Method for adjusting the color of natural L-cysteine ​​hydrochloride crystals

Abstract

This invention relates to a method for producing L-cysteine ​​hydrochloride crystals with low yellowness from an L-cysteine ​​process solution derived from a fermentation liquid.

Description

[Technical Field]

[0001] [Cross-reference to Related Application] The present application claims the benefit of priority based on Korean Patent Application No. 10-2023-0126192 filed on September 21, 2023, and all contents disclosed in the document of said Korean patent application are incorporated as a part of the present specification.

[0002] The present invention relates to a method for adjusting the chromaticity of natural L-cysteine hydrochloride crystals. [Background Art]

[0003] L-cysteine is an amino acid that plays an important role in sulfur metabolism of all living organisms. It is used not only for biosynthesis of proteins such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites, but also acts as a precursor of coenzyme A. Industrially, L-cysteine and derivatives thereof can also be used in various fields including the pharmaceutical industry (for treatment of bronchial diseases), the cosmetics industry (for hair shampoos and permanent wave compositions), and the food industry (as antioxidants and flavor enhancers).

[0004] L-cysteine ​​is generally produced by decomposing animal-derived L-cystine, such as duck feathers or human hair, or fermentation-derived L-cystine, which is made from microbial metabolites, into L-cysteine ​​using an electrochemical reduction reaction. In contrast, methods for producing L-cysteine ​​using microorganisms have been disclosed, including a fermentation step (US8802399B) in which natural L-cysteine ​​is produced using a strain of microorganisms with modified O-acetyltransferase in a culture medium containing sulfides, and a step (WO2013-089478A3) in which O-phosphohomoserine produced by a microbial culture method is mixed with sulfides, and an enzyme-catalyzed reaction is induced using O-phosphoserine sulfhydrase to produce natural L-cysteine. Furthermore, a process for producing L-cysteine ​​hydrochloride hydrate (WO2019-151769A1) is disclosed, which separates and purifies the natural state of the L-cysteine ​​ferment broth using a continuous chromatography process without the use of chemical reactions or artificial synthetic compounds, and which has the advantages of high yield and purity, as well as efficient productivity improvements and low water usage.

[0005] However, when using a continuous chromatography process, separation of the final product from the fermentation medium or pigments produced during fermentation is required, and additional purification steps may be necessary. While activated carbon is mainly used to remove pigments, in the case of fermentation liquid produced from L-cysteine ​​fermentation liquid using the aforementioned method, pigments and impurities that are not removed by activated carbon may develop color under the conditions for producing cysteine ​​hydrochloride crystals, resulting in coloration of the final product and a decrease in color quality.

[0006] Against this backdrop, the object of the present invention is to provide a method for culturing microorganisms and a method for producing L-cysteine ​​hydrochloride crystals of uniform color from L-cysteine ​​fermentation liquid produced by an enzyme-catalyzed reaction. [Overview of the project] [Problems that the invention aims to solve]

[0007] An example of this application is (a) A step of obtaining an L-cysteine ​​separatory from a fermentation liquid containing L-cysteine; (b) A step of filtering the L-cysteine ​​separation to obtain the filtrate; (c) A step of concentrating the filtrate to obtain a concentrate; (d) Adding hydrochloric acid to the filtrate or concentrate before or after step (c); and (e) A method for producing L-cysteine ​​crystals is provided, comprising the step of cooling the concentrated solution to recover L-cysteine ​​crystals.

[0008] Another example of this application provides an L-cysteine ​​crystal characterized by a yellowness of 4.0 or less. [Means for solving the problem]

[0009] The present invention will be described in more detail below. Each description and embodiment disclosed in the application may also apply to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not considered to be limited by the specific descriptions set forth below.

[0010] This application, in one aspect, (a) A step of obtaining an L-cysteine ​​separatory from a fermentation liquid containing L-cysteine; (b) A step of filtering the L-cysteine ​​separation to obtain the filtrate; (c) A step of concentrating the filtrate to obtain a concentrate; (d) Adding hydrochloric acid to the filtrate or concentrate before or after step (c); and (e) A method for producing L-cysteine ​​crystals is provided, comprising the step of cooling the concentrated solution to recover L-cysteine ​​crystals.

[0011] The method for producing L-cysteine ​​crystals according to this application will be described in detail step by step as follows.

[0012] (a) Step: Obtaining L-cysteine ​​separation solution The method for producing L-cysteine ​​crystals according to this application may include step (a) obtaining an L-cysteine ​​separatory from a fermentation liquid containing L-cysteine.

