Method for preparing L-valine crystals and / or particles using pH adjustment and PVA addition

Abstract

The present disclosure relates to methods of making valine crystals and / or valine particles using pH adjustment and PVA addition.

Description

Technical Field

[0001] This invention relates to a method for preparing valine crystals and granules, which reduces the amount of steam required in the production of valine granules and improves the efficiency of valine granule production by forming valine crystals and mixed granules with low moisture content. Background Technology

[0002] In order to obtain high-purity amino acids through fermentation, it is necessary to perform amino acid separation or purification steps after the fermentation process to remove byproducts.

[0003] However, for feed-grade amino acid products, it is not necessary to produce products with high amino acid content (98% or higher), and it may be more appropriate to produce pellet products with high content (70% or higher) that contain other valuable nutrients from the fermentation broth without generating waste in the purification step.

[0004] In conventional techniques (US 7,514,111 B2), to produce granulated amino acid products for use as feed additives, a large amount of water in the fermentation broth is evaporated, and then granules are produced using a fluidized bed granulator. In the case of highly soluble amino acids, crystals do not form even when the water content is evaporated to approximately 60% to 65% (moisture content 35% to 40%), thus granulation can be achieved using fluidized bed granulation, in which the fermentation broth is sprayed through nozzles. However, due to the low solubility of valine, crystals form even when the solid content in the fermentation broth is as low as approximately 12% to 15% (moisture content 85% to 88%), so reducing the moisture content of the fermentation broth cannot prevent crystal formation. Therefore, a large amount of water must be evaporated during the drying process, thus requiring a significant amount of energy for drying.

[0005] [Existing Technical Documents]

[0006] [Patent Documents]

[0007] (Patent Document 1) US 7,514,111 B2

[0008] (Patent Document 2) US 10,072,278 B2 Summary of the Invention

[0009] [Technical Issues]

[0010] One object of this disclosure is to provide a method for preparing valine crystals, comprising: a step of preparing a fermentation broth containing valine; a step of adjusting the pH of the fermentation broth; a step of adding polyvinyl alcohol (PVA) to the fermentation broth; and a step of separating valine crystals containing water from the fermentation broth.

[0011] Another object of this disclosure is to provide a method for preparing valine particles, comprising: a step of preparing a fermentation broth containing valine; a step of adjusting the pH of the fermentation broth; a step of adding polyvinyl alcohol (PVA) to the fermentation broth; a step of separating valine crystals containing water from the fermentation broth; a step of preparing mixed particles by mixing the valine crystals with seed crystals; and a step of drying the valine mixed particles.

[0012] [Technical Solution]

[0013] This disclosure provides a method for preparing valine crystals, comprising: preparing a fermentation broth containing valine; adjusting the pH of the fermentation broth; adding polyvinyl alcohol (PVA) to the fermentation broth; and separating valine crystals containing water from the fermentation broth.

[0014] This disclosure provides a method for preparing valine particles, comprising: a step of preparing a fermentation broth containing valine; a step of adjusting the pH of the fermentation broth; a step of adding polyvinyl alcohol (PVA) to the fermentation broth; a step of separating valine crystals containing water from the fermentation broth; a step of preparing mixed particles by mixing the valine crystals with seed crystals; and a step of drying the valine mixed particles.

[0015] [Beneficial Effects]

[0016] According to the present invention, by adjusting the pH of the fermentation broth containing valine and adding PVA, the moisture content of valine crystals or valine mixed particles can be reduced, thereby reducing the energy required for drying. Attached Figure Description

[0017] Figure 1 SEM images of valine crystals prepared according to embodiments of the present disclosure are shown. Figure 1 (a) and Figure 1 (b) Each represents valine crystals prepared by adding 2% PVA to the culture medium without PVA or with PVA. Detailed Implementation

[0018] This disclosure will be described in detail below. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of this disclosure. Moreover, the scope of this disclosure is not limited by the specific descriptions below.

[0019] Furthermore, numerous academic papers and patent documents are cited throughout the specification. The content of these cited papers and patent documents is incorporated herein by reference in its entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0020] Furthermore, those skilled in the art can recognize or identify many equivalents of the specific embodiments of this disclosure described herein through conventional experiments. Moreover, these equivalent substitutions are intended to be included in this disclosure.

[0021] As used in the specification and appended claims of this disclosure, the singular articles (“a,” “an,” and “the”) include plural referents unless the context clearly indicates otherwise. Furthermore, unless the context otherwise indicates, singular terms include their plural forms, and plural terms include their singular forms. As used in the specification and appended claims of this disclosure, the use of “or” can include the meaning of “and / or” unless otherwise stated.

[0022] As used herein, the term "about" may precede a specific numerical value. As used herein, the term "about" includes not only the exact numerical value specified after the term, but also a range that approximates or approximates that value. Considering the context in which the number appears, it can be determined whether the specific number mentioned is close to or approximates that number. For example, the term "about" may refer to a range of -10% to +10% of a value. As another example, the term "about" may refer to a range of -5% to +5% of a given value, but is not limited to this.

[0023] The method for preparing valine crystals according to this disclosure includes the steps of preparing a fermentation broth containing valine; adjusting the pH of the fermentation broth; adding polyvinyl alcohol (PVA) to the fermentation broth; and separating valine crystals containing water from the fermentation broth. Each step is described in detail below.

[0024] The step of preparing the fermentation broth is the step of preparing a fermentation broth containing valine. Valine contained in the fermentation broth is an amino acid with low solubility in water. Specifically, low solubility does not mean solubility equal to or less than a specific standard. The fermentation broth prepared in the above steps can be a fermentation product prepared by the metabolism of a microbial strain. As used herein, the term "fermentation product" can refer to a product formed by the enzymatic or metabolic decomposition of organic matter using microorganisms, such as those producing valine. For example, a fermentation product can include the culture itself obtained by culturing microorganisms in a culture medium, or a culture obtained after removing the microbial strain, its concentrate, dried product, or lyophilized product. Furthermore, in this case, the fermentation broth can contain the entire fermentation product containing valine, or it can be a fermentation product containing valine in which impurities have been removed.

[0025] The fermentation broth contains valine as the main component, but may also contain other components. For example, in addition to valine, other amino acids, organic acids, and inorganic substances may be provided in the fermentation broth, and these substances may be in a water-soluble state. The concentration of valine in the fermentation broth may be from about 1 g / L to about 200 g / L. However, the above-mentioned concentration of valine is merely illustrative, and fermentation broth with concentrations outside the above range may also be used in the method for producing valine crystals according to embodiments of this disclosure.

[0026] The "valine-producing microorganism" or "valine-producing microorganism" used in the step of preparing the fermentation broth of this disclosure includes wild-type microorganisms naturally capable of valine production and microorganisms that have undergone natural or artificial genetic modification, wherein the ability to produce valine is conferred by a parent strain that does not have the ability to produce valine. It can be a microorganism containing genetic modifications for the production of the amino acid (i.e., valine), wherein a specific mechanism is weakened or enhanced due to factors such as the insertion of a foreign gene or the enhancement or inactivation of an endogenous gene. Specifically, in this disclosure, a valine-producing microorganism or a valine-producing microorganism can be a microorganism in which some genes in the valine biosynthesis pathway are enhanced or weakened, or some genes in the valine degradation pathway are enhanced or weakened. "Enhancing" or "increasing" the valine production capacity of the microorganisms of this disclosure means that the valine production capacity of the microorganisms of this disclosure is improved compared to the valine production capacity of microorganisms other than those of this disclosure, parent strains, or unmodified microorganisms. For example, compared to the valine production capacity of another microorganism, the microorganism of this disclosure may have an increased valine production capacity of about 1% or more, 10% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 1100% or more, 1200% or more, or 1300% or more, and the microorganism of this disclosure may have an increased valine production capacity of about 1.01 times or more, 2 times or more, 5 times or more, 10 times or more, 11 times or more, 12 times or more, or 13 times or more, but is not limited thereto. The term "about" refers to a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., which includes all values ​​equal to or similar to the value following the term "about," but is not limited thereto.

[0027] The microorganisms used in the step of preparing the fermentation broth of this disclosure may be at least one selected from the group consisting of: yeast Candida famata, Ascomycetes Eremothecium ashbyii and Ashbya gossypii, and bacteria Bacillus subtilis and Corynebacterium.

[0028] For example, when the microorganisms used in the preparation of fermentation broth in this disclosure are Corynebacterium species, the specific microorganisms may be Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium efficiens, Corynebacterium callunae, Corynebacterium stationis, Corynebacterium singulare, Corynebacterium halotolerans, Corynebacterium striatum, Corynebacterium ammoniagenes, Corynebacterium pollutisoli, Corynebacterium imitans, and Corynebacterium testis. Corynebacterium testudinoris, Corynebacterium crenatum, or Corynebacterium flavescens, or more specifically, Corynebacterium glutamicum, but not limited thereto.

[0029] Corynebacterium species, particularly *Corynebacterium glutamicum*, are Gram-positive microorganisms widely used for the production of L-amino acids and other useful substances. Various studies have been conducted to develop microorganisms and fermentation processes with high production efficiency for the production of L-amino acids and other useful substances. For example, substance-specific methods are primarily employed, such as increasing the expression of genes encoding enzymes involved in L-valine biosynthesis or eliminating genes unnecessary for this biosynthesis. In this disclosure, fermentation broths containing amino acids can be prepared using strains of the *Corynebacterium* genus.

[0030] According to one embodiment of this disclosure, the *Corynebacterium glutamicum* used to prepare the fermentation broth containing valine can be a strain overexpressing L-valine biosynthesis, wherein the regulatory region of L-valine biosynthesis is inactivated by transformation of *Corynebacterium glutamicum* ATCC13032, but is not limited thereto. Alternatively, *Corynebacterium glutamicum* can be a strain transformed with the vector pDZDvalL, pDZDvalP, or pDZDvalS, using the chromosome of *Corynebacterium glutamicum* KCCM11201P, *Corynebacterium glutamicum* KCCM11336P, *Corynebacterium glutamicum* KCCM11337P, *Corynebacterium glutamicum* KCCM11338P, or *Corynebacterium glutamicum* KCCM11201P_DvalA as a template. In another instance, *Corynebacterium glutamicum* may be *Corynebacterium glutamicum* ATCC13032_DvalL, *Corynebacterium glutamicum* ATCC13032_DvalP, or *Corynebacterium glutamicum* ATCC13032_DvalS, but is not limited thereto, and includes, but is not limited to, microorganisms capable of producing fermentation broths containing valine (US 10,072,278 B2).

[0031] The preparation of the fermentation broth may further include culturing "microorganisms that produce valine". The cultivation of these microorganisms can be carried out in suitable culture media and under suitable culture conditions known in the art. This cultivation process can be readily adapted and used by those skilled in the art based on the selected strain. Specifically, the cultivation can be batch culture, continuous culture, or fed-batch culture, but is not limited to these.

[0032] As used herein, the term "culture medium" refers to a mixture containing nutrients required for the cultivation of microorganisms as its main components, and said culture medium provides nutrients, growth factors, etc., including water, which is essential for survival and development. Specifically, any culture medium and culture conditions can be used to cultivate the microorganisms of this disclosure without particular limitation, as long as the culture medium is used for the general cultivation of microorganisms. The microorganisms of this disclosure can be cultured under aerobic conditions in a common culture medium containing suitable carbon sources, nitrogen sources, phosphorus sources, inorganic compounds, amino acids, and / or vitamins, while controlling temperature, pH, etc.

[0033] As used herein, the term "fermentation broth containing valine" may be used interchangeably with "fermentation broth containing valine" or "valine fermentation broth".

[0034] Subsequently, the pH of the fermentation broth is adjusted. In this document, the pH of the fermentation broth may be adjusted to a pH greater than about 3.0 and less than 6.0. In some cases, the pH of the fermentation broth may be adjusted to about 3.5 to less than 6.0, about 4.0 to less than 6.0, about 4.5 to less than 6.0, about 5.0 to less than 6.0, about 5.5 to less than 6.0, greater than about 3.0 to about 5.5, about 3.5 to about 5.5, about 4.0 to about 5.5, about 4.5 to about 5.5, about 5.0 to about 5.5, about 3.0 to about 5.0, about 3.5 to about 5.0, about 4.0 to about 5.0, about 4.5 to about 5.0, greater than about 3.0 to about 4.5, about 3.5 to about 4.5, about 4.0 to about 4.5, greater than about 3.0 to about 4.0, about 3.5 to about 4.0, or greater than about 3.0 to about 3.5.

[0035] By adjusting the pH of the fermentation broth, the size and hardness of the valine crystals formed in the subsequent concentration step can be controlled. Specifically, when the pH of the fermentation broth is adjusted to the aforementioned range before concentration, large and hard valine crystals can be formed, thereby reducing the water content of the separated valine crystals. When the pH of the fermentation broth is outside the aforementioned range, the valine crystals precipitated after concentration are small, making it difficult to separate valine crystals from the mother liquor of the fermentation broth. Furthermore, since the valine crystals contain a relatively large amount of water, subsequent drying requires a significant amount of energy.

[0036] In the step of adjusting the pH of the fermentation broth, conventional acidic or alkaline substances can be used for pH adjustment. Simultaneously, substances added to the fermentation broth for pH adjustment can be selected, as long as they do not affect the physical properties or chemical structure of the valine contained in the fermentation broth. Furthermore, considering that valine products can be used in food or feed, edible acidic or alkaline substances can be added.

[0037] After the pH adjustment described above, if necessary, a process including heating the fermentation broth can be performed to completely dissolve any valine that may have precipitated in the broth. For example, the fermentation broth can be heated to a temperature of approximately 60°C to approximately 90°C.

[0038] After adjusting the pH of the fermentation broth, a step of adding polyvinyl alcohol (PVA) to the prepared fermentation broth is performed. For example, in the method of this disclosure, the addition of PVA is intended to adjust the crystallinity of the L-valine crystals to be formed, wherein the addition of PVA allows the formation of L-valine crystals exhibiting uniform and excellent crystal properties, unaffected by the concentration rate during concentration.

[0039] Specifically, PVA may be added in an amount of 0.1% to 5% by weight based on the total weight of the fermentation broth. In some cases, PVA may be added in amounts of 0.3% to 4% by weight, 0.5% to 3% by weight, 0.5% to 2.5% by weight, 1% to 3% by weight, 1% to 2.8% by weight, 1.5% to 3% by weight, 1.5% to 2.5% by weight, 1% to 2.5% by weight, or 1.8% to 2.3% by weight.

[0040] For example, L-valine crystals prepared by adding PVA to a culture medium and concentrating according to the method of this disclosure can be crystals in which the water content is reduced by 8% to 40% compared to crystals prepared according to a method that does not include adding PVA in a second step.

[0041] For example, the method of this disclosure may also include a concentration step after the step of adding PVA to the fermentation broth. The concentration step is intended to form L-valine crystals, and the concentration may be carried out at a solids content of 15% to 30%, but is not limited thereto.

[0042] The concentration step removes water from the fermentation broth, precipitating valine in crystalline form. Concentration can be carried out by various methods. In the judgment of someone skilled in the art, concentration can be performed in conventional concentrators (e.g., paddle dryers, slurry dryers, vacuum concentrators, forced circulation concentrators, thin-film concentrators, or rotary concentrators).

[0043] In the concentration step, concentration can be achieved by removing water from the fermentation broth at a rate of 30 g / L / hr to 150 g / L / hr (meaning 30 g to 150 g of water is removed per liter of fermentation broth per hour). In some cases, concentration can be achieved by removing water from the fermentation broth at rates of 90 g / L / hr to 150 g / L / hr, 120 g / L / hr to 150 g / L / hr, 60 g / L / hr to 120 g / L / hr, 90 g / L / hr to 120 g / L / hr, or 60 g / L / hr to 90 g / L / hr. In this disclosure, the concentration rate is a factor affecting the size of valine crystals. When concentrated at the above rates, relatively large valine crystals are formed, and the crystals can be easily separated from the mother liquor of the fermentation broth. Furthermore, the water content in the valine crystals is correspondingly reduced.

[0044] Subsequently, a step is performed to separate valine crystals containing water from the fermentation broth. The valine crystals may be valine crystals that have had their water content reduced through concentration.

[0045] In the separation step, valine crystals containing moisture are separated from the fermentation broth, and in particular, a solid-liquid separator, such as a vacuum membrane filter, a pressure membrane filter, or a centrifuge, can be used. However, the above method is merely illustrative, and the method of performing the separation step is not limited to the examples described above.

[0046] In the separation step, the mother liquor remaining after separating valine crystals can be reused for concentration. Therefore, valine that failed to form water-containing crystals or precipitated as crystals smaller than a certain size during the concentration step, and which remains in the mother liquor after the valine crystal separation step, can be recovered. Furthermore, after mother liquor recycling, if necessary, additional processes such as heating the fermentation broth can be performed to redissolve the valine precipitated as fine crystals.

[0047] According to the method for preparing valine crystals according to the embodiments of this disclosure, valine crystals with low moisture content and easy separation can be obtained by adjusting the pH of the fermentation broth containing valine and adding an appropriate amount of PVA to the fermentation broth.

[0048] The valine crystals produced according to embodiments of this disclosure can have a viscosity greater than 0 cP and less than 80 cP. In some cases, the viscosity of the valine crystals can be greater than 20 cP to less than 80 cP, greater than 40 cP to less than 80 cP, greater than 60 cP to less than 80 cP, greater than 0 cP to 70 cP, greater than 20 cP to 70 cP, greater than 40 cP to 70 cP, greater than 60 cP to 70 cP, greater than 0 cP to 50 cP, greater than 20 cP to 50 cP, greater than 40 cP to 50 cP, greater than 0 cP to 30 cP, greater than 20 cP to 30 cP, or greater than 0 cP to 10 cP. Valine crystals with viscosities within the above ranges can be easily separated from the mother liquor because they have low water content and suitable size.

[0049] The valine crystals prepared according to embodiments of this disclosure may contain 10% to 55% by weight of water. In some cases, the valine crystals may contain water in amounts of 20% to 55% by weight, 30% to 55% by weight, 40% to 55% by weight, 50% to 55% by weight, 10% to 50% by weight, 20% to 50% by weight, 30% to 50% by weight, 40% to 50% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 10% to 30% by weight, 20% to 30% by weight, or 10% to 20% by weight. Valine crystals with the above-mentioned amounts of water have a relatively low moisture content and can be used directly without a separate drying step, or can be used after minimal drying, thereby reducing the energy required for the process.

[0050] Furthermore, following the step of separating L-valine crystals, the method disclosed herein may additionally include a drying step, a sieving step, or both. Each step may be performed using methods known in the art, without limitation (e.g., US 2021-0094903 A1). Specific conditions may be appropriately modified to optimize the process, but are not limited thereto.

[0051] This disclosure provides L-valine crystals containing PVA and L-valine, and with a moisture content reduced by 5% to 40% compared to L-valine crystals prepared without PVA during concentration.

[0052] For example, L-valine crystals can exist in the form of spherical microparticles.

[0053] Specifically, the L-valine crystals of this disclosure can be prepared by the method described above. Furthermore, the L-valine crystals of this disclosure can be used in feed or as a feed additive, but their uses are not limited thereto.

[0054] A method for preparing valine particles includes: preparing a fermentation broth containing valine; adjusting the pH of the fermentation broth; adding polyvinyl alcohol (PVA) to the fermentation broth; separating valine crystals containing water from the fermentation broth; preparing mixed particles by mixing the valine crystals with seed crystals; and drying the valine mixed particles.

[0055] Specifically, the steps of preparing the fermentation broth, adjusting the pH of the fermentation broth, adding PVA to the fermentation broth, and separating the valine crystals can be performed in the same manner as the method for producing valine crystals described above. Therefore, to avoid repetition, the following description focuses on the steps of preparing mixed particles by mixing the valine crystals with seed crystals and drying the mixed particles.

[0056] In the step of preparing mixed particles, the valine crystals obtained in the step of separating valine crystals are mixed with seed crystals to prepare valine particles.

[0057] The seed crystals used in the step of preparing the mixed particles, also known as seed crystals or nuclei, can refer to a substance used as a catalyst for liquid crystallization or granulation. In this disclosure, the seed crystals can be valine crystals. When in contact with the fermentation broth, the seed crystals can form particles because the solid components present in the fermentation broth bind with the seed crystals, thereby causing aggregation.

[0058] The seed crystals used in the step of preparing the mixed particles can have an average particle size of 150 μm to 300 μm. Specifically, seed crystals with an average particle size of 150 μm to 250 μm, 200 μm to 300 μm, or 200 μm to 250 μm can be used, but are not limited thereto. According to this disclosure, the particle size of the seed crystals used will affect the production capacity of the particles, and therefore those skilled in the art can make appropriate selections considering factors such as the required moisture content.

[0059] In the step of preparing mixed granules, a mixing granulator can be used. The mixing granulator obtains granules by supplying valine crystals obtained in the preceding separation step while simultaneously feeding seed crystals into the mixing granulator at a constant rate via a feeder. The mixing granulator may include a feeder for supplying seed crystals into the interior and components for supplying valine crystals. Furthermore, the interior of the mixing granulator may be equipped with gas nozzles for generating airflow within the chamber, paddles for the seed crystals and valine crystals supplied within the mixing chamber, etc. Valine mixed granules can be formed by agglomerating the seed crystals with the valine crystals added into the chamber.

[0060] According to one embodiment of this disclosure, valine mixed particles with low moisture content and large crystal size can be formed by mixing seed crystals and valine crystals containing moisture. As described above, valine crystals with large crystal size can be precipitated according to this disclosure, and valine crystals with large crystal size can be easily separated from the fermentation broth; therefore, the moisture content in the valine crystals is relatively low. Furthermore, in the step of preparing the mixed particles, valine crystals with low moisture content are granulated together with seed crystals in a mixing granulator, and the resulting valine mixed particles also have low moisture content and large size.

[0061] Specifically, “particle” refers to macroscopic particles, which are large permanent aggregates formed by the aggregation of smaller particles (such as powder), and can be particles with an average particle diameter of 50 μm to 5 mm, 75 μm to 4 mm, or 100 μm to 3 mm.

[0062] The valine mixed particles obtained in the step of preparing the mixed particles may contain 10% to 50% by weight of moisture. In some cases, the valine mixed particles may contain moisture in amounts of 20% to 50% by weight, 30% to 50% by weight, 40% to 50% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 10% to 30% by weight, 20% to 30% by weight, 10% to 40% by weight, 20% to 40% by weight, 30% to 40% by weight, 10% to 30% by weight, 20% to 30% by weight, or 10% to 20% by weight. These moisture contents are low compared to the moisture content of valine particles obtained according to conventional techniques. Therefore, the energy required to dry the valine mixed particles obtained according to this disclosure can be reduced.

[0063] Subsequently, a drying step is performed on the mixed valine particles.

[0064] In the drying step, the valine mixture particles obtained above are dried. The drying method is not limited.

[0065] One aspect of this disclosure is to provide a valine product comprising valine mixed granules. As used herein, the term "valine product" refers to a product in which valine contained in a fermentation broth is formulated into various dosage forms. For example, a valine product may refer to a mixture containing valine in granular form. However, the dosage form of the valine product may be varied if desired, as long as it does not alter the spirit of this disclosure. Furthermore, as described above, further subsequent processes can be carried out to achieve various forms of valine products. The aforementioned valine products can be used as animal feed additives, etc., and their use is not limited.

[0066] According to this disclosure, valine mixed particles with large size and low moisture content can be prepared by concentrating a fermentation broth containing valine, separating valine crystals, and mixing the separated valine crystals with seed crystals. The low moisture content of the valine mixed particles has the advantage of reducing the required drying energy. In one example, according to this disclosure, the moisture content of the valine mixed particles can be further reduced by adjusting the pH of the fermentation broth or controlling the concentration rate.

[0067] The L-valine crystals or granules disclosed herein are suitable for use as feed additives in animal feed preparation. For example, as a feed additive, the L-valine crystals can be mixed alone with feed materials, as part of an animal feed premix, or as a precursor to an animal feed. Feed compositions containing L-valine crystals or granules can be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed compositions can be conveniently administered to animals as a top dressing by directly mixing them into animal feed or as a standalone oral formulation.

[0068] [Modes for Implementing the Invention]

[0069] The present disclosure is explained in more detail below through the following embodiments. However, these embodiments are set forth to illustrate the present disclosure, and the scope of the present disclosure is not limited thereto. Furthermore, each description and embodiment described herein can be applied to other descriptions and embodiments. That is, all combinations of the various elements described herein fall within the scope of the present disclosure. In addition, numerous papers and patent documents are cited throughout the specification. The contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which this disclosure pertains and the content of this disclosure.

[0070] Experimental Example 1: Preparation of Valine Fermentation Broth by Culturing Corynebacterium spp.

[0071] In this experimental example, to prepare particles containing L-valine, the L-valine-producing strain *Corynebacterium glutamicum* KCCM11338P (US 10072278B2) was cultured for 72 hours at approximately 30°C in a medium consisting of 5% glucose, 2% ammonium sulfate, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate, 2.0% corn steep liquor (CSL), and 200 μg / L biotin, thereby obtaining a fermentation broth with the following components. The fermentation broth contained the culture medium and the microorganism, and its moisture content and composition were analyzed. Table 1 shows the results of the component analysis of the valine fermentation broth.

[0072] Table 1

[0073]

[0074] Experimental Example 2: Comparing the water content in valine crystals based on the pH of the fermentation broth

[0075] The fermentation broth with an L-valine concentration of 80 g / L prepared in Example 1 was divided into 2 L portions, and sulfuric acid was added to each portion to prepare process solutions with different pH values ​​(7.5 to 3.0). To precipitate valine crystals in the prepared process solutions, the solution was concentrated using a vacuum concentrator at a vacuum of 120 torr and a rate of 60 g / L / hr until the valine concentration in the process solution reached 200 g / L. The concentrated crystal slurry containing valine crystals was then subjected to solid-liquid separation using a basket centrifuge to obtain L-valine crystals and mother liquor.

[0076] The moisture content of valine crystals was compared according to the pH of the fermentation broth using LOD analysis (Table 2).

[0077] Table 2

[0078]

[0079] LOD moisture measurements confirmed that the moisture content in valine crystals was lowest when the pH of the fermentation broth was greater than 3.0 and less than 6.0. Furthermore, viscosity analysis of the concentrated slurry confirmed that the viscosity was significantly lower when the pH of the fermentation broth was greater than 3.0 and less than 6.0. The lower viscosity of the concentrated slurry indicates that the valine crystals contained in the concentrated slurry are larger in size, and almost no small crystals are formed. In contrast, in the fermentation broths with high concentrated slurry viscosity at pH 7.0, pH 6.0, and pH 3.0, analysis showed that a large number of small valine crystals formed, leading to the high slurry viscosity.

[0080] Therefore, it was found that the pH of the fermentation broth affects the formation of valine crystals. Specifically, it was found that large-sized valine crystals form within a certain pH range, resulting in low viscosity of the concentrated slurry and low moisture content of the separated crystals.

[0081] Experiment Example 3: Comparing the moisture content in valine crystals based on the type of additives

[0082] To verify the effect of the presence and / or type of additives in the preparation of valine crystals from a valine-containing culture medium, a culture medium was prepared by adding 2% by weight (based on the weight of L-valine) of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), or monosodium glutamate (MSG) to the fermentation broth prepared in Example 1, wherein the pH was adjusted to 3.8 by adding sulfuric acid. These cultures were concentrated to a concentration of 200 g / L using a vacuum concentrator at a vacuum of 120 torr and a rate of 30 g / L / hr. Subsequently, solid-liquid separation was performed using a basket centrifuge at 1800 rpm for 10 minutes to obtain L-valine crystals from the concentrated culture medium obtained above, and the moisture content of the separated L-valine crystals was determined by LOD analysis and compared (Table 3).

[0083] Table 3

[0084]

[0085] As shown in Table 3, compared with the control group without additives, the use of other additives did not cause a significant change in the water content of the crystals, while the crystals obtained using PVA showed a reduction in water content of 30% or more. This indicates that adding PVA to the culture medium before concentration can effectively reduce the water content in the final valine crystals.

[0086] Experiment Example 4: Comparison of water content in valine crystals based on the amount of PVA used

[0087] Using PVA, which showed the most outstanding effect in Experimental Example 3, the effect was examined by varying the input amount. Specifically, culture media prepared with different input amounts of 0.5 wt%, 1.5 wt%, 2.0 wt%, and 2.5 wt% (based on the weight of L-valine) were concentrated and separated to obtain crystals as in Experimental Example 3, and the moisture content of the separated L-valine crystals was determined by LOD analysis (Table 4).

[0088] Table 4

[0089]

[0090] Compared to the control sample with no added PVA (0% by weight), all samples within the PVA input range showed a reduction in moisture content in the crystals, with a reduction rate observed ranging from approximately 9.1% to 34.7%.

[0091] Furthermore, the morphology of crystals produced in samples treated with 2% PVA and in controls without PVA was observed using SEM (×30, JEOL, JCM-6000PLUS). The results showed that while the control group without PVA produced small, plate-like crystals, the group treated with 2% PVA formed larger, more spherical particles. Figure 1 ).

[0092] Experimental Example 5: Preparation of valine particles from isolated crystals

[0093] Valine crystals were produced in the same manner as in Experimental Example 4, and the separated valine crystals were mixed with pre-dried valine seed crystals (with a valine content of 75%) to prepare crystals with a moisture content of 15% to 47%, and then a valine mixed granule crystal was produced using a mixing granulator.

[0094] It has been confirmed that, using a mixing granulator, mixed granules can be produced from all crystals with a moisture content ranging from 15% to 47%.

[0095] Table 5

[0096]

[0097] As described above, those skilled in the art will understand that this disclosure may be implemented in other specific forms without departing from the technical spirit or essential features of this disclosure. In this regard, the embodiments disclosed herein are for illustrative purposes only and should not be construed as limiting the scope of this disclosure. The scope of this disclosure should be understood to include the meaning and scope of the appended claims rather than the specific embodiments, and all changes or modifications derived from equivalent concepts fall within the scope of this disclosure.

Claims

1. A method of preparing valine crystals, comprising: a step of preparing a fermentation broth comprising valine; a step of adjusting pH of the fermentation broth; a step of adding polyvinyl alcohol (PVA) to the fermentation broth; and a step of isolating valine crystals comprising moisture from the fermentation broth.

2. The method of claim 1, wherein in the step of adjusting pH of the fermentation broth, the pH of the fermentation broth is adjusted to a pH greater than 3 and less than 6.

3. The method of claim 1, wherein PVA is added in an amount of 0.1 wt% to 5 wt% based on the total weight of the fermentation broth.

4. The method of claim 1, wherein the method further comprises a step of concentrating the fermentation broth after the step of adding PVA to the fermentation broth.

5. The method of claim 4, wherein the concentration is performed by removing moisture from the fermentation broth at a rate of 30 g / L / hr to 150 g / L / hr.

6. The method of claim 1, wherein the valine crystals have a viscosity greater than 0 cP and less than 80 cP.

7. The method of claim 6, wherein the valine crystals comprise 10 wt% to 55 wt% moisture.

8. The method of claim 1, wherein the valine crystals have a spherical microparticle form.

9. A method of preparing valine particles, comprising: a step of preparing a fermentation broth comprising valine; a step of adjusting pH of the fermentation broth; a step of adding polyvinyl alcohol (PVA) to the fermentation broth; a step of isolating valine crystals comprising moisture from the fermentation broth; a step of preparing mixed particles by mixing the valine crystals with seed crystals; and a step of drying the mixed valine particles.

10. The method of claim 9, wherein the method further comprises a step of concentrating the fermentation broth after the step of adding PVA to the fermentation broth.

Images