Method for controlling chromaticity of natural l-cysteine hydrochloride crystals

Abstract

The present invention relates to a method for producing L-cysteine hydrochloride crystals with reduced yellowness from an L-cysteine process liquid derived from a fermentation liquid. The method for producing L-cysteine crystals comprises the steps of: (a) obtaining an L-cysteine separation solution from a fermentation solution containing L-cysteine; (b) filtering the L-cysteine separation solution to obtain a filtrate; (c) concentrating the filtrate to obtain a concentrate; (d) adding hydrochloric acid to the filtrate or concentrate before or after step (c); and (e) cooling the concentrate to recover L-cysteine crystals.

Description

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

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0126192, filed September 21, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a method for controlling the color of natural L-cysteine ​​hydrochloride crystals.

[0004]

[0005] L-cysteine ​​is an amino acid that plays a crucial role in sulfur metabolism in all living organisms. It is used in the biosynthesis of proteins such as hair keratin, glutathione, biotin, methionine, and other sulfur-containing metabolites, as well as serving as a precursor to coenzyme A. Furthermore, L-cysteine ​​and its derivatives have diverse industrial applications, including in the pharmaceutical industry (for treating bronchial diseases), cosmetics (hair shampoos, permanent makeup compositions), and food (as an antioxidant and flavor enhancer).

[0006] L-cysteine ​​is generally manufactured by decomposing animal-derived L-cysteine, such as duck feathers or human hair, or fermentation-derived L-cysteine, such as microbial metabolites, into L-cysteine ​​using an electrochemical reduction reaction. In contrast, known methods for manufacturing L-cysteine ​​using microorganisms include a fermentation process that produces natural L-cysteine ​​using a strain with modified O-acetyltransferase in a medium containing sulfide (US 8802399 B), and a process that mixes O-phosphohomoserine manufactured by a microbial culture method with sulfide and induces an enzyme-catalyzed reaction using O-phosphoserine sulfhydrylase to produce natural L-cysteine ​​(WO 2013-089478 A3). In addition, a process for manufacturing L-cysteine ​​hydrochloride hydrate is known (WO 2019-151769 A1), which separates and purifies the natural state of L-cysteine ​​fermentation broth using a continuous chromatography process without using chemical reactions or artificial synthetic compounds, and has the advantages of high yield and purity as well as efficient productivity increase and low water usage.

[0007] However, when using a continuous chromatography process, separation of the final product and the fermentation medium or pigment substances produced during fermentation is required, and additional purification steps may be necessary. While pigment substances are typically removed using activated carbon, in the case of fermentation broth produced from L-cysteine ​​fermentation broth using the above method, pigment substances and impurities that are not removed by activated carbon may develop color under the conditions used to produce cysteine ​​hydrochloride crystals, causing coloration in the final product and deteriorating color quality.

[0008] Against this backdrop, the purpose of the present invention is to provide a method for producing L-cysteine ​​hydrochloride crystals having a uniform color from an L-cysteine ​​fermentation broth produced by a microbial culture method and an enzyme-catalyzed reaction.

[0009]

[0010] An example of this application is

[0011] (a) a step of obtaining an L-cysteine ​​separation solution from a fermentation solution containing L-cysteine;

[0012] (b) a step of filtering the L-cysteine ​​separation solution to obtain a filtrate;

[0013] (c) a step of concentrating the filtrate to obtain a concentrate;

[0014] (d) a step of adding hydrochloric acid to the filtrate or concentrate before or after the step (c); and

[0015] (e) a step of cooling the concentrate to recover L-cysteine ​​crystals; and a method for producing L-cysteine ​​crystals is provided.

[0016] Another example of the present application provides L-cysteine ​​crystals characterized by a yellowness of 4.0 or less.

[0017]

[0018] Hereinafter, the present invention will be described in more detail.

[0019]

[0020] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. In other words, 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 limited by the specific descriptions described below.

[0021]

[0022] This application, in one aspect,

[0023] (a) a step of obtaining an L-cysteine ​​separation solution from a fermentation solution containing L-cysteine;

[0024] (b) a step of filtering the L-cysteine ​​separation solution to obtain a filtrate;

[0025] (c) a step of concentrating the filtrate to obtain a concentrate;

[0026] (d) a step of adding hydrochloric acid to the filtrate or concentrate before or after the step (c); and

[0027] (e) a step of cooling the concentrate to recover L-cysteine ​​crystals; a method for producing L-cysteine ​​crystals is provided.

[0028]

[0029] The method for manufacturing L-cysteine ​​crystals of the present application is described in detail step by step as follows.

