Method for preparing purified polyhydroxyalkanoates

Abstract

The process for preparing purified polyhydroxyalkanoates of the present invention can reduce the b* value of the final product beyond that of conventional methods by using two oxidizing agents in a decolorization step in sequence. Furthermore, the present invention uses sodium chlorite (NaClO) as the primary oxidizing agent, which significantly reduces coloration and causes almost no decrease in molecular weight.

Description

[Technical Field]

[0001] The present disclosure relates to a process for preparing purified polyhydroxyalkanoates, and more particularly to a process for preparing purified polyhydroxyalkanoates with excellent color by a decolorization process using an oxidizing agent. [Background technology]

[0002] Polyhydroxyalkanoates (PHAs) are biodegradable polymers composed of several hydroxylcarboxylic acids that are produced by many microorganisms and used as intracellular storage materials. Polyhydroxyalkanoates have similar physical properties to conventional petroleum-derived synthetic polymers such as polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), and are completely biodegradable and highly biocompatible.

[0003] Specifically, polyhydroxyalkanoates are natural thermoplastic polyester polymers that accumulate within microbial cells. As biodegradable materials, they can be composted and ultimately decomposed into carbon dioxide, water, and organic waste without producing harmful waste. In particular, polyhydroxyalkanoates are biodegradable in both soil and marine environments, making them environmentally friendly. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 2001-0009719 Summary of the Invention [Problem to be solved by the invention]

[0005] To obtain polyhydroxyalkanoates (PHAs), fermentation broth from microorganisms is subjected to cell disruption and separation to prepare a crude product. This is followed by purification processes such as decolorization to remove impurities and reduce the color of the final PHA. However, for flexible and sticky PHAs, impurities attached to the PHA particles are not removed, resulting in a small particle size during the purification process, which makes subsequent repeated solid-liquid separation and washing difficult, resulting in a high impurity content in the final PHA. As mentioned above, conventional PHA purification methods are unable to effectively reduce the impurity content in some PHAs, resulting in the b* value of the purified final PHA increasing to 15 or higher, making it difficult to commercialize. This problem is more severe for some PHAs with low or no crystallinity.

[0006] As a result of the inventors' investigations, they found that the color of the final product can be improved by sequentially performing decolorization using two types of oxidizing agents during the purification process of polyhydroxyalkanoate, thereby solving the problems of conventional purification methods.

[0007] Therefore, an object of the present disclosure is to provide a process for preparing a purified polyhydroxyalkanoate having excellent color, which process includes a decolorization step using an oxidizing agent. [Means for solving the problem]

[0008] According to one aspect of the present disclosure, there is provided a process for preparing a purified polyhydroxyalkanoate, the process comprising: preparing a crude product comprising polyhydroxyalkanoate (PHA); subjecting the crude product to a first bleaching treatment to obtain a first product; and subjecting the first product to a second bleaching treatment to obtain a second product, wherein the first bleaching treatment is performed with a first oxidizing agent comprising sodium chlorite (NaClO).

[0009] In one embodiment, the second bleaching may be performed with a second oxidizing agent comprising hydrogen peroxide (H2O2).

[0010] In another embodiment, the first bleaching may be carried out at a pH of 2-7, and the second bleaching may be carried out at a pH of 8-13.

[0011] In another embodiment, in the first bleaching, sodium chlorite may be used in an amount of 0.01 to 1% by weight, based on the total weight of the crude product.

[0012] In another embodiment, in the second bleaching, hydrogen peroxide may be used in an amount of 0.01 to 1% by weight, based on the total weight of the first product.

[0013] In another embodiment, the lipid content of the first product may be less than 1% by weight based on the weight of the total solids.

[0014] In another embodiment, the process may further comprise deproteinizing the second product with a protease.

[0015] In another embodiment, in the deproteinization, the protease may be used in an amount of 0.001 to 0.1% by weight, based on the total weight of the second product.

[0016] In another embodiment, deproteinization may be carried out at a pH of 7-11.