[0013] In this application, the term "L-cysteine" refers to one of the amino acids that make up proteins, and is the only sulfur-containing amino acid among L-amino acids that has a thiol group (R-SH). L-cysteine ​​is obtained by chemical synthesis or biological production through microbial fermentation, but is not limited to these. Specifically, in this application, L-cysteine ​​may be L-cysteine ​​biologically produced through microbial fermentation, or it may be natural L-cysteine ​​obtained by inducing an enzymatic catalytic reaction between O-phosphohomoserine, a precursor produced through microbial fermentation, and a sulfide in the presence of phosphoserine sulfhydrylase. The natural L-cysteine ​​may be L-cysteine ​​that has not undergone a chemical reaction, chemical adsorption, or elution step in the manufacturing process.

[0014] In this application, the term "natural" means not produced by a chemical reaction. According to the publication of the European Union Flavorings Regulation 1334 / 2008, only substances obtained by physical, enzymatic, or microbial processes are defined as "natural" flavorings. From this perspective, L-cysteine ​​produced by the electrochemical reduction reaction of L-cystine, regardless of whether it is of animal origin or microbial fermentation origin, cannot be called natural.

[0015] In this application, the term "fermentation liquid" means a culture medium obtained by culturing microorganisms that produce L-cysteine, a culture containing microorganisms cultured together with the culture medium, or an enzyme conversion solution containing a precursor and enzyme capable of producing L-cysteine. Specifically, the fermentation liquid containing L-cysteine ​​may be a culture medium or enzyme conversion solution containing natural L-cysteine. More specifically, it may be an L-cysteine ​​culture or culture medium biologically produced through microbial fermentation having L-cysteine-producing properties, or it may be an enzyme conversion solution of natural L-cysteine ​​obtained by inducing an enzyme-catalyzed reaction between O-phosphohomoserine, a precursor produced through microbial fermentation, and a sulfide in the presence of phosphoserine sulfhydrylase. L-cysteine ​​crystals produced using the fermentation liquid as a raw material can be considered natural L-cysteine, as they are not produced by a chemical reaction.

[0016] In this application, the step of obtaining the separated liquid in step (a) may be carried out using continuous chromatography. In one example, the fermentation liquid can be used as the raw material liquid for the continuous chromatography process. That is, it can be injected into the continuous chromatography apparatus in step (a).

[0017] The pH of the fermentation liquid injected into the continuous chromatography apparatus varies depending on the manufacturing method, but may be 6.0-11.5, 6.5-11.0, 7.0-10.5, 7.5-10.0, or 8.0-9.5. The fermentation liquid itself can also be used as a raw material for the continuous chromatography process, and may further include a step of adjusting the pH of the fermentation liquid containing L-cysteine ​​to 6.0-11.5, 6.5-11.0, 7.0-10.5, 7.5-10.0, or 8.0-9.5 prior to step (a). This adjustment can be made by adding, but is not limited to, an acid such as sulfuric acid or hydrochloric acid, or a base such as sodium hydroxide (caustic soda), ammonia, lithium hydroxide, or potassium hydroxide. The pH adjusting agent can be appropriately selected and used by those skilled in the art, as long as it does not affect the structure of L-cysteine ​​and ultimately yields L-cysteine ​​crystals.

[0018] The term "Continuous Chromatography" as used in this application refers to a process that has been developed from the conventional batch chromatography process into a continuous process. Specifically, the solid phase and liquid phase may be continuously supplied into the chromatography apparatus, and since the solid phase and liquid phase move in opposite directions to cause countercurrent contact, it enables more efficient separation of substances. In this application, the term can be used as a concept that includes mobile bed (TMB, True Moving Bed) chromatography and simulated mobile bed (SMB, Simulated Moving Bed) chromatography. Furthermore, since the two aforementioned mobile bed chromatography processes and simulated mobile bed chromatography are based on the same principle, those skilled in the art can appropriately select and use them considering productivity and other factors.

[0019] The continuous chromatography process used in this application eliminates the need for adsorption / elution processes, resulting in higher productivity per unit time and reduced water usage compared to ion exchange processes. Furthermore, while obtaining L-cysteine ​​powder products in high yield from process solutions containing L-cysteine ​​obtained through ion exchange or conventional chromatography processes requires significant energy in the concentration and crystallization process, the method described in this application can reduce energy costs.

[0020] The stationary phase of the continuous chromatography apparatus is an ion exchange resin, specifically a strongly acidic cation exchange resin. The functional group of the strongly acidic cation exchange resin is an acidic group, but is not limited thereto. Furthermore, the parent material of the strongly acidic cation exchange resin used in this application is not limited as long as it has a strongly acidic functional group attached to it. For example, a styrene-divinylbenzene copolymer is used, but is not limited thereto. As a specific example, the strongly acidic cation exchange resin is a styrene sulfate-divinylbenzene copolymer, but is not limited thereto.