[0030]

[0031] (a) Step: L-cysteine ​​separation solution obtaining step

[0032] The method for producing L-cysteine ​​crystals of the present application may include step (a), a step of obtaining an L-cysteine ​​separation solution from a fermentation solution containing L-cysteine.

[0033] In this application, the term “L-cysteine” refers to one of the amino acids constituting proteins, and is the only sulfur-containing amino acid among L-amino acids that has a thiol group (R-SH). L-cysteine ​​may be chemically synthesized or biologically produced through microbial fermentation, but is not limited thereto. Specifically, L-cysteine ​​in this application may be L-cysteine ​​biologically produced through microbial fermentation, or may be natural L-cysteine ​​obtained by inducing an enzymatically catalyzed 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.

[0034] In this application, the term "natural" means not produced by chemical reactions. According to the EU Flavorings Regulation 1334 / 2008, only substances obtained through physical, enzymatic, or microbial processes are defined as "natural" flavorings. In this regard, regardless of whether L-cysteine ​​is of animal or microbial fermentation origin, all L-cysteine ​​produced by electrochemical reduction cannot be considered natural.

[0035] In this application, the term “fermentation solution” refers to a culture solution obtained by culturing a microorganism that produces L-cysteine, a culture containing a microorganism cultured with the medium, or an enzymatic conversion solution containing a precursor and an enzyme capable of producing L-cysteine. Specifically, the fermentation solution containing L-cysteine ​​may be a culture solution or an enzymatic conversion solution containing natural L-cysteine. More specifically, it may be an L-cysteine ​​culture or culture solution biologically produced through fermentation of a microorganism having L-cysteine-producing ability, or it may be an enzymatic conversion solution of natural L-cysteine ​​obtained by inducing an enzyme-catalyzed reaction between O-phosphohomoserine and sulfide, which are precursors produced through microbial fermentation, in the presence of phosphoserine sulfhydrylase. L-cysteine ​​crystals produced using the fermentation solution as a raw material solution may be natural L-cysteine ​​as they are not produced by a chemical reaction.

[0036] In the present application, the step of obtaining the separated solution in step (a) may be performed using continuous chromatography. In one example, the fermentation solution may be used as a raw material for the continuous chromatography process. That is, it may be injected into the continuous chromatography device in step (a).

[0037] The pH of the fermentation solution injected into the continuous chromatography device may vary depending on the manufacturing method, but may be a pH of 6.0 to 11.5, 6.5 to 11.0, 7.0 to 10.5, 7.5 to 10.0, or 8.0 to 9.5. The fermentation solution itself may be used as a raw material solution for the continuous chromatography process, and a step of adjusting the pH of the fermentation solution containing L-cysteine ​​to 6.0 to 11.5, 6.5 to 11.0, 7.0 to 10.5, 7.5 to 10.0, or 8.0 to 9.5 prior to step (a) may additionally be included. For example, the adjustment may be effected by adding an acid such as sulfuric acid or hydrochloric acid, or a base such as sodium hydroxide (caustic soda), ammonia, lithium hydroxide, or potassium hydroxide, but is not limited thereto. The above pH adjusting agent can be appropriately selected and used by a person skilled in the art as long as it does not affect the structure of L-cysteine ​​and can ultimately obtain crystals of L-cysteine.

[0038] The term "continuous chromatography" used in this application refers to a process that develops the existing batch chromatography process into a continuous process. Specifically, solid phases and liquid phases can be continuously supplied into a chromatography device, and the solid phases and liquid phases move in opposite directions to create countercurrent contact, thereby enabling more efficient separation of substances. In this application, it can be used as a concept that includes true moving bed (TMB) chromatography and simulated moving bed (SMB) chromatography. In addition, since the two true moving bed chromatography and simulated moving bed chromatography have the same principles, those skilled in the art can select and use them appropriately, taking productivity and other factors into consideration.

[0039] The continuous chromatography process used in the present application eliminates the need for an adsorption / elution process, thereby offering advantages over the ion exchange process, including higher hourly productivity and reduced process water usage. Furthermore, to obtain a high yield of L-cysteine ​​powder from a process solution containing L-cysteine ​​obtained through an ion exchange process or a general chromatography process, a significant amount of energy is required for the concentration and crystallization process. The method of the present application, however, can reduce energy costs.

[0040] The stationary phase of the above continuous chromatography device may be an ion exchange resin, and specifically, may be a strongly acidic cation exchange resin. The functional group of the strongly acidic cation exchange resin may be a sulfuric acid group, but is not limited thereto. In addition, the core material of the strongly acidic cation exchange resin used in the present invention is not limited as long as a strongly acidic functional group can be attached. For example, a styrene-divinylbenzene copolymer may be used, but is not limited thereto. For a specific example, the strongly acidic cation exchange resin may be a sulfuric acid styrene-divinylbenzene copolymer, but is not limited thereto.