[0017] In another embodiment, a crude product comprising polyhydroxyalkanoate can be prepared by a method comprising performing solid-liquid separation of a fermentation broth comprising polyhydroxyalkanoate (PHA) to obtain biomass, mixing an additive comprising a surfactant with the biomass to obtain a suspension, disrupting the biomass in the suspension to obtain a slurry, and performing solid-liquid separation of the slurry.

[0018] According to another aspect of the present disclosure, there is provided a polyhydroxyalkanoate prepared by the above process, which has a b* value of less than 15 on the CIELAB color coordinates.

[0019] In one embodiment, the crystallization temperature (Tc) of the polyhydroxyalkanoate may not be measured or may be measured to be between 60°C and 120°C.

[0020] In another embodiment, the repeating units constituting the polyhydroxyalkanoate may include at least one selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxybutyrate (4-HB), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH). [Effects of the Invention]

[0021] According to the process of the present disclosure, the decolorization step using two oxidizing agents in sequence reduces the b* value of the final product to less than 15, thereby solving the problem of increased color that can occur in conventional aqueous purification processes for polyhydroxyalkanoates (PHAs). Furthermore, as described above, the use of two oxidizing agents in sequence to solve the conventional color problem can reduce the amount of enzyme used, thereby reducing production costs and being more economical.

[0022] In particular, sodium chlorite (NaClO2), used as the first oxidizing agent in the present disclosure, produces a remarkable effect of improving coloration, while, unlike other chlorine-based oxidizing agents (such as NaClO), it hardly causes changes in polymer properties due to molecular weight reduction. Therefore, the purified polyhydroxyalkanoate obtained by the process of the present disclosure has improved coloration and purity while also maintaining excellent molecular weight compared to conventional polyhydroxyalkanoates.

[0023] In particular, the effects of the present disclosure can be more pronounced in the purification process of amorphous PHA. When the process of the present disclosure is applied to the purification process of other crystalline and semi-crystalline PHAs, the effects of improving color and purity, and maintaining molecular weight can be obtained. [Brief explanation of the drawings]

[0024] [Figure 1] 1 illustrates a process for preparing purified polyhydroxyalkanoates according to one embodiment of the present disclosure. [Figure 2] An example of a process for preparing a crude polyhydroxyalkanoate product is shown below. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present disclosure will now be described in more detail with reference to various embodiments. In this specification, the terms of each component are used to distinguish them from one another and are not intended to limit the scope of the present disclosure. Also, in this specification, unless the context clearly indicates otherwise, the singular forms "a," "the," and "the" are to be construed as including the plural forms.

[0026] As used herein, the term "comprises" is intended to identify certain features, regions, steps, processes, elements, and / or components, and does not exclude the presence or addition of any other features, regions, steps, processes, elements, and / or components, unless specifically stated to the contrary.

[0027] In this specification, terms such as "first," "second," etc. are used to describe various components, but these components should not be limited to these terms. These terms are used to distinguish one element from another.

[0028] As used herein, molecular weight or weight average molecular weight is not usually stated with units, but may be understood to have units of g / mol or Da.

[0029] Preparation process of purified polyhydroxyalkanoates In one embodiment, a process for preparing a purified polyhydroxyalkanoate includes preparing a crude product comprising polyhydroxyalkanoate (PHA), subjecting the crude product to a first bleaching treatment to obtain a first product, and subjecting the first product to a second bleaching treatment to obtain a second product, wherein the first bleaching treatment is performed with a first oxidizing agent comprising sodium chlorite (NaClO).

[0030] The process may further include deproteinizing the second product with a protease.

[0031] FIG. 1 illustrates a process for preparing purified polyhydroxyalkanoates according to one embodiment of the present disclosure.

[0032] Referring to FIG. 1, according to one embodiment, a crude polyhydroxyalkanoate product is prepared (S100). An acid is added to the crude product to adjust the pH to about 3-5, and sodium chlorite (NaClO2) is added to perform a first decolorization process at about 50-70°C (S210). An alkaline component is added to the product to adjust the pH to about 8-10, and hydrogen peroxide (H2O2) is added to perform a second decolorization process at about 50-70°C (S220). An alkaline component is added to the decolorized product to adjust the pH to about 8-9.5, and deproteinization is performed using a protease (S310). After deproteinization, the PHA solid is recovered (410). The product is washed with water and dehydrated (S430), yielding a purified final polyhydroxyalkanoate (S500).