[0021] In the present application, other types of stationary phases commonly used for amino acid separation and purification in the art include non-functionalized exchange resins such as non-functionalized styrene-divinylbenzene copolymers and non-functionalized methacrylate polymers; strongly basic anion exchange resins such as trimethylamine styrene-divinylbenzene copolymers; weakly basic anion exchange resins such as tertiary amine styrene-divinylbenzene copolymers; weakly acidic cation exchange resins such as carboxylated methacrylate polymers. It is difficult for these to purify L-cysteine to a content of 50% (w / w) or more in the solid content obtained from the separated liquid obtained through a continuous chromatography process. On the other hand, when a strongly acidic cation exchange resin such as sulfonated styrene-divinylbenzene copolymer is used, the L-cysteine content in the dry solid content obtained from the separated liquid obtained through a continuous chromatography process can be purified to 80% (w / w) or more, specifically 90% (w / w) or more.

[0022] In the chromatography apparatus, as the mobile phase, water without added chemical compounds (for example, organic solvents such as methanol, isopropyl alcohol, acetonitrile, etc.), diluted caustic soda solution, diluted sulfuric acid solution, diluted phosphoric acid solution, diluted hydrochloric acid solution, diluted potassium hydroxide solution, or mixtures thereof can be used, but the mobile phase is not limited thereto. As a specific example, water can be used as the mobile phase for continuous chromatography. When a mobile phase containing a chemical compound is used, the chemical compound may remain in the final product, which may result in the residual content of the chemical compound exceeding the standard limit, making it impossible to sell the product or distribute it as a natural product. In addition, cost reduction effects can be expected by not adding any chemical substances other than water to the process water. In the ion exchange process, a solvent such as hydrochloric acid or sulfuric acid must be used as an eluent to elute the adsorbed L-cysteine, which inevitably leads to an increase in product cost associated with the use and disposal of such chemical substances.

[0023] When the fermentation broth containing L-cysteine in step (a) is injected into a continuous chromatography apparatus, L-cysteine is separated by a continuous chromatography step, and a separated solution containing L-cysteine can be obtained. In the present application, the separated solution from which L-cysteine is separated and which contains L-cysteine may be abbreviated and expressed as "separated solution", "chromatographic separated solution" or "process solution".

[0024] Step (b): Step of obtaining L-cysteine filtrate Thereafter, the method of the present application may include step (b), a step of filtering the L-cysteine separated solution obtained in step (a) to obtain a filtrate.

[0025] In step (b), methods known in the art are applied for filtering the L-cysteine separated solution. For example, a filtration method using a Nutsche filter, a Filter press, or a Housing filter with a filter made of CA, PVDF, PES or PTFE material can be used. In one embodiment, filtration can be performed using a Nutsche filter, but the method is not limited thereto.

[0026] The filtrate acquisition step in step (b) above may be carried out with the addition of activated carbon. For example, the activated carbon may be in amounts of 0.01-50 wt%, 0.1-50 wt%, 1-50 wt%, 5-50 wt%, 0.01-40 wt%, 0.1-40 wt%, 1-40 wt%, 5-40 wt%, 0.01-30 wt%, 0.1-30 wt%, 1-30 wt%, 5-30 wt%, 0.01-20 wt%, 0.1-20 wt%, 1-20 wt%, 2-20 wt%, 3-20 wt%, 4-20 wt%, 5-20 wt%, 6-20 wt%, 7-20 wt%, 8-20 wt%, and 9% relative to the total weight of L-cysteine. ~20wt%, 0.01~15wt%, 0.1~15wt%, 1~15wt%, 2~15wt%, 3~15wt%, 4~15wt%, 5~15wt%, 6~15wt%, 7~15wt%, 8~15wt%, 9~15wt%, 0.01~12wt%, 0.1~12wt%, 1~12wt%, 2~12wt%, 3~12wt%, 4~12wt%, 5~12wt%, 6~12wt%, 7~12wt%, 8~12wt%, or 9~12wt% may be added, but is not limited to these amounts.

[0027] (c) Step: Step to obtain L-cysteine ​​concentrate Subsequently, the method of this application may include step (c), a step of concentrating the L-cysteine ​​filtrate obtained in step (b) to obtain a concentrate.

[0028] The concentration in step (c) above can be carried out in a conventional concentrator, such as a forced-circulation concentrator, a thin-film concentrator, or a rotary concentrator. For example, it can be carried out in a rotary concentrator.

[0029] The vacuum level of the concentrator in the concentration step of step (c) above is 90-130 mmHg, 95-125 mmHg, or 100-120 mmHg, for example, 110 mmHg. This is not limited to the above. Furthermore, the external temperature of the concentrator may be 50-90°C, 55-85°C, or 60-80°C, for example, 70°C, but is not limited to these.