[0041] In the present application, it is difficult to purify the L-cysteine ​​content of the solid fraction obtained through a continuous chromatography process to 50% or more (w / w) in the separated liquid using other types of stationary phases frequently used in the art for amino acid separation and purification, such as 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; and weakly basic cation exchange resins such as carboxyl methacrylate polymers. On the other hand, when a strongly acidic cation exchange resin such as a styrene-divinylbenzene copolymer is used, the L-cysteine ​​content of the solids excluding moisture in the separated liquid obtained through a continuous chromatography process can be purified to 80% (w / w) or more, specifically 90% (w / w) or more.

[0042] The above chromatography apparatus may use water, diluted caustic soda, diluted sulfuric acid, diluted phosphoric acid, diluted hydrochloric acid, diluted potassium hydroxide, or a mixture thereof as the mobile phase without chemical compounds (e.g., organic solvents such as methanol, isopropyl alcohol, and acetonitrile), but is not limited thereto. For example, water may be used as the mobile phase for continuous chromatography. When a mobile phase containing chemical compounds is used, the chemical compounds may remain in the final product, which may result in the product not being able to be sold or distributed as a natural product due to exceeding the residue standard for chemical compounds. In addition, since no chemicals other than water are added to the process water, a product cost reduction effect can be expected. 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 entails an increase in product cost related to the use and disposal of the chemical compounds.

[0043] In the above step (a), when the fermentation solution containing L-cysteine ​​is injected into a continuous chromatography device, a separated solution containing L-cysteine ​​can be obtained by separating the solution according to the continuous chromatography process. In the present invention, the separated solution containing L-cysteine ​​by separating the L-cysteine ​​can be simply expressed as "separated solution", "chromatography separated solution", or "process solution".

[0044]

[0045] (b) Step: L-cysteine ​​filtrate obtaining step

[0046] Thereafter, the method of the present application may include a step (b), a step of filtering the L-cysteine ​​separation solution obtained in step (a) to obtain a filtrate.

[0047] In the above step (b), a method known in the art can be applied for filtering the L-cysteine ​​separation solution. For example, a filtration method using a Nutsche filter, a filter press, or a housing filter using a filter made of CA, PVDF, PES, or PTFE material can be used, and in one embodiment, filtration can be performed using a Nutsche filter, but is not limited thereto.

[0048] The step of obtaining the filtrate in step (b) above may be performed by adding activated carbon. In one example, the activated carbon comprises 0.01 to 50 wt%, 0.1 to 50 wt%, 1 to 50 wt%, 5 to 50 wt%, 0.01 to 40 wt%, 0.1 to 40 wt%, 1 to 40 wt%, 5 to 40 wt%, 0.01 to 30 wt%, 0.1 to 30 wt%, 1 to 30 wt%, 5 to 30 wt%, 0.01 to 20 wt%, 0.1 to 20 wt%, 1 to 20 wt%, 2 to 20 wt%, 3 to 20 wt%, 4 to 20 wt%, 5 to 20 wt%, 6 to 20 wt%, 7 to 20 wt%, 8 to 20 wt%, 9 to 20 wt%, 0.01 to 15 wt%, 0.1 to 15 wt%, 1 to 15 wt%, 2 to 15 wt%, 3 to 15 wt%, 4 to 15 wt%, 5 to 15 wt%, 6 to 15 wt%, 7 to 15 wt%, 8 to 15 wt%, 9 to 15 wt%, 0.01 to 12 wt%, 0.1 to 12 wt%, 1 to 12 wt%, 2 to 12 wt%, 3 to 12 wt%, 4 to 12 wt%, 5 to 12 wt%, 6 to 12 wt%, 7 to 12 wt%, 8 to 12 wt%, or It may be added in an amount of 9 to 12 wt%, but is not limited thereto.

[0049]

[0050] (c) Step: Obtaining L-cysteine ​​concentrate

[0051] Thereafter, the method of the present application may include a step (c), a step of concentrating the L-cysteine ​​filtrate obtained in step (b) to obtain a concentrate.

[0052] The concentration in step (c) above can be performed in a conventional concentrator, such as a forced circulation concentrator, a thin film concentrator, or a rotary concentrator. In one example, it can be performed in a rotary concentrator.

[0053] The vacuum of the concentrator in the concentration step of the above step (c) may be, but is not limited to, 90 to 130 mmHg, 95 to 125 mmHg, or 100 to 120 mmHg, for example, 110 mmHg. In addition, the external temperature of the concentrator may be, but is not limited to, 50 to 90°C, 55 to 85°C, or 60 to 80°C, for example, 70°C.

