Amino acid production method with improved filtration flow rate

Abstract

The present application relates to a method for producing an amino acid, which comprises adding a coagulant and / or a flocculating agent to a culture medium containing an amino acid.

Description

[Technical Field]

[0001] The present application relates to a method for producing amino acids with improved filtration flow rate, which includes adding a coagulant and / or a flocculating agent to a culture medium containing amino acids. [Background technology]

[0002] Amino acids are the basic building blocks of proteins and are used as important components in pharmaceutical raw materials, food additives, animal feed, nutrients, insecticides, fungicides, etc. In particular, branched chain amino acids (BCAAs) are a collective term for the essential amino acids valine, leucine, and isoleucine, and are known to have antioxidant effects and to directly promote protein synthesis in muscle cells.

[0003] Meanwhile, production of branched-chain amino acids using microorganisms is mainly carried out by microorganisms of the genus Escherichia or Corynebacterium, and it is known that they are biosynthesized from pyruvate through various steps to 2-ketoisocaproic acid, a precursor. However, the production of branched-chain amino acids using such microorganisms has the problem that industrial mass production is not easy.

[0004] Meanwhile, a typical amino acid production process involves a series of steps, including i) microbial cultivation, ii) filtration of the culture solution containing amino acids, iii) decolorization, iv) filtration, v) concentration, and vi) crystallization. These steps may be adjusted depending on the product characteristics. Among these steps, the "filtration process" uses a membrane filter to remove bacteria, proteins, colloidal substances, natural organic matter (NOM), and low-molecular-weight substances contained in the culture solution. When the culture solution is filtered through a membrane in this filtration process, impurities accumulate on the membrane over time, forming a gel-like cake layer on the membrane surface. Such membrane fouling reduces the membrane permeation flux. This reduced membrane permeation flux reduces daily production, lengthens the membrane cleaning cycle, and shortens the membrane replacement cycle, resulting in increased maintenance and production costs and reduced price competitiveness. Therefore, in order to suppress the formation of a cake layer due to impurities on the membrane surface, which reduces the membrane permeation flow rate, a method for removing impurities from the culture solution and a method for reducing membrane contamination due to impurities during the filtration process are needed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 8,465,962 [Patent Document 2] U.S. Patent No. 9,885,093 [Patent Document 3] U.S. Patent No. 10,351,859 [Patent Document 4] U.S. Patent No. 7,863,435 [Patent Document 5] U.S. Patent No. 10,787,692 [Patent Document 6] U.S. Patent No. 9,029,105 [Patent Document 7] US Patent Application Publication No. 2021 / 0094903 [Patent Document 8] U.S. Patent No. 9,587,261 Summary of the Invention [Problem to be solved by the invention]

[0006] The present application aims to provide a method for producing an amino acid from a culture solution containing the amino acid, comprising: (a) adding an organic coagulant, a cationic polymer flocculant, or both to the culture solution containing the amino acid to precipitate impurities; and (b) filtering the culture solution in which the impurities have been precipitated through a membrane.

[0007] Another object of the present application is to provide a feed composition containing the amino acid produced by the method of the present application. [Means for solving the problem]

[0008] These will be described in detail below. Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in this application are included in this application. Furthermore, this application is not limited to the following specific descriptions.

[0009] Furthermore, throughout this specification, many papers and patent documents are referenced and citations are provided, the disclosures of which are incorporated herein by reference in their entirety to more clearly explain the state of the art to which this application pertains and the contents of this application.

[0010] In order to achieve the above object, one aspect of the present application provides a method for producing an amino acid from a culture solution containing the amino acid, the method comprising: (a) adding an organic coagulant, a cationic polymer flocculant, or both to the culture solution containing the amino acid to precipitate impurities; and (b) filtering the culture solution in which the impurities have been precipitated through a membrane.

[0011] In this application, the term "coagulant" refers to a substance that curdles or clots with particles in a liquid, causing them to separate from the liquid. Specifically, it refers to a substance that promotes the formation of larger clumps of fine particles in a liquid. For example, particles in a liquid carry surface charges that repel each other, preventing them from settling and maintaining a floating state in the water. However, by transferring opposite charges to these particles, coagulants destabilize the charges, resulting in the particles clinging together. Coagulants are used in a variety of industries, from food, beverage, chemical, and pharmaceutical production to drinking water, municipal wastewater treatment, and oil and gas drilling. They are primarily used to precipitate relatively small particles of a few microns in size, e.g., around 2 microns.

