted in a higher recovery rate and higher purity of L-citrulline in the process liquid. In the separation process using the cation exchange resin of Comparative Example 1, it has the disadvantage of difficulty in removing organic acids and the need for acid eluent. Furthermore, as in Comparative Example 2, when a single column was used instead of a continuous column to purify the fermentation broth, the resolution was relatively low, resulting in a co-elution fraction of L-citrulline and impurities. To obtain high-purity L-citrulline, L-citrulline can only be obtained from the fraction where impurities were not eluted, and not from the fraction where L-citrulline and impurities were co-eluted. Therefore, a large amount of L-citrulline is discarded along with impurities, leading to a decrease in the recovery rate of L-citrulline.

[0110] However, when continuous chromatography is used as in Example 1 of this disclosure, the separation between L-citrulline and impurities is improved, thus significantly reducing the co-eluting fraction of L-citrulline and impurities. Therefore, high-purity L-citrulline can be obtained from the majority of the eluted fraction, thereby improving the purity and recovery rate of L-citrulline.

[0111] Experimental Example 2. L-Citrine Crystallization Process from Purified Citrulline Process Liquid

[0112] In Experiment 2, the purified citrulline process liquid obtained according to Example 1 was concentrated and crystallized, and the results obtained according to one embodiment of the present disclosure with temperature regulation (Example 2) were compared with the results obtained without temperature regulation (Comparative Example 3).

[0113] Example 2. Temperature-controlled concentration and crystallization

[0114] First, the purified citrulline process liquid obtained using resin for continuous chromatography is decolorized. For decolorization, activated carbon is added at 3% of the weight of citrulline, and decolorization is carried out at approximately 60°C for about 1 hour. After decolorization, the activated carbon is filtered off to obtain the decolorized process liquid. During filtration, the activated carbon is completely removed using a glass filter (Merck) with a size of approximately 0.7 μm. The decolorized process liquid is placed in a crystallization vessel (DAIHAN) and concentrated to form L-citrulline crystals. Concentration is carried out at a high internal temperature of approximately 50°C to approximately 90°C, and the solution is concentrated to approximately 500 g / L. Subsequently, the temperature is cooled to approximately 5°C to approximately 30°C at a rate of approximately 5°C per hour. As the temperature gradually decreases, small rectangular anhydrous crystals form from the initial amorphous crystals. The crystals thus obtained have a purity of 98.5% or higher and a moisture content of 5% or lower. This process yields crystals that meet food-grade citrulline content specifications, and because the moisture content of the crystals is 5% or lower, the steam cost required for drying in the process is also reduced. The results are shown in Table 3.

[0115] [Table 3]

[0116]

[0117] Comparative Example 3. Concentration and Crystallization without Temperature Control

[0118] The purified citrulline process liquid obtained using resins for continuous chromatography was decolorized. For decolorization, activated carbon was added at 3% of the weight of citrulline, and decolorization was carried out at 60°C for 1 hour. After decolorization, the activated carbon was filtered off to obtain the decolorized process liquid. The purity of the process liquid was 97.5%. During filtration, activated carbon was completely removed using a glass filter (Merck) with a size of approximately 0.7 μm. The decolorized process liquid was placed in a crystallization vessel (DAIHAN) and concentrated to form crystals. The solution was concentrated to 500 g / L at an internal temperature of 50°C or lower, and then the crystals were separated. Unlike the examples, no additional cooling was performed, and the crystals were amorphous with no discernible shape. Since the mother liquor could not be separated during separation, the purity of the crystals was 97.1%, and the water content was 22%. The results are shown in Table 4.

[0119] [Table 4]

[0120]

[0121] like Figure 5 It has been confirmed that L-citrulline crystals can have multiple crystalline forms. The L-citrulline crystals obtained in Example 2 were found to be anhydrous, and these anhydrous crystals had a relatively low moisture content of 5% or less. In contrast, the amorphous crystals of Comparative Example 3 were found to have a moisture content of 22%, which is significantly higher than the moisture content of the amorphous body. Furthermore, the purity of the crystals in Comparative Example 3 was found to be lower than the purity of the amorphous body prepared in Example 2. Therefore, it is confirmed that L-citrulline crystals with low moisture content and high purity can be prepared by a crystallization method according to one embodiment of this disclosure.

[0122] As described above, those skilled in the art will understand that the present invention can be implemented in other specific forms without departing from the technical spirit or essential characteristics of the invention. Therefore, the above embodiments should be interpreted as exemplary, not as limiting the scope of this disclosure. It should be understood that all variations or modifications derived from the definitions and scope of the claims and their equivalents fall within the scope of this disclosure.

Claims

1. A method for preparing L-citrulline, comprising: Step 1: Preparing a fermentation broth containing L-citrulline; Step 2: Perform continuous chromatography on the fermentation broth to obtain purified citrulline process liquid; and Step 3: Crystallization and separation of L-citrulline from the citrulline process liquid.

2. The method according to claim 1, wherein step 3 further comprises concentrating and purifying the citrulline process liquid.

3. The method according to claim 2, wherein step 3 further comprises, after concentrating and purifying the citrulline process liquid, converting the amorphous crystal form of L-citrulline crystals into an amorphous crystal form by a cooling process.

4. The method according to claim 3, wherein the concentration and purification of the citrulline process liquid is carried out in the range of 50°C to 90°C, and the cooling process after concentration of the purified citrulline process liquid is carried out by cooling to a temperature range of 5°C to about 45°C.

5. The method according to claim 1, wherein step 2 further comprises: Step 2-1: Introducing the fermentation broth into multiple resin columns to separate L-citrulline from impurities; and Step 2-2 involves introducing water into a resin column that retains L-citrulline to obtain a purified citrulline process liquid containing L-citrulline and water.

6. The method of claim 5, wherein the resin column is provided with a strongly acidic cation exchange resin.

7. The method according to claim 1, further comprising filtering and decolorizing the purified citrulline process liquid between step 2 and step 3.

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