[0054]

[0055] (d) Step: Hydrochloric acid addition step

[0056] The method of the present application may include a step of adding hydrochloric acid to the filtrate or concentrate before or after step (d), or before or after step (c).

[0057] The amount of hydrochloric acid added in the above step (d) may be such that the hydrochloric acid concentration of the concentrate after addition may be less than 6.5, less than 6.0 N, less than 5.5 N, less than 4.5 N, 4.5 to less than 5.5 N, or 5.5 N to less than 6.5 N, but is not limited thereto.

[0058] The hydrochloric acid addition step of step (d) may be performed before performing step (e), may be performed after step (b), and specifically, may be performed before step (c), after step (c), or simultaneously with step (c).

[0059] The method for producing L-cysteine ​​crystals of the present application may further include a step of controlling the retention time of the concentrate at a temperature of 10°C to 70°C after step (d).

[0060] The term “residence time” of a concentrate in the present application may mean the time for which the concentrate is maintained in a liquid state before crystallization, and may be used interchangeably with the terms “storage time”, “concentration time”, “holding time” or “holding time”.

[0061] In the present application, the retention time of the concentrate may vary depending on the hydrochloric acid concentration and retention temperature of the concentrate after the addition of hydrochloric acid in step (d).

[0062] In one example,

[0063] The retention time of the above concentrate at a retention temperature of 10℃ or more and less than 30℃

[0064] (i) within 24 hours when the hydrochloric acid concentration of the concentrate is 4.5 N or higher after the addition of hydrochloric acid in step (d);

[0065] The retention time of the above concentrate at a retention temperature of 30℃ or more and less than 50℃

[0066] (i) within 720 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d);

[0067] (ii) within 480 minutes when the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 4.5 N to 5.5 N; and / or

[0068] (iii) within 240 minutes when the hydrochloric acid concentration of the concentrate is less than 5.5 N to 6.5 N after the addition of hydrochloric acid in step (d);

[0069] The retention time of the above concentrate at a retention temperature of 50℃ or higher is

[0070] (i) within 240 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d);

[0071] (ii) within 180 minutes when the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 4.5 N to 5.5 N; and / or

[0072] (iii) If the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 5.5 N to 6.5 N, it may be within 120 minutes, but is not limited thereto.

[0073] The residence time of the above concentrate is specifically,

[0074] When the residence temperature is between 15℃ and 25℃,

[0075] (i) within 540 minutes when the hydrochloric acid concentration of the concentrate is 4.5 N or higher after the addition of hydrochloric acid in step (d);

[0076] When the residence temperature is 35℃ to 45℃,

[0077] (i) within 540 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d);

[0078] (ii) within 420 minutes when the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 4.5 N to 5.5 N; and / or

[0079] (iii) within 210 minutes when the hydrochloric acid concentration of the concentrate is less than 5.5 N to 6.5 N after the addition of hydrochloric acid in step (d);

[0080] When the residence temperature is 55℃ to 65℃,

[0081] (i) within 210 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d);

[0082] (ii) within 150 minutes when the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 4.5 N to 5.5 N; and / or

[0083] (iii) If the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 5.5 N to 6.5 N, it may be within 90 minutes, but is not limited thereto.

[0084]

[0085] In the present application, the retention temperature of the concentrate may mean a temperature that maintains the concentrate constant during the retention time of the concentrate, and may mean the temperature of the concentrate.

[0086] The retention temperature of the above concentrate is 10℃, 15℃ or higher, 20℃ or higher, 25℃ or higher, 30℃ or higher, 40℃ or higher, 45℃ or higher, 50℃ or higher, 55℃ or higher, 10 to 70℃, 15 to 70℃, 20 to 70℃, 25 to 70℃, 30 to 70℃, 35 to 70℃, 40 to 70℃, 45 to 70℃, 50 to 70℃, 55 to 70℃, 10 to 65℃, 15 to 65℃, 20 to 65℃, 25 to 65℃, 30 to 65℃, 35 to 65℃, 40 to 65℃, 45 to 65℃, 50 to 65℃, 10℃ or higher and less than 30℃, It may be 15°C to 25°C, 30°C to 50°C, 35°C to 45°C, or 55 to 65°C.

[0087] After adjusting the retention time of the above concentrate, the 430 nm absorbance of the L-cysteine ​​concentrate may be, but is not limited to, 0.65 or less, 0.60 or less, or 0.55 or less.

[0088] The L-cysteine ​​concentrate after adjusting the retention time of the above concentrate can exhibit a relatively low absorbance value due to a reduced reddening phenomenon compared to the L-cysteine ​​concentrate that has not undergone the above process, by a step of adding hydrochloric acid before or after the concentration step and / or a step of adjusting the retention time according to the hydrochloric acid concentration of the concentrate after adding hydrochloric acid.

[0089] In the present application, the 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 retention time according to the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid may contain a “color-inducing substance” that is a substance causing the reddish phenomenon, and the method of the present application includes, as a step of reducing the “color-inducing substance,” the step of adding hydrochloric acid before or after the concentration step and / or the step of adjusting the retention time according to the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid.

[0090] The term “color inducing substance” in this application means a substance that induces color, which is a substance that causes a reddish phenomenon, and may be used interchangeably with the terms “color inducing substance” or “color developing substance.”

[0091] In the present application, the color-inducing substance that causes a reddish phenomenon and is included in the L-cysteine ​​concentrate may be, but is not limited to, PLP (pyridoxal-5'-phosphate).

[0092]

[0093] (e) Step: Cooling of concentrate and recovery of crystals

[0094] Thereafter, the method of the present application may include step (e), a step of cooling the concentrate to which hydrochloric acid has been added to recover L-cysteine ​​crystals.

[0095] A step of crystallizing L-cysteine ​​may be additionally included after cooling according to the above step (e).

[0096] The term “crystallization” in this application refers to a phenomenon in which a liquid or amorphous solid forms a crystal, which occurs through two phenomena: the generation of crystal nuclei and the growth of crystal nuclei.

[0097] The above concentrate may be cooled and / or aged prior to recovery to form and / or grow crystal nuclei. Furthermore, even if L-cysteine ​​crystals are not precipitated in the concentrate, crystals may be formed when the concentrate is cooled and / or aged.

[0098] The above cooling step may specifically mean cooling to a temperature of -10 to 55°C over a period of 2 to 6 hours, specifically to a temperature of 0 to 45°C over a period of 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 a period of 2 to 6 hours.

[0099] The above-mentioned maturing step may refer to leaving the solution without temperature change. In the context of the present application, it may also refer to maintaining the cooled temperature at a constant level, or even maintaining the temperature of the concentrate at a constant level even when cooling is not performed. Specifically, maturing may be performed over a period of 1 to 3 hours.

[0100] The above step (e) can recover L-cysteine ​​crystals precipitated from the concentrate. Specifically, the concentrate can be subjected to solid-liquid separation to recover L-cysteine ​​crystals from the slurry. This can be performed using a solid-liquid separator such as a pressure-reducing membrane filtration device, a pressure-reducing membrane filtration device, or a centrifugal separator, but is not limited thereto. The slurry and / or the precipitated L-cysteine ​​crystals can additionally undergo a washing or drying step.

[0101]

[0102] In the present application, the L-cysteine ​​crystals manufactured through steps (a) to (e) can maintain uniform yellowness.

[0103] The yellowness of the above L-cysteine ​​crystal is one of the chromaticities measured by a colorimeter, and may be used interchangeably with the b or b* value in the present application.

[0104] The yellowness of the above L-cysteine ​​crystals is 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 It could be 4.0.

[0105] The term “color difference meter” in this application means a device that measures the color of an object, and the color difference meter used for color measurement in this application may be CHROMA METER (CR-410, KONICA MINOLTA), but is not limited thereto.

[0106] In the present application, the L-cysteine ​​concentrate after controlling the retention time of the L-cysteine ​​concentrate is manufactured through a step of adding hydrochloric acid before or after the concentration step and / or a step of controlling the retention time according to the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid, and the L-cysteine ​​crystals produced can exhibit a relatively low yellowness due to a reduced reddening phenomenon compared to L-cysteine ​​crystals that have not undergone the above process.

[0107] In the present application, L-cysteine ​​crystals that are not subjected to the step of adding hydrochloric acid before or after the concentration step and / or the step of controlling the retention time according to the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid may contain a “color-inducing substance” that is a substance causing the reddish phenomenon, and the method of the present application includes, as a step of reducing the “color-inducing substance,” a step of adding hydrochloric acid before or after the concentration step and / or a step of controlling the retention time according to the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid.

[0108] In the present application, the color-inducing substance that causes a reddish phenomenon and is included in the L-cysteine ​​concentrate may be, but is not limited to, PLP (pyridoxal-5'-phosphate).

[0109]

[0110] In addition, the present application provides, in another aspect, L-cysteine ​​crystals characterized by a yellowness of 4.0 or less.

[0111] The above L-cysteine ​​crystals are

[0112] (a) a step of obtaining an L-cysteine ​​separation solution from a fermentation solution containing L-cysteine;

[0113] (b) a step of filtering the L-cysteine ​​separation solution to obtain a filtrate;

[0114] (c) a step of concentrating the filtrate to obtain a concentrate;

[0115] (d) a step of adding hydrochloric acid to the filtrate or concentrate before or after the step (c); and

[0116] (e) a step of cooling the concentrate to which hydrochloric acid has been added to recover L-cysteine ​​crystals; It may be manufactured by a method for manufacturing L-cysteine ​​crystals, including the step of:

[0117] The method for producing the above L-cysteine ​​crystals is as described above.

[0118]

[0119] Figure 1 is a diagram showing the color change of L-cysteine ​​solutions according to the concentration of pyridoxal 5′-phosphate (PLP) added and the retention time. (a) shows the color of each L-cysteine ​​solution 0 hours after the addition of PLP, (b) shows the color of each L-cysteine ​​solution 2 hours after the addition of PLP, and (c) shows the color of each L-cysteine ​​solution 4 hours after the addition of PLP.

[0120]

[0121] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these examples.

[0122]

[0123] Example 1. Comparison of color of fermentation-derived hydrochloride crystals containing L-cysteine ​​according to hydrochloric acid concentration.

[0124] Example 1-1. Production of fermentation-derived process solution containing L-cysteine ​​using microbial fermentation.

[0125] A natural L-cysteine ​​fermentation solution was obtained by inducing an enzyme-catalyzed reaction using O-phosphohomoserine and sulfide, precursors produced through microbial fermentation, using O-phosphoserine sulfhydrase (OPS sulfhydrase) and pyridoxal 5′-phosphate (PLP). The pH of the L-cysteine ​​fermentation solution was 8-9.5.

[0126] Specifically, the KCCM 11103P (CA07-0022 / pCL-prmf-serA*(G336V)-serC; Republic of Korea Patent No. 10-1381048) strain, which has OPS production ability due to the deletion of serB and the introduction of a mutant serA* in the E. coli W3110 strain, was cultured on an MMYE agar plate at 33°C for 24 hours, and 1 / 10 of the cells on the plate were scraped from one plate and seeded in 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 of glycine) and seed cultured at 30°C at 200 rpm for 6 hours. After completion of seed culture, seed culture medium corresponding to 16% of the volume of the main culture medium was inoculated into a 1 L small fermenter filled with 300 ml of the main culture medium, and culture was performed at 33°C at pH 7.0 to obtain an OPS fermentation broth. The 50 mM OPS fermentation broth was reacted with 50 mg / ml Msm-T enzyme derived from Mycobacterium tuberculosis H37Rv in the presence of 100 mM Na2S and 0.2 mM pyridoxal 5′-phosphate (PLP) to obtain a fermentation broth containing L-cysteine ​​(US 8557549 B2).

[0127] The resulting L-cysteine ​​fermentation broth was filtered through a 0.14 μm membrane to separate the cells, yielding a filtrate. The pH of the filtrate was adjusted to pH 2-7 using 98% sulfuric acid. A continuous chromatography process (US 11427537 B2) using a strongly acidic cation exchange resin was then used to obtain a separated solution containing L-cysteine.

[0128] Activated carbon was added at 10 wt% based on the total weight of L-cysteine ​​to the separated solution from which L-cysteine ​​was separated, and stirred at 40°C for 1 hour. The activated carbon was separated using a Nutsche Filter to obtain a filtrate.

[0129]

[0130] Example 1-2. Comparison of color of hydrochloride crystals containing L-cysteine ​​according to hydrochloric acid concentration.

[0131] In order to compare the color difference between the L-cysteine ​​concentrate and the hydrochloride crystals containing L-cysteine ​​recovered by cooling the concentrate according to the concentration of hydrochloric acid when hydrochloric acid was added to the filtrate obtained by separation using a Nutsche filter in the above Example 1-1, the following experiment was performed.

[0132] 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 so that the hydrochloric acid concentrations were 4, 5, 6, and 6.5 N, respectively, and after being kept at 60°C for 1 hour, the change in absorbance of the concentrate was measured.

[0133] Each L-cysteine ​​concentrate (preparation solution) containing the above-described various concentrations of hydrochloric acid was cooled to 15℃ at a constant cooling rate for 4 hours in a jacket tank with stirring. Then, L-cysteine ​​crystals were separated from the L-cysteine ​​crystal slurry using a basket separator at a rotation speed of 3,000 rpm for 15 minutes. After separation, drying was performed in an oven dryer at 40℃ for more than 2 hours to reduce the residual moisture to 0.5% or less, and finally L-cysteine ​​hydrochloride crystals were manufactured. The yellowness (b* value) of the manufactured L-cysteine ​​hydrochloride crystals was measured using a CHROMA METER (CR-410, KONICA MINOLTA) colorimeter, and the results are shown in Table 1 below. The closer the yellowness (b* value) is to 0, the closer it is to white, and the higher the value, the more yellow it becomes.