[0012] In this application, the term "flocculant (also known as a flocculating agent or a flocking agent)" refers to a substance used to remove suspended solids from a liquid by inducing coagulation, such as by causing solids to aggregate and begin to form flakes or settle to the bottom. The resulting relatively large and / or relatively heavy particles are also referred to as flocs. Unlike coagulants, which induce chemical coagulation by destabilizing electrical charges, flocculants promote physical coagulation by agglomerating colloids and other suspended particles in a liquid to form flocs. Such flocculants are also used in filtration processes, such as water treatment processes, to precipitate small particles and improve filterability. They are primarily used to precipitate relatively large particles of 10 μm or larger.

[0013] For example, the coagulant or flocculant used in the manufacturing method of the present application may be an organic coagulant, a cationic polymer flocculant, or a combination thereof.

[0014] Specifically, the organic coagulant contains components such as poly(dimethylamine-co-epichlorohydrin) (EPI-AMINE) and poly(diallyldimethylammonium chloride) (polyDADMAC), and the cationic polymer flocculant contains long chain polymer components such as polyacrylamide (PAM), polyethylenimine (PEI), and polyvinylamine (PVAM), but is not limited to these.

[0015] For example, the production method of the present application may further include a step of adjusting the culture solution containing the amino acid to an acidic pH of 3 to 5. For example, the culture solution containing the amino acid may be adjusted to, but not limited to, pH 3.0 to 4.5, pH 3.5 to 4.0, pH 3.7 to 4.3, or pH 3.8 to 4.2. In specific examples of the present application, it has been confirmed that the flocculation efficiency of a coagulant or flocculant is affected by the pH conditions of the solution, and in particular, improved flocculation efficiency is observed under acidic conditions compared to neutral conditions. However, the flocculation efficiency of a coagulant or flocculant is affected not only by pH conditions but also by the concentration of the coagulant or flocculant, the type of amino acid being produced, and the type, size, and / or amount of impurities in the culture solution, and is therefore not limited to the pH conditions.

[0016] The method for producing amino acids according to the present application is characterized by an improved filtration flow rate in membrane filtration compared to when no coagulant and / or flocculating agent is used.

[0017] Furthermore, the method for producing amino acids of the present application has the effect of improving the color of the membrane-filtered filtrate and reducing the content of impurities.

[0018] For example, a culture medium containing amino acids may contain at least one impurity selected from the group consisting of bacterial cells, proteins, colloidal substances, natural organic matter (NOM), and low-molecular-weight substances. For example, a culture medium for producing amino acids contains various impurities, including suspension materials such as bacterial cells, as well as colloidal substances derived from fermentation by-products such as fermentation media and cell lysates. These particles adhere to membranes in the filtration step used in the subsequent amino acid production process, forming cakes that not only reduce filtration efficiency but also foul the membranes and shorten the membrane replacement cycle, thereby adversely affecting productivity. In addition, the use of a coagulant and / or flocculant in the production method of the present application aggregates / flocculates and precipitates these impurities, thereby significantly reducing membrane fouling, such as cake formation, improving the membrane permeation flow rate, extending the membrane life, and improving productivity as well as production economy.

[0019] In the present application, the culture solution containing the amino acids is obtained by microbial fermentation, but is not limited thereto. Here, any method known in the art for microbial fermentation may be used.

[0020] For example, the production method of the present application may further include a pretreatment step of separating bacterial cells from a culture solution containing amino acids. For example, the bacterial cell separation can be performed using, but is not limited to, a mechanical separator (MS) or a membrane filter (MF). Here, bacterial cell separation using a mechanical separator is mainly performed by centrifugation, but in this case, colloidal substances cannot be separated and often transfer to the filtrate. Therefore, when combined with the production method of the present application, a particularly significant improvement effect can be obtained.

[0021] In the present application, the term "culture medium" refers to a culture obtained by culturing a microorganism. The culture medium contains the cultured microorganism. Furthermore, the culture medium is also referred to as a "fermentation medium."

[0022] In the present application, the term "culture medium containing amino acids" is used interchangeably with "culture medium containing amino acids" or "amino acid culture medium."

[0023] In the present application, the culture solution containing the amino acid may be a fermented product containing bacterial cells obtained by a known microbial fermentation method (Patent Documents 1, 2, 3, 4, 5, 6, and 7), the liquid obtained by removing bacterial cells from the fermented product, or a concentrated liquid obtained by concentrating the fermented product, but is not limited to these.

[0024] Specifically, the fermentation broth containing the amino acid of the present application can be obtained by culturing or fermenting a microorganism that produces the amino acid, and those skilled in the art can select and use the microorganism and its culturing or fermentation method from known types and methods. For example, the microorganism includes all wild-type microorganisms and microorganisms that have been genetically modified, either naturally or artificially, and is a microorganism in which a specific mechanism has been weakened or strengthened by inserting an exogenous gene or by strengthening or inactivating the activity of an endogenous gene, etc., and which has undergone genetic mutation or enhanced activity to produce the target L-amino acid (e.g., Patent Documents 4 and 8). Specifically, the microorganism is not particularly limited as long as it produces the desired amino acid, and includes microorganisms belonging to the genera Enterobacter, Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, and Brevibacterium. More specifically, it is a microorganism belonging to the genus Corynebacterium or Escherichia.The microorganisms of the genus Corynebacterium include Corynebacterium glutamicum, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium efficiens, Corynebacterium stationis, Corynebacterium phocae, Corynebacterium flavescens, Corynebacterium humireducens, Corynebacterium halotolerans, Corynebacterium Examples of Escherichia microorganisms include, but are not limited to, Corynebacterium halotolerans, Corynebacterium pollutisoli, Corynebacterium marinum, Corynebacterium freiburgense, Corynebacterium cystitidis, Corynebacterium durum, Corynebacterium pilosum, and Corynebacterium testudinoris. Examples of Escherichia microorganisms include, but are not limited to, Escherichia coli.

[0025] Furthermore, the production method of the present application may optionally further include at least one step selected from the group consisting of a decolorization step, a filtrate concentration step, a crystallization step, a crystal separation step, a drying step, a sieving step, and a commercialization step after the membrane filtration step. Any method known in the art (e.g., Patent Document 7) may be used for each of the above steps. Furthermore, specific conditions may be appropriately changed to optimize the process, but are not limited thereto.

[0026] For example, the amino acids that can be produced by applying the production method of the present application include, but are not limited to, isoleucine, valine, and leucine. The type and / or size of impurities contained in the culture medium varies depending on the type of amino acid to be produced. Therefore, taking into consideration the above factors, a coagulant or flocculating agent suitable for improving the membrane permeation flow rate may be selected and used for the amino acid to be produced, or both may be used in combination.

[0027] Another aspect of the present application provides a feed composition comprising the amino acids produced by the methods of the present application.

[0028] The amino acids of the present application are suitable for use as feed additives in the manufacture of animal feed. For example, the amino acids as feed additives can be part of an animal feed premix or a precursor to the animal feed and can be mixed with the feed material itself. The feed compositions containing the amino acids can be administered to animals alone or in combination with other feed additives in an edible carrier. The feed compositions can also be easily administered to animals as a top dressing, by mixing them directly with the animal feed, or in an oral dosage form separate from the feed. [Effects of the Invention]

[0029] In the manufacturing method of the present application, impurities contained in the culture solution are precipitated by adding a coagulant and / or flocculant to the culture solution, which reduces membrane fouling in the subsequent filtration process and improves the membrane permeation flow rate, thereby not only improving productivity but also improving the quality of the produced product by reducing the content of proteins and coloring substances in the filtrate. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present application will be described in more detail below with reference to examples, but these examples are merely illustrative of the present application and are not intended to limit the present application. [Example]

[0031] Effect of adding coagulant / flocculant in flocculation experiments The process liquid was an amino acid culture liquid produced using a microorganism. The amino acid culture liquids used were an isoleucine culture liquid, a valine culture liquid, and a leucine culture liquid. To test the membrane filtration efficiency, a large number of bacterial cells were centrifuged and removed using a mechanical separator (Alfa Laval Seperator), and the supernatant was then used as the feed liquid for the membrane filtration test. The pH of the feed liquid was 6, and the pH was adjusted using 98% H2SO4 and 50% NaOH.

[0032] Specifically, in the membrane filtration experiment, 200 mL of amino acid culture solution was prepared by first removing the bacterial cells, and then the pH was adjusted with 98% H2SO4 and 50% NaOH. To each solution whose pH had been adjusted as described above, 200 ppm of each of the seven coagulants / flocculants shown in Table 1 was added. After 10 seconds of rapid stirring and 30 seconds of slow stirring, the solution was allowed to settle for 30 minutes. The solution was then poured into a Buchner funnel with a diameter of 3.6 cm and a membrane area of ​​0.001 m2. 2 The process liquid was passed through the filter cloth and separated into a solid cake, which was a precipitated coagulation, and a liquid filtrate. The average membrane flow rate was measured when 200 mL of filtrate had been discharged. The unit membrane area (m) per unit time (hr) was 2The flow rate (L) passing through the tube was calculated.

[0033]

number

[0034] [Table 1] [Example]

[0035] Effect of pH on flocculation experiments To confirm the effect of pH on the flocculation experiment, the same experiment as in Example 1 was conducted, but the pH was adjusted to 4 in the acidic range, 6 in the neutral range, and 8 in the basic range, and flocculation was confirmed for each. The results are also shown in Tables 2 to 7. In summary, regardless of the type of coagulant / flocculant, no precipitate appeared at pH 6 or 8, while at an acidic pH of 4, precipitate sometimes formed and sometimes did not, depending on the zone / component and / or type of coagulant / flocculant. This indicates that the combination of appropriate coagulant / flocculant dosage and acidic pH promotes precipitation, further improving the filtration flow rate and filtrate quality. [Example]

[0036] Effect of coagulant / flocculant addition and pH on isoleucine production. As in Example 1, membrane filtration experiments were performed using the filtrate from the amino acid culture medium, from which the bacterial cells had been primarily removed, as the feed solution. However, the amino acid culture medium was a culture medium containing isoleucine, and the pH was adjusted to 4, 6, or 8. The experiments were performed without or with the coagulant / flocculant listed in Table 1. As in Example 1, each sample was observed for the presence or absence of precipitate formation, and the permeation flow rate was calculated. The results are shown in Tables 2 and 3. In addition, the absorbance at 280 nm and 420 nm, which are used as a protein content indicator and a color indicator, respectively, was measured for the filtrate obtained from each sample. The results are also shown in Tables 2 and 3.

[0037] [Table 2]

[0038] [Table 3]

[0039] As shown in Tables 2 and 3, in the isoleucine production process tested, no precipitate was observed regardless of pH in the coagulant / flocculant-free samples, and the permeation rate was low. Among the coagulant / flocculant-added groups, APAM addition, like the no-addition control, did not result in any precipitate formation regardless of pH, and no improvement in permeation rate was observed compared to the no-addition control. On the other hand, the inorganic coagulant PAC addition group formed a precipitate at pH 4, but only slightly improved the permeation rate, and no precipitate even formed at other pH ranges. In contrast, in the EPI-AMINE, polyDADMAC, PAM, PEI, or PVAM addition groups, no precipitate formed in any of the experimental groups adjusted to pH 6 and 8, and no improvement in permeation rate was observed. However, the experimental group adjusted to pH 4 showed a precipitate and a significant increase in permeation rate. In particular, PAM improved the filtration rate by approximately 5.7 times. Moreover, these experimental groups showed a significant reduction in absorbance at 420 nm, a color index, indicating that the process improved the quality of the filtrate. [Example]

[0040] Effect of coagulant / flocculant addition and pH on valine production. As in Example 1, membrane filtration experiments were performed using the lysate of an amino acid culture solution from which the bacterial cells had been primarily removed as a feed solution. However, a culture solution containing valine was used as the amino acid culture solution, the pH was adjusted to 4 or 6, and the experiment was performed without or with the coagulant / flocculant listed in Table 1. As in Example 1, each sample was observed for the presence or absence of precipitate formation, and the permeation flow rate was calculated. The results are shown in Tables 4 and 5. In addition, the absorbance at 280 nm and 420 nm, which are used as a protein content index and a color index, respectively, of the filtrate obtained from each sample was measured. The results are also shown in Tables 4 and 5.

[0041] [Table 4]

[0042] [Table 5]

[0043] As shown in Tables 4 and 5, in the valine production process tested, no precipitate was observed regardless of pH in the coagulant / flocculant-free samples, and the permeation rate was low. Among the coagulant / flocculant-added groups, APAM addition, like the no-addition control, did not result in any precipitate formation regardless of pH, and no improvement in permeation rate was observed compared to the no-addition control. On the other hand, in the group containing the inorganic coagulant PAC, precipitate formed at pH 4, but the improvement in permeation rate was minimal, and no precipitate even formed at other pH ranges. In contrast, in the groups containing EPI-AMINE, polyDADMAC, PAM, PEI, or PVAM, no precipitate formed in any of the experimental groups adjusted to pH 6, and no improvement in permeation rate was observed. However, precipitate was observed in the experimental group adjusted to pH 4, and the permeation rate showed a significant increase. In particular, polyDADMAC showed an approximately 4.0-fold improvement in filtration rate. Furthermore, these experimental groups showed a significant decrease in absorbance at 420 nm, a color index. This indicates that the process improves the quality of the filtrate. [Example]