[0033] On the other hand, the crude polyhydroxyalkanoate used as a starting material in the present disclosure may be prepared from the fermentation broth by processes such as filtration, crushing, and purification.

[0034] FIG. 2 shows an example of a process for preparing a crude polyhydroxyalkanoate.

[0035] Referring to Figure 2, according to one embodiment, a fermentation broth of polyhydroxyalkanoate is prepared (S10). Cells are separated by centrifugation or the like (S20). A surfactant or the like is added (S30). The cells are disrupted using a high-pressure homogenizer (S40). Solid-liquid separation is performed by centrifugation or the like (S50). A crude product of polyhydroxyalkanoate is obtained (S100).

[0036] Each step of the preparation process according to the present disclosure is described in detail below.

[0037] Polyhydroxyalkanoates Polyhydroxyalkanoates (PHAs) can be classified according to their molecular structure as crystalline, semi-crystalline, or amorphous polyhydroxyalkanoates.

[0038] According to one embodiment, the polyhydroxyalkanoate may include amorphous polyhydroxyalkanoate. Due to its inherent flexibility and stickiness, amorphous polyhydroxyalkanoate is difficult to repeatedly separate solids and liquids and wash during the aqueous purification process, while the content of impurities in the purified final PHA remains high, making its commercialization difficult. However, according to the present disclosure, during the purification process of amorphous PHA, decolorization using two oxidizing agents is sequentially performed, which can significantly improve the color of the final product.

[0039] According to another embodiment, the polyhydroxyalkanoate may include a semi-crystalline polyhydroxyalkanoate. According to another embodiment, the polyhydroxyalkanoate may include a crystalline polyhydroxyalkanoate. According to another embodiment, the polyhydroxyalkanoate may include two or more of an amorphous polyhydroxyalkanoate, a semi-crystalline polyhydroxyalkanoate, and a crystalline polyhydroxyalkanoate.

[0040] The polyhydroxyalkanoate may comprise at least one repeat unit selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxybutyrate (4-HB), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH).

[0041] Furthermore, polyhydroxyalkanoates may include isomers. For example, polyhydroxyalkanoates may include structural isomers, enantiomers, or geometric isomers. Specifically, polyhydroxyalkanoates may include structural isomers.

[0042] The polyhydroxyalkanoates may be homopolymers or copolymers. According to one embodiment, the polyhydroxyalkanoates may include copolymers, specifically copolymers containing two or more different repeat units randomly distributed in the polymer chain.

[0043] As an example, the polyhydroxyalkanoate may contain 4-hydroxybutyrate (4-HB) repeat units. For example, the content of hydroxybutyrate (4-HB) repeat units in the polyhydroxyalkanoate may be 0.1 wt% or more, 1 wt% or more, 5 wt% or more, 10 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, or 60 wt% or more, and 100 wt% or less, 99 wt% or less, 90 wt% or less, 80 wt% or less, 70 wt% or less, or 60 wt% or less, based on the total weight of the polyhydroxyalkanoate. Specifically, the polyhydroxyalkanoate may contain 4-hydroxybutyrate (4-HB) repeat units in an amount of 25 wt% or more.

[0044] Specifically, the polyhydroxyalkanoate may be a copolymer containing 4-HB repeating units. Examples of repeating units that the polyhydroxyalkanoate may contain in addition to 4-HB include at least one selected from the group consisting of lactic acid, glycolic acid, 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH).

[0045] More specifically, the polyhydroxyalkanoate may contain, in addition to 4-HB repeating units, one or more repeating units selected from the group consisting of 3-HB, 3-HP, 3-HH, 3-HV, 4-HV, 5-HV, and 6-HH. As an example, the polyhydroxyalkanoate may include a copolymer containing 4-HB repeating units and 3-HB repeating units. Specifically, it may include poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P3HB-co-4HB).