[0030] (d) Step: Adding hydrochloric acid The method of this application may include, before or after step (d) and step (c), adding hydrochloric acid to the filtrate or concentrate.

[0031] The amount of hydrochloric acid added in step (d) above may be less than 6.5, less than 6.0 N, less than 5.5 N, less than 4.5 N, between 4.5 and 5.5 N, or between 5.5 N and 6.5 N, but is not limited to these.

[0032] The hydrochloric acid addition step of step (d) may be performed before step (e) or after step (b), but more specifically, it may be performed before step (c), after step (c), or simultaneously with step (c).

[0033] The method for producing L-cysteine ​​crystals according to this application may further include a step after step (d) in which the residence time of the concentrate is adjusted at a temperature of 10°C to 70°C.

[0034] The term "residence time" of a concentrate, as used in this application, means the time the concentrate is maintained in a liquid state before crystallization, and may be used interchangeably with the terms "storage time," "concentration time," "maintenance time," or "mooring time."

[0035] In this application, the residence time of the concentrate varies depending on the hydrochloric acid concentration and residence temperature of the concentrate after the addition of hydrochloric acid in step (d). for example, When the residence temperature is 10°C or higher and less than 30°C, the residence time of the concentrated liquid is (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is 4.5 N or higher, within 24 hours; When the residence temperature is 30°C or higher and less than 50°C, the residence time of the concentrated liquid is: (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 720 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 4.5 N to 5.5 N, within 480 minutes; and / or (iii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 5.5 N to 6.5 N, within 240 minutes; If the residence temperature is 50°C or higher, the residence time of the concentrated liquid is (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 240 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 4.5 N to 5.5 N, within 180 minutes; and / or (iii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is between 5.5N and 6.5N, the time limit is 120 minutes or less, but is not limited to this.

[0036] The residence time of the aforementioned concentrated liquid is, specifically, When the dwell temperature is between 15°C and 25°C, (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is 4.5 N or higher, within 540 minutes; When the dwell temperature is between 35°C and 45°C, (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 540 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 4.5 N to 5.5 N, within 420 minutes; and / or (iii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 5.5N to 6.5N, within 210 minutes; When the dwell temperature is between 55°C and 65°C, (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 210 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is less than 4.5 N to 5.5 N, within 150 minutes; and / or (iii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is 5.5N to less than 6.5N, the time is 90 minutes or less, but is not limited to this.

[0037] In this application, the residence temperature of the concentrate means the temperature at which the concentrate is maintained constant during the residence time of the concentrate, and can mean the temperature of the concentrate.

[0038] The residence temperature of the concentrated liquid may be 10°C, 15°C or higher, 20°C or higher, 25°C or higher, 30°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, 10-70°C, 15-70°C, 20-70°C, 25-70°C, 30-70°C, 35-70°C, 40-70°C, 45-70°C, 50-70°C, 55-70°C, 10-65°C, 15-65°C, 20-65°C, 25-65°C, 30-65°C, 35-65°C, 40-65°C, 45-65°C, 50-65°C, 10°C or higher but less than 30°C, 15-25°C, 30°C or higher but less than 50°C, 35-45°C, or 55-65°C.

[0039] After adjusting the residence time of the concentrate, the 430 nm absorbance of the L-cysteine ​​concentrate is 0.65 or less, 0.60 or less, or 0.55 or less, but is not limited to these values.

[0040] The L-cysteine ​​concentrate, after adjusting the residence time of the concentrate, can exhibit a reduced reddening phenomenon and a relatively lower absorbance value compared to the L-cysteine ​​concentrate that has not undergone the aforementioned steps, by adding hydrochloric acid before or after the concentration step and / or adjusting the residence time according to the hydrochloric acid concentration of the concentrate after hydrochloric acid addition.

[0041] In this application, an L-cysteine ​​concentrate that has not undergone the step of adding hydrochloric acid before or after the concentration step and / or the step of adjusting the residence time according to the hydrochloric acid concentration of the concentrate after hydrochloric acid addition may contain a "chromaticity-inducing substance" which is a substance that causes redness, and the method of this application includes, as a step of reducing the "chromaticity-inducing substance", the step of adding hydrochloric acid before or after the concentration step and / or the step of adjusting the residence time according to the hydrochloric acid concentration of the concentrate after hydrochloric acid addition.

[0042] The term "chromaticity-inducing substance" in this application refers to a substance that induces color, and is a substance that causes reddening, and can be used interchangeably with the terms "color value-inducing substance" or "chromaticity-exhibiting substance."

[0043] In this application, the chromaticity-inducing substance contained in the L-cysteine ​​concentrate that causes the reddening phenomenon is PLP (pyridoxal-5'-phosphate), but is not limited to this.

[0044] (e) Step: Cooling of the concentrate and crystal recovery step Subsequently, the method of this application may include step (e) a step of cooling the concentrated solution to which hydrochloric acid has been added to recover L-cysteine ​​crystals.

[0045] The cooling process described in (e) may further include a step of crystallizing L-cysteine.

[0046] In this application, the term "crystallization" refers to the phenomenon in which a liquid or amorphous solid forms crystals, and this occurs in conjunction with two phenomena: the generation of crystal nuclei and the growth of crystal nuclei.

[0047] The concentrate can be cooled and / or aged before collection to form and / or grow crystal nuclei. Furthermore, even if L-cysteine ​​crystals do not precipitate in the concentrate, crystal formation can occur when the concentrate is cooled and / or aged.

[0048] The aforementioned cooling step specifically means cooling to a temperature of -10 to 55°C over 2 to 6 hours, more specifically to a temperature of 0 to 45°C over 2 to 6 hours, more specifically to a temperature of 0 to 30°C, and even more specifically to a temperature of 15°C over 2 to 6 hours.

[0049] The aforementioned maturation step means leaving the product undisturbed without temperature changes. In this application, it means maintaining a constant cooled temperature, or maintaining a constant temperature of the concentrate even if it is not cooled. Specifically, maturation can take 1 to 3 hours.

[0050] Step (e) allows for the recovery of L-cysteine ​​crystals precipitated from the concentrate. Specifically, the concentrate can be separated into solid and liquid to recover the L-cysteine ​​crystals from the slurry. This is done using, but is not limited to, a solid-liquid separator such as a vacuum membrane filter, a pressure membrane filter, or a centrifuge. The slurry and / or the precipitated L-cysteine ​​crystals may be further washed or dried.

[0051] In this application, the L-cysteine ​​crystals produced through steps (a) to (e) above can maintain a uniform yellowness.

[0052] The yellowness of the L-cysteine ​​crystal is one of the chromatic values ​​measured by a colorimeter and can be used interchangeably with the b or b* value in this application.

[0053] The yellowness of the L-cysteine ​​crystal may be less than 7.1, 7.0 or less, 6.5 or less, 5.0 or less, 4.5 or less, 4.0 or less, 1.5 to 7.0, 1.5 to 6.5, 1.5 to 5.0, 1.5 to 4.5, 1.5 to 4.0, 2.0 to 7.0, 2.0 to 6.5, 2.0 to 5.0, 2.0 to 4.5, 2.0 to 4.0, 2.1 to 7.0, 2.1 to 6.5, 2.1 to 5.0, 2.1 to 4.5, 2.1 to 4.0, 2.5 to 7.0, 2.5 to 6.5, 2.5 to 5.0, 2.5 to 4.5, or 2.5 to 4.0.

[0054] In this application, the term "color difference meter" refers to a device for measuring the color of an object. The color difference meter used for chromaticity measurement in this application is a CHROMA METER (CR-410, KONICA MINOLTA), but is not limited to this.

[0055] In this application, L-cysteine ​​crystals produced by adjusting the residence time of the L-cysteine ​​concentrate, through a step of adding hydrochloric acid before or after the concentration step and / or a step of adjusting the residence time according to the hydrochloric acid concentration of the concentrate after hydrochloric acid addition, can exhibit a reduced reddening phenomenon and a relatively lower yellowness compared to L-cysteine ​​crystals that have not undergone the aforementioned steps.

[0056] In this application, L-cysteine ​​crystals that have not undergone the step of adding hydrochloric acid before or after the concentration step and / or the step of adjusting the residence time according to the hydrochloric acid concentration of the concentrated solution after hydrochloric acid addition may contain a "chromaticity-inducing substance" which is a substance that causes the reddening state, and the method of this application includes, as a step of reducing the "chromaticity-inducing substance", the step of adding hydrochloric acid before or after the concentration step and / or the step of adjusting the residence time according to the hydrochloric acid concentration of the concentrated solution after hydrochloric acid addition.

[0057] In this application, the chromaticity-inducing substance contained in the L-cysteine ​​concentrate that causes the reddening phenomenon is PLP (pyridoxal-5'-phosphate), but is not limited to this.

[0058] Furthermore, in another aspect, this application provides an L-cysteine ​​crystal characterized by having a yellowness of 4.0 or less.

[0059] The L-cysteine ​​crystal is (a) A step of obtaining an L-cysteine ​​separatory from a fermentation liquid containing L-cysteine; (b) A step of filtering the L-cysteine ​​separation to obtain the filtrate; (c) A step of concentrating the filtrate to obtain a concentrate; (d) Adding hydrochloric acid to the filtrate or concentrate before or after step (c); and (e) The L-cysteine ​​crystals may be produced by a method for producing L-cysteine ​​crystals, which includes the step of (e) cooling the concentrated solution to which hydrochloric acid has been added to recover the L-cysteine ​​crystals.

[0060] The method for producing the L-cysteine ​​crystals is as described above. [Brief explanation of the drawing]

[0061] [Figure 1] This diagram shows the color change of L-cysteine ​​solutions according to the concentration of pyridoxal 5'-phosphate (PLP) added and the residence time. (a) shows the color of each L-cysteine ​​solution at 0 hours after PLP addition, (b) shows the color at 2 hours after PLP addition, and (c) shows the color at 4 hours after PLP addition. [Modes for carrying out the invention]

[0062] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

[0063] Example 1. Comparison of the chromaticity of fermentation-derived hydrochloride crystals containing L-cysteine ​​according to hydrochloric acid concentration. Example 1-1. Production of a fermentation-derived process liquid containing L-cysteine ​​using microbial fermentation. A natural L-cysteine ​​ferment broth was obtained by inducing an enzymatic catalyzed reaction between O-phosphohomoserine, a precursor produced through microbial fermentation, and sulfide using O-phosphoserine sulfhydrase (OPS) and pyridoxal 5'-phosphate (PLP). The pH of the L-cysteine ​​ferment broth was 8-9.5.

[0064] Specifically, the KCCM 11103P (CA07-0022 / pCL-prmf-serA*(G336V)-serC; Korean Registered Patent No. 10-1381048) strain, which is a modified E. coli W3110 strain lacking serB and introduced with mutant serA* to produce OPS, was cultured on MMYE agar plates at 33°C for 24 hours. One-tenth of the cells from each plate were scraped out and inoculated into a baffle flask with flask seed medium (10 g / L glucose, 0.5 g / L magnesium sulfate, 3 g / L potassium dihydrogen phosphate, 10 g / L yeast extract, 0.5 g / L sodium chloride, 1.5 g / L ammonium chloride, 12.8 g / L sodium pyrophosphate, 1 g / L glycine), and seed cultured at 30°C at 200 rpm for 6 hours. After seed culture was completed, seed culture medium equivalent to 16% of the main culture medium volume was inoculated into a 1L small fermenter filled with 300ml of main culture medium, and cultivation was carried out at 33°C and pH 7.0 to obtain OPS fermentate. Under conditions of 100mM Na2S and 0.2mM pyridoxal phosphate (pyridoxal 5'-phosphate, PLP), the 50mM OPS fermentate was reacted with 50mg / ml Msm-T enzyme derived from Mycobacterium tuberculosis H37Rv to obtain a fermentate containing L-cysteine ​​(US8557549B2).

[0065] The obtained L-cysteine ​​fermentation liquid was separated from the bacterial cells using a 0.14 μm membrane to obtain a filtrate. The pH of the filtrate was lowered to pH 2-7 using 98% sulfuric acid. Subsequently, the separated filtrate containing L-cysteine ​​was obtained by continuous chromatography (US11427537B2) using a strongly acidic cation exchange resin.

[0066] To the separated liquid from which L-cysteine ​​had been separated, 10 wt% activated carbon relative to the total weight of L-cysteine ​​was added and the mixture was stirred at 40°C for 1 hour. The activated carbon was separated using a Nutsche filter to obtain a filtrate.

[0067] Examples 1-2. Comparison of the chromaticity of L-cysteine-containing hydrochloride crystals according to hydrochloric acid concentration. In Example 1-1, when hydrochloric acid was added to the filtrate obtained by separation using a Nutsche filter, the following experiment was conducted to compare the color difference between the L-cysteine ​​concentrate and the L-cysteine-containing hydrochloride crystals recovered after cooling the concentrate, depending on the concentration of hydrochloric acid.

[0068] Specifically, the filtrate obtained in Example 1-1 was concentrated in a rotary concentrator (EYELA N-1200B) at a vacuum of 110 mmHg and an external temperature of 70°C. After concentration, hydrochloric acid was added to achieve concentrations of 4, 5, 6, and 6.5 N, respectively, and the concentrate was left to stand at 60°C for 1 hour. The change in absorbance of the concentrated solution was then measured.

[0069] Each L-cysteine ​​concentrate (prepared solution) containing hydrochloric acid of various concentrations was stirred and cooled in a jacketed tank to 15°C at a constant cooling rate for 4 hours. Then, L-cysteine ​​crystals were separated from the L-cysteine ​​crystal slurry using a basket separator at a rotational speed of 3,000 rpm for 15 minutes. After separation, the mixture was dried in an oven dryer at 40°C for at least 2 hours to reduce the residual moisture content to 0.5% or less, ultimately producing L-cysteine ​​hydrochloride crystals. The yellowness (b* value) of the produced L-cysteine ​​hydrochloride crystals was measured using a CHROMA METER (CR-410, KONICA MINOLTA) colorimeter and is shown in Table 1 below. A yellowness (b* value) closer to 0 indicates a whiter color, while an increasing value indicates a more yellowish color.

[0070] [Table 1]

[0071] As a result, as shown in Table 1, it can be seen that the increase in absorbance increases as the hydrochloric acid concentration of the concentrated solution (prepared solution) increases. Furthermore, it was confirmed that the chromaticity value of the L-cysteine ​​hydrochloride crystals is determined according to the final absorbance value of the concentrated solution (prepared solution). It was confirmed that when the absorbance value is 0.55 or less, L-cysteine ​​hydrochloride crystals with a yellowness (b* value) of 4.0 or less can be obtained.

[0072] Example 2. Comparison of absorbance of fermentation-derived concentrate containing L-cysteine ​​according to its residence temperature. The following experiment was conducted to investigate the effect of residence temperature on the absorbance of a fermentation-derived concentrate containing L-cysteine.

[0073] Specifically, the L-cysteine ​​separated in the continuous chromatography step of Example 1-1 was separated into a filtration solution, and 5 wt% activated carbon relative to the L-cysteine ​​was added and the mixture was stirred at 40°C for 1 hour. The activated carbon was separated using a Nutsche filter to obtain a filtrate.

[0074] The filtrate was concentrated in a rotary concentrator at a vacuum of 110 mmHg and an external temperature of 70°C. Then, hydrochloric acid was added to produce concentrated solutions with hydrochloric acid concentrations of 4.5, 5.5, and 6.5 N.

[0075] Each L-cysteine ​​concentrate, to which hydrochloric acid of various concentrations was added, was allowed to stand for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours at residence temperatures of 20°C, 40°C, and 60°C, respectively. The UV / Vis absorbance at 430 nm was then measured to confirm the change in absorbance over time. The results of the absorbance measurements over time are shown in Table 2 below.

[0076] [Table 2]

[0077] As a result, as shown in Table 2, it was confirmed that the increase in absorbance was greater as the retention temperature of the concentrate increased and as the hydrochloric acid concentration increased. When the concentrate was retained (stored) at 20°C, unlike the results of Examples 1-2 above, the change in absorbance per hour remained almost unchanged at 0.003-0.004 even as the hydrochloric acid concentration and retention time increased. At 40°C, it was confirmed that the change in absorbance per hour increased linearly from 0.04 to 0.13 as the hydrochloric acid concentration increased from 4.5N to 6.5N, and at 60°C, it was confirmed that the change in absorbance per hour increased linearly from 0.12 to 0.43 as the hydrochloric acid concentration increased from 4.5N to 6.5N. Therefore, it was confirmed that the change in absorbance of the concentrate was influenced by specific substances derived from the fermentation liquid, depending on the retention temperature and hydrochloric acid concentration conditions.

[0078] Comparative Example 1. Confirmation of the effect of activated carbon input amount on the removal of chromaticity-inducing substances. In addition to the concentration of hydrochloric acid added to the concentrate and the residence temperature of the concentrate, which are conditions for adjusting the chromaticity of L-cysteine ​​hydrochloride crystals derived in Examples 1 and 2, the following experiment was conducted to confirm the effect of the amount of activated carbon added on the removal of chromaticity-inducing substances.

[0079] Specifically, 0, 5, and 10 wt% activated carbon were added to the separation solution from which L-cysteine ​​had been separated by continuous chromatography, relative to the total weight of L-cysteine, and the mixture was stirred at 40°C for 1 hour. After separating the activated carbon with a Nutsche filter to obtain a filtrate, the filtrate was concentrated in a rotary concentrator at a vacuum of 110 mmHg and an external temperature of 70°C. After concentration, hydrochloric acid was added to prepare a concentrate with a hydrochloric acid concentration of 5.5 N, and the absorbance change of the concentrate over time was measured at 60°C every 2 hours. The results are shown in Table 3 below.

[0080] [Table 3]

[0081] As a result, as shown in Table 3, after decolorization by adding 10 wt% activated carbon, the increase in absorbance per unit time of the concentrated solution was confirmed to be at a similar level of 0.25 to 0.35. Therefore, although a small amount of chromaticity-inducing substances are removed by the decolorization process using activated carbon, the effect is not significant. Thus, it was confirmed that the concentration of hydrochloric acid added to the concentrated solution and the retention temperature conditions of the concentrated solution, as determined in Examples 1 and 2, are the most important factors for adjusting the chromaticity of the L-cysteine ​​hydrochloride crystals.