[0134] Hydrochloric acid concentration (N)Starting absorbance (430 nm)Final absorbance (430 nm)Determination b* value40.020.132.150.020.352.560.020.5546.50.020.657.1

[0135] As a result, as shown in Table 1, it can be seen that the absorbance increase value increases as the hydrochloric acid concentration of the concentrate (preparation) increases. In addition, it was confirmed that the color value of the L-cysteine ​​hydrochloride crystals is determined according to the final absorbance value of the concentrate (preparation). It was confirmed that when the absorbance value is 0.55 or less, L-cysteine ​​hydrochloride crystals having a yellowness (b* value) of 4.0 or less can be obtained.

[0136]

[0137] Example 2. Comparison of absorbance according to retention temperature of fermentation-derived concentrate containing L-cysteine

[0138] To determine the effect of retention temperature on the absorbance of fermentation-derived concentrate containing L-cysteine, the following experiment was performed.

[0139] Specifically, activated carbon at 5 wt% of L-cysteine ​​was added to the separated solution containing L-cysteine ​​separated by the continuous chromatography process of Example 1-1, and stirred at 40°C for 1 hour. The activated carbon was separated using 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, and then a concentrate was prepared by adding hydrochloric acid so that the hydrochloric acid concentration was 4.5, 5.5, and 6.5 N. The retention temperatures of each L-cysteine ​​concentrate with the hydrochloric acid of various concentrations were set to 20°C, 40°C, and 60°C, respectively, and the concentrate was retained for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, and 8 hours, and then the UV / Vis absorbance at 430 nm was measured to confirm the change in absorbance over time. The results of the absorbance measurement over time are shown in Table 2 below.

[0140] Absorbance (430 nm) change rate according to retention temperature (℃) and hydrochloric acid concentration (N) and retention time 0 hr1 hr2 hr3 hr4 hr6 hr8 hr absorbance / hr 20 4.50.150.150.150.160.160.170.170.00 35.50.140.150.150.150.150.170.170.00 46.50.150.150.150.150.160.160.170.180.00 4404.50.150.190.210.240.300.360.430.045.50.140.180.220.300.350.500.620.066.50.150.29 0.350.450.680.951.210.13604.50.150.280.370.410.610.861.110.125.00.150.270.450.620.911.261.540.175.50.140.360.740.781.131.742.340.286.00.140.440.911.031.582.272.870.346.50.150.471.111.382.072.693.630.43

[0141] As a result, as shown in Table 2, it was confirmed that the higher the retention temperature of the concentrate and the higher the hydrochloric acid concentration, the greater the increase in absorbance. Unlike the results of Examples 1-2 above, when retained (stored) at 20℃, the change in absorbance per hour hardly changed at 0.003-0.004 even when the hydrochloric acid concentration and retention time increased. At 40℃, it was confirmed that when the hydrochloric acid concentration increased from 4.5 N to 6.5 N, the change in absorbance per hour increased linearly from 0.04 to 0.13, and at 60℃, when the hydrochloric acid concentration increased from 4.5 N to 6.5 N, the change in absorbance per hour increased linearly from 0.12 to 0.43. Therefore, it was confirmed that the change in absorbance of the concentrate was influenced by a specific substance derived from the fermentation liquid depending on the retention temperature and hydrochloric acid concentration conditions.

[0142]

[0143] Comparative Example 1. Confirmation of the effect of activated carbon input on the removal of color-causing substances.

[0144] In order to confirm the influence of the conditions for controlling the color of the L-cysteine ​​hydrochloride crystals derived from the above Examples 1 and 2, including the concentration of hydrochloric acid added to the concentrate and the retention temperature of the concentrate, on the removal of color-causing substances, the following experiment was conducted.

[0145] Specifically, activated carbon was added at 0, 5, and 10 wt% based on the total weight of L-cysteine ​​to the separated solution from which L-cysteine ​​was separated by a continuous chromatography process, and 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 so that the hydrochloric acid concentration became 5.5 N to make a concentrate, and the results of measuring the change in absorbance of the concentrate over time at 60°C every 2 hours are shown in Table 3 below.

[0146] Absorbance (430 nm) change rate with retention time of activated carbon wt% 0 hr 2 hr 4 hr 8 hr Absorbance / hr 0 0.34 0.97 1.85 3.12 0.35 5 0.14 0.74 1.13 2.34 0.28 10 0.02 0.65 0.94 2.04 0.25

[0147] As a result, as shown in Table 3, the increase in absorbance per hour of the concentrate after decolorization by adding 10 wt% activated carbon was confirmed to be at a similar level of 0.25 to 0.35. Therefore, although a small amount of color-causing substances can be removed by the decolorization process using activated carbon, the effect is not great, so it was confirmed that the concentration of hydrochloric acid added to the concentrate derived from Examples 1 and 2 and the retention temperature conditions of the concentrate are the most important factors for controlling the color of L-cysteine ​​hydrochloride crystals.