[0046] The PHA may include one or more PHAs. For example, it may include a first PHA, a second PHA, or a mixture thereof. For example, the first PHA may be an amorphous PHA, and the second PHA may be a semi-crystalline PHA. The first PHA may include 4-HB repeat units in an amount of, for example, 15% to 60% by weight, 15% to 55% by weight, 20% to 55% by weight, 25% to 55% by weight, 30% to 55% by weight, 35% to 55% by weight, 20% to 50% by weight, 25% to 50% by weight, 30% to 50% by weight, 35% to 50% by weight, or 20% to 40% by weight. The second PHA may include 4-HB repeat units in an amount of, for example, 0.1% to 30% by weight, 0.5% to 30% by weight, 1% to 30% by weight, 3% to 30% by weight, 1% to 28% by weight, 1% to 25% by weight, 1% to 24% by weight, 1% to 20% by weight, 1% to 15% by weight, 2% to 25% by weight, 3% to 25% by weight, 3% to 24% by weight, 5% to 24% by weight, 5% to 20% by weight, greater than 5% to less than 20% by weight, 7% to 20% by weight, 10% to 20% by weight, 15% to 25% by weight, or 15% to 24% by weight. The PHA may also be a mixture of a first PHA and a second PHA in a weight ratio of 20:80 to 80:20 or 30:70 to 70:30.

[0047] Crude polyhydroxyalkanoate In this step, a crude product of polyhydroxyalkanoate is prepared.

[0048] Generally, polyhydroxyalkanoates can be produced by a fermentation process. A crude product of polyhydroxyalkanoates can also be obtained by fermentation.

[0049] The method for producing polyhydroxyalkanoate (PHA) by fermentation may be carried out by a known method, for example, by using a wild-type or genetically modified microorganism cultured on a specific substrate to produce polyhydroxyalkanoate by fermentation, and is not particularly limited.

[0050] During fermentation, the polyhydroxyalkanoate thus produced accumulates within the cells of the microorganisms, and therefore a process for separating and purifying it is absolutely necessary.

[0051] For example, biomass containing polyhydroxyalkanoates is separated from fermentation broth, a surfactant is added, and the microbial cells are disrupted to isolate only the polyhydroxyalkanoates.

[0052] As an example, a crude product containing polyhydroxyalkanoate can be prepared by a method including performing solid-liquid separation of a fermentation broth containing polyhydroxyalkanoate (PHA) to obtain biomass, mixing an additive including a surfactant with the biomass to obtain a suspension, crushing the biomass in the suspension to obtain a slurry, and performing solid-liquid separation of the slurry.

[0053] First, the fermentation broth formed by the microorganism is subjected to solid-liquid separation by mechanical separation such as centrifugation and membrane separation, or other known methods, to separate the biomass containing cells in which polyhydroxyalkanoates (PHAs) have accumulated.

[0054] The separated biomass may be mixed with a solvent to adjust the solids concentration. The biomass may be mixed with a surfactant or the like. For example, an anionic surfactant or a nonionic surfactant may be used as the surfactant. Specifically, sodium laureth sulfate, sodium lauryl sulfate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate may be used. As a result, a suspension containing biomass, a surfactant, and the like may be obtained.

[0055] The biomass in the suspension is then pulverized. The pulverization may be performed by mechanical pulverization (physical pulverization) or chemical pulverization (non-mechanical pulverization). For example, the pulverization may be performed by ultrasonic pulverization, high-pressure pulverization, or mill pulverization. Ultrasonic pulverization may be performed, for example, at an energy level of 20 Hz or higher for 10 to 60 minutes. High-pressure pulverization may be performed, for example, at a pressure of 10 bar or higher for 1 to 60 minutes. Mill pulverization may be performed using a colloid mill, bead mill, or ball mill for 1 to 60 minutes.