[0082] Experimental Example 1. Confirmation of absorbance changes in L-cysteine ​​reagent solutions according to PLP concentration. Pyridoxal 5'-phosphate (PLP) is one of the coenzymes used in a method (US8557549B2) to produce cysteine ​​or its derivatives by reacting O-phosphoserine (OPS) with a sulfide in the presence of O-phosphoserinesulfhydrylase (OPSS) or a microorganism expressing it.

[0083] To confirm the effect of the concentration of the coenzyme PLP (pyridoxal-5'-phosphate) on the change in the chromaticity of the L-cysteine ​​solution, the following experiment was conducted.

[0084] Specifically, 100 g / L of L-cysteine ​​reagent was mixed with 6.0 N hydrochloric acid, and either no PLP was added, or PLP was added at concentrations of 1 wt%, 5 wt%, and 10 wt%, respectively. Subsequently, the change in UV / Vis absorbance at 430 nm was measured every 2 hours at a residence temperature of 60°C for each sample solution, and the results are shown in Figure 1 and Table 4 below.

[0085] [Table 4]

[0086] As a result, as shown in Figure 1 and Table 4, under conditions without PLP addition, there was no change in the absorbance of the L-cysteine ​​sample solution with increasing residence time. In contrast, under conditions with PLP addition, it was confirmed that the rate of change in the absorbance of the L-cysteine ​​sample solution with respect to time increased as the concentration of PLP increased. Furthermore, it was confirmed that the color of the L-cysteine ​​solution changed from yellow to orange as the residence time increased (as the heating time increased) at a residence temperature of 60°C (see Figure 1).

[0087] From the results described above, it was confirmed that PLP may be included in the specific substance derived from the fermentation liquid in Example 2, which affects absorbance depending on the retention temperature and hydrochloric acid concentration conditions.

Claims

1. (a) A step of obtaining an L-cysteine ​​separatory from a fermentation liquid containing L-cysteine; (b) A step of filtering the L-cysteine ​​separation liquid to obtain the filtrate; (c) A step of concentrating the filtrate to obtain a concentrate; (d) Adding hydrochloric acid to the filtrate or concentrate before or after step (c); and A method for producing L-cysteine ​​crystals, comprising the step of (e) cooling the concentrated solution to recover L-cysteine ​​crystals.

2. The method for producing L-cysteine ​​crystals according to claim 1, wherein the step of obtaining the separated liquid in step (a) is performed using continuous chromatography.

3. The method for producing L-cysteine ​​crystals according to claim 1, wherein the step of obtaining the filtrate in step (b) is carried out by adding activated carbon.

4. The method for producing L-cysteine ​​crystals according to claim 3, wherein the activated carbon is added in an amount of 0.01 to 50 wt% relative to the total weight of L-cysteine.

5. The method for producing L-cysteine ​​crystals according to claim 1, wherein the concentration in step (c) is carried out under conditions of a vacuum of 100 to 120 mmHg and an external temperature of 60 to 80°C.

6. The method for producing L-cysteine ​​crystals according to claim 1, wherein the amount of hydrochloric acid added in step (d) is such that the hydrochloric acid concentration of the concentrated solution after addition is less than 6.5 N.

7. A method for producing L-cysteine ​​crystals according to claim 1, further comprising the step of adjusting the residence time of the concentrate at a temperature of 10°C to 70°C after step (d).

8. The method for producing L-cysteine ​​crystals according to claim 7, wherein the residence time is within 24 hours when the residence temperature is 10°C or higher and less than 30°C, and the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is 4.5 N or higher.

9. The residence time is when the residence temperature is 30°C or higher and less than 50°C. (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 720 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is between 4.5 N and less than 5.5 N, within 480 minutes; or (iii) The method for producing L-cysteine ​​crystals according to claim 7, wherein the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is 5.5 N to less than 6.5 N, and the time is within 240 minutes.

10. The residence time is when the residence temperature is 50°C or higher. (i) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) above is less than 4.5 N, within 240 minutes; (ii) If the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is between 4.5 N and less than 5.5 N, within 180 minutes; or (iii) The method for producing L-cysteine ​​crystals according to claim 7, wherein the hydrochloric acid concentration of the concentrated solution after adding hydrochloric acid in step (d) is 5.5 N to less than 6.5 N, and the time is within 120 minutes.

11. The method for producing L-cysteine ​​crystals according to claim 7, wherein the absorbance of the concentrated solution at 430 nm after adjusting the residence time is 0.55 or less.

12. The method for producing L-cysteine ​​crystals according to claim 1, wherein the concentrated liquid from step (e) is cooled to a temperature of 0 to 30°C.

13. The method for producing L-cysteine ​​crystals according to any one of claims 1 to 12, wherein the L-cysteine ​​crystals have a yellowness of 4.0 or less.

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