[0148]

[0149] Experimental Example 1. Confirmation of the change in absorbance of L-cysteine ​​reagent solution according to PLP concentration.

[0150] Pyridoxal 5'-phosphate (PLP) is one of the coenzymes used in a method for producing cysteine ​​or a derivative thereof by reacting O-phosphoserine (OPS) as a substrate with sulfide in the presence of O-phosphoserine sulfhydrylase (OPSS) or a microorganism expressing the same (US 8557549 B2). The following experiment was performed to determine the effect of the concentration of the coenzyme PLP (pyridoxal-5'-phosphate) on the color change of an L-cysteine ​​solution.

[0151] Specifically, 6.0 N hydrochloric acid was added to 100 g / L of L-cysteine ​​reagent, and PLP was not added or PLP was added at concentrations of 1 wt%, 5 wt%, and 10 wt%, respectively. Then, the change in UV / Vis absorbance at 430 nm of each sample solution was measured every 2 hours at a retention temperature of 60°C, and the results are shown in Fig. 1 and Table 4 below.

[0152] PLP concentration absorbance (430 nm) wt% 0 hr2 hr4 hr0000100.130.02500.160.281000.560.98

[0153] As a result, as shown in Fig. 1 and Table 4, under the condition without PLP addition, there was no change in the absorbance of the L-cysteine ​​sample solution over time, whereas under the condition with PLP addition, it was confirmed that the rate of change in the absorbance of the L-cysteine ​​sample solution over time increased as the concentration of PLP increased. In addition, it was confirmed that the color of the L-cysteine ​​solution changed from yellow to orange as the retention time increased (as the heating time increased) at a retention temperature of 60℃ (see Fig. 1).

[0154] Through the above results, it was confirmed that a specific substance derived from the fermentation broth that affects absorbance depending on the residence temperature and hydrochloric acid concentration conditions in Example 2 may include PLP.

Claims

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

2. A method for producing L-cysteine ​​crystals, wherein the step of obtaining the separated solution in step (a) is performed using continuous chromatography in the first paragraph.

3. A method for producing L-cysteine ​​crystals, wherein the step of obtaining a filtrate in step (b) is performed by adding activated carbon.

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

5. A method for producing L-cysteine ​​crystals, wherein the concentration in step (c) is performed under conditions of a vacuum of 100 to 120 mmHg and an external temperature of 60 to 80°C in the first paragraph.

6. A method for producing L-cysteine ​​crystals, wherein the amount of hydrochloric acid added in step (d) is such that the hydrochloric acid concentration of the concentrate after addition is less than 6.5 N.

7. A method for producing L-cysteine ​​crystals, further comprising a step of controlling the retention time of the concentrate at a temperature of 10°C to 70°C after step (d) in the first paragraph.

8. A method for producing L-cysteine ​​crystals in the 7th paragraph, wherein the retention time is within 24 hours when the retention temperature is 10°C or higher and less than 30°C, and the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is 4.5 N or higher.

9. In paragraph 7, the residence time is when the residence temperature is 30℃ or more and less than 50℃, (i) within 720 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d); (ii) within 480 minutes when the hydrochloric acid concentration of the concentrate after the addition of hydrochloric acid in step (d) is less than 4.5 N to 5.5 N; or (iii) A method for producing L-cysteine ​​crystals, wherein the concentration of hydrochloric acid in the concentrate after addition of hydrochloric acid in step (d) is less than 5.5 N to 6.5 N, within 240 minutes; 10. In paragraph 7, when the residence time is at a residence temperature of 50°C or higher, (i) within 240 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N after the addition of hydrochloric acid in step (d); (ii) within 180 minutes when the hydrochloric acid concentration of the concentrate is less than 4.5 N to 5.5 N after the addition of hydrochloric acid in step (d); or (iii) A method for producing L-cysteine ​​crystals, wherein the concentration of hydrochloric acid in the concentrate is less than 5.5 N to 6.5 N after addition of hydrochloric acid in step (d), within 120 minutes; 11. A method for producing L-cysteine ​​crystals, wherein, in the 7th paragraph, after adjusting the retention time, the 430 nm absorbance of the concentrate is 0.55 or less.

12. A method for producing L-cysteine ​​crystals, wherein the concentrate of step (e) is cooled to a temperature of 0 to 30°C in the first paragraph.

13. A 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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