[0056] As a result of the grinding, a slurry containing fine particles of polyhydroxyalkanoate and cell-derived impurities such as cell walls may be obtained, which is then subjected to solid-liquid separation using mechanical separation methods such as centrifugation and membrane separation, or other known methods, to obtain a crude polyhydroxyalkanoate product.

[0057] The crude polyhydroxyalkanoate product contains trace amounts of cell-derived impurities (proteins, lipids, etc.), and therefore requires further purification.

[0058] For example, the protein content in the crude product may be 3.5 wt % or less, 3 wt % or less, 2.7 wt % or less, or 2.5 wt % or less, or 0.1 wt % or more, 1 wt % or more, or 1.5 wt % or more, based on the total weight of solids in the crude product.

[0059] Furthermore, the lipid content in the crude product may be 1.5 wt % or less, 1.3 wt % or less, 1 wt % or less, or 0.7 wt % or less, or 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more, based on the total solid weight of the crude product.

[0060] Furthermore, the solid content in the crude product may be 1% by weight or more, 3% by weight or more, 5% by weight or more, 7% by weight or more, or 10% by weight or more, and may be 80% by weight or less, 50% by weight or less, 30% by weight or less, 20% by weight or less, or 15% by weight or less, based on the total weight of the crude product.

[0061] First bleaching In this step, a crude product containing polyhydroxyalkanoate is subjected to a first decolorization to obtain a first product.

[0062] The first decolorization is performed using a first oxidizing agent containing sodium chlorite (NaClO2). When decolorization is performed using sodium chlorite as the oxidizing agent, the color improvement effect is significant, and unlike other chlorine-based oxidizing agents (e.g., NaClO), there is almost no decrease in molecular weight. This is thought to be because cell-derived impurities (proteins, lipids, etc.) in the crude product are decomposed by reaction with sodium chlorite, while PHA is difficult to decompose.

[0063] The amount of sodium chlorite used in the first bleaching step may be 0.01 wt% or more, 0.02 wt% or more, 0.03 wt% or more, 0.04 wt% or more, 0.05 wt% or more, 0.06 wt% or more, 0.1 wt% or more, 0.2 wt% or more, 0.3 wt% or more, 0.4 wt% or more, or 0.5 wt% or more, based on the total weight of the crude product (solution), and may be 1.3 wt% or less, 1.2 wt% or less, 1.1 wt% or less, 1 wt% or less, 0.9 wt% or less, 0.8 wt% or less, 0.7 wt% or less, or 0.6 wt% or less. As a specific example, in the first bleaching step, sodium chlorite may be used in an amount of 0.01 to 1 wt% or 0.05 to 1 wt% based on the total weight of the crude product. When the amount of sodium chlorite used is within the above preferred range, sufficient bleaching effect can be achieved while minimizing molecular weight reduction.

[0064] In the first decolorization, an additional oxidizing agent may be used in combination with sodium chlorite (NaClO) as the first oxidizing agent, for example, chlorine dioxide (ClO) in an amount of 0.05 to 1 wt % based on the total weight of the crude product.

[0065] The first bleaching may be performed under acidic pH conditions. For example, the pH range for the first bleaching may be 7 or less, 6.5 or less, 6 or less, 5.5 or less, 5 or less, 4.5 or less, or 4 or less, or may be 1 or more, 1.5 or more, 2 or more, 2.5 or more, 3 or more, or 3.5 or more. As a specific example, the first bleaching may be performed at a pH of 1 to 7 or a pH of 2 to 7. As a more specific example, the first bleaching may be performed at a pH of 3 to 6 or a pH of 3 to 5.

[0066] An acid component may be used to adjust the acidic pH. Examples of acid components used to adjust the pH in the first decolorization step include, but are not limited to, phosphoric acid, sulfuric acid, hydrochloric acid, and nitric acid. By adding an appropriate amount of such an acid component, the crude product can be adjusted to a desired acidic pH.

[0067] The temperature in the first bleaching may be 40° C. or higher, 45° C. or higher, 50° C. or higher, 55° C. or higher, or 60° C. or higher, and may be 90° C. or lower, 80° C. or lower, 75° C. or lower, 70° C. or lower, or 65° C. or lower. As a specific example, the first bleaching may be performed at a temperature of 40 to 90° C. or 50 to 70° C.