[0044] Effect of coagulant / flocculant addition and pH on leucine production. As in Example 1, membrane filtration experiments were performed using the filtrate of the amino acid culture medium from which the bacterial cells had been primarily removed as the feed solution. However, the amino acid culture medium was a culture medium containing leucine, the pH was adjusted to 4 or 6, and the experiments were performed without or with the coagulant / flocculant listed in Table 1. As in Example 1, each sample was observed for the presence or absence of precipitate formation, and the permeation flow rate was calculated. The results are shown in Tables 6 and 7. In addition, the absorbance at 280 nm and 420 nm, which are used as a protein content indicator and a color indicator, respectively, was measured for the filtrate obtained from each sample. The results are also shown in Tables 6 and 7.

[0045] [Table 6]

[0046] [Table 7]

[0047] As shown in Tables 2–7, in the leucine production process tested, no precipitate was observed regardless of pH in the coagulant / flocculant-free samples, and the permeation flow rate was low. In the coagulant / flocculant-added groups, APAM addition, like the no-addition control, did not result in any precipitate formation regardless of pH, and no improvement in permeation flow rate was observed compared to the no-addition control. On the other hand, in the group containing the inorganic coagulant PAC, precipitate formed at pH 4, but the improvement in permeation flow rate was minimal, and no precipitate even formed at other pH ranges. In contrast, in the groups containing EPI-AMINE, polyDADMAC, PAM, PEI, or PVAM, no precipitate formed in any of the experimental groups adjusted to pH 6, and no improvement in permeation flow rate was observed. However, precipitate was observed in the experimental group adjusted to pH 4, and the permeation flow rate increased significantly. In particular, PAM demonstrated an approximately 4.0-fold improvement in filtration flow rate. Furthermore, these experimental groups also demonstrated a significant decrease in absorbance at 420 nm, a color index. This indicates that the process improves the quality of the filtrate.

[0048] In summary, the production method of the present application, which further includes a step of adding a coagulant / flocculant before membrane filtration, can significantly improve the average membrane permeation flow rate while maintaining the same concentration rate compared to when a coagulant / flocculant is not added, thereby significantly increasing the daily amino acid production volume, suggesting that an improved process with excellent price competitiveness can be provided. Furthermore, the production method of the present application has been confirmed to efficiently remove impurities including proteins as well as coloring substances, significantly reducing the absorbance at 420 nm, which is a product specification requirement in the food amino acid industry, and is therefore also useful for improving the quality of amino acid products.

[0049] From the above description, those skilled in the art to which the present application pertains will understand that the present application can be implemented in other specific forms without changing the technical idea or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present application should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalent concepts.

Claims

1. A method for producing amino acids from a culture medium containing amino acids, comprising: (a) adding an organic coagulant, a cationic polymer flocculant, or both to a culture solution containing amino acids to precipitate impurities; (b) filtering the culture solution from which the impurities have been precipitated through a membrane; Amino acid production method.

2. The organic coagulant is poly(dimethylamine-co-epichlorohydrin) (EPI-AMINE) or poly(diallyldimethylammonium chloride) (polyDADMAC), and the cationic polymer flocculant is polyacrylamide (PAM), polyethylenimine (PEI), or polyvinylamine (PVAM). The method for producing an amino acid according to claim 1.

3. The culture medium containing the amino acid further comprises adjusting the pH to an acidic range of 3 to 5. The method for producing an amino acid according to claim 1.

4. The filtration flow rate of the membrane filtration is improved. The method for producing an amino acid according to claim 1.

5. The color of the membrane-filtered filtrate is improved and the content of impurities is reduced. The method for producing an amino acid according to claim 1.

6. The impurities are at least one selected from the group consisting of bacterial cells, proteins, colloidal substances, natural organic matter (NOM), and low molecular weight substances. The method for producing an amino acid according to claim 1.

7. The culture solution containing the amino acid is obtained by fermentation using a microorganism. The method for producing an amino acid according to claim 1.

8. Further comprising a pretreatment step of separating bacterial cells from the culture solution containing the amino acids. The method for producing an amino acid according to claim 1.

9. After the membrane filtration step, optionally, at least one step selected from the group consisting of a decolorization step, a filtrate concentration step, a crystallization step, a crystal separation step, a drying step, a sieving step and a productization step is further included; The method for producing an amino acid according to claim 1.

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