[0068] The time required for the first bleaching may be 10 minutes or more, 30 minutes or more, or 50 minutes or more, and may be 3 hours or less, 2 hours or less, or 1 hour 30 minutes or less.Specific examples include 30 minutes to 1 hour 30 minutes.

[0069] The content of impurities in the first product obtained by the first decolorization may be reduced compared to the previous step.

[0070] For example, the protein content in the first product may be 3.5% by weight or less, 3% by weight or less, or 2.7% by weight or less, and may be 1% by weight or more, 1.5% by weight or more, or 0.1% by weight or more, based on the total weight of solids in the first product.

[0071] The lipid content in the first product may be 1.5 wt% or less, 1.3 wt% or less, 1 wt% or less, or 0.7 wt% or less, or 0.1 wt% or more, 0.2 wt% or more, or 0.3 wt% or more, based on the total solid weight of the first product. Specifically, the lipid content of the first product may be less than 1 wt% based on the total solid weight, which is significantly reduced compared to the previous process.

[0072] Furthermore, the solid content in the first product may be 1% by weight or more, 3% by weight or more, 5% by weight or more, 7% by weight or more, or 10% by weight or more, and may be 80% by weight or less, 50% by weight or less, 30% by weight or less, 20% by weight or less, or 15% by weight or less, relative to the total weight of the first product.

[0073] Second bleaching In this step, the first product obtained by the first bleaching is subjected to a second bleaching to obtain a second product.

[0074] The second decolorization step may be performed using a second oxidizing agent containing hydrogen peroxide (HO). Using sodium chlorite as the oxidizing agent in the first decolorization step, compared to using hydrogen peroxide as the oxidizing agent in the second decolorization step, can significantly reduce the content of impurities (proteins and lipids) after the second decolorization step. This can improve the efficiency of impurity dissolution during the recovery of the PHA solids, thereby reducing the re-recovery rate of the impurities. Therefore, the amount of enzyme used for protein degradation in the subsequent deproteinization step can be reduced, thereby reducing production costs.

[0075] The amount of hydrogen peroxide used in the second bleaching step may be 0.01 wt% or more, 0.02 wt% or more, 0.03 wt% or more, 0.04 wt% or more, 0.05 wt% or more, 0.06 wt% or more, 0.1 wt% or more, 0.2 wt% or more, 0.3 wt% or more, 0.4 wt% or more, or 0.5 wt% or more, based on the total weight of the first product (solution), and may be 1.3 wt% or less, 1.2 wt% or less, 1.1 wt% or less, 1 wt% or less, 0.9 wt% or less, 0.8 wt% or less, 0.7 wt% or less, or 0.6 wt% or less. As a specific example, hydrogen peroxide may be used in the second bleaching step in an amount of 0.01 to 1 wt% or 0.05 to 1 wt% based on the total weight of the first product. The amount of hydrogen peroxide used in the present disclosure is less than that used in conventional methods, and is possible by sequential use with sodium chlorite (first oxidizing agent).

[0076] The second bleaching may be performed under basic pH conditions. For example, the pH range for the second bleaching may be 7 or more, 7.5 or more, 8 or more, or 8.5 or more, and may be 13 or less, 12 or less, 11 or less, 10.5 or less, 10 or less, 9.5 or less, or 9 or less. As a specific example, the second bleaching may be performed at a pH of 7 to 13 or a pH of 8 to 13. As a more specific example, the second bleaching may be performed at a pH of 8 to 11 or a pH of 8 to 10.

[0077] An alkaline component may be used to adjust the pH to a basic level. Examples of alkaline components used to adjust the pH in the second bleaching step include, but are not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, and sodium carbonate. By adding an appropriate amount of such an alkaline component, the first product can be adjusted to a desired basic pH.

[0078] The temperature for the second bleaching may be 40° C. or higher, 45° C. or higher, 50° C. or higher, 55° C. or higher, or 60° C. or higher, and may be 90° C. or lower, 80° C. or lower, 75° C. or lower, 70° C. or lower, or 65° C. or lower. As a specific example, the second bleaching may be performed at a temperature of 40° C. to 90° C. or 50° C. to 70° C.

[0079] The time required for the second bleaching may be 10 minutes or more, 30 minutes or more, or 50 minutes or more, and may be 3 hours or less, 2 hours or less, or 1 hour 30 minutes or less.Specific examples include 30 minutes to 1 hour 30 minutes.

[0080] The content of impurities in the second product obtained by the second decolorization may be reduced compared to the previous step.

[0081] For example, the protein content in the second product may be 2.5 wt % or less, 2 wt % or less, or 1.5 wt % or less, and may be 0.1 wt % or more, 0.5 wt % or more, or 1 wt % or more, based on the total weight of solids in the second product.

[0082] Furthermore, the lipid content in the second product may be 1 wt % or less, 0.8 wt % or less, 0.6 wt % or less, or 0.4 wt % or less, or 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more, based on the total solid weight in the second product.

[0083] Furthermore, the solid content in the second product may be 1 wt % or more, 3 wt % or more, 5 wt % or more, 7 wt % or more, or 10 wt % or more, relative to the total weight of the second product, and may be 80 wt % or less, 50 wt % or less, 30 wt % or less, 20 wt % or less, or 15 wt % or less.

[0084] Protein removal A step of deproteinizing the second product using a protease may then be carried out.

[0085] Examples of proteases include alcalase, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase, and one or more of these may be used.

[0086] The amount of protease used in deproteinization may be 0.001 wt% or more, 0.002 wt% or more, 0.003 wt% or more, 0.004 wt% or more, 0.005 wt% or more, 0.006 wt% or more, 0.01 wt% or more, 0.015 wt% or more, 0.02 wt% or more, or 0.025 wt% or more, relative to the total weight of the second product (solution), and may be 0.2 wt% or less, 0.15 wt% or less, 0.1 wt% or less, 0.05 wt% or less, 0.045 wt% or less, or 0.04 wt% or less. For example, in deproteinization, the protease may be used in an amount of 0.001 to 0.1 wt% or 0.005 to 0.1 wt% relative to the total weight of the second product.

[0087] In addition to proteases, lipolytic enzymes, cell wall degrading enzymes, DNA degrading enzymes, etc. may be added to the second product. Lipolytic enzymes include, for example, lipase, phospholipase, cholinesterase, and phosphatase. Cell wall degrading enzymes include, for example, lysozyme, amylase, cellulase, maltase, saccharase, α-glycosidase, β-glycosidase, and N-glycosidase. DNA degrading enzymes include, for example, ribonuclease.

[0088] The deproteinization may be carried out under basic pH conditions. For example, the pH range for deproteinization may be 7 or more, 7.5 or more, 8 or more, or 8.5 or more, and may be 12 or less, 11 or less, 10.5 or less, 10 or less, 9.5 or less, or 9 or less.

[0089] An alkaline component may be used to adjust the pH to a basic level. Examples of alkaline components used to adjust the pH in deproteinization include, but are not limited to, sodium hydroxide, potassium hydroxide, lithium hydroxide, and sodium carbonate. By adding an appropriate amount of such alkaline component, the second product can be adjusted to a desired basic pH.

[0090] Furthermore, the temperature during deproteinization may be 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher, and may be 90°C or lower, 80°C or lower, 75°C or lower, 70°C or lower, or 65°C or lower.

[0091] As a specific example, deproteinization may be carried out at a pH of 7 to 11, more specifically at a pH of 8 to 10, and at a temperature of 50 to 70°C.

[0092] Deproteinization can significantly reduce the protein content, for example, the protein content may be 1 wt % or less, 0.8 wt % or less, 0.6 wt % or less, or 0.4 wt % or less, or 0.1 wt % or more, 0.15 wt % or more, 0.2 wt % or more, or 0.25 wt % or more, based on the total weight of solids in the product after deproteinization.

[0093] Furthermore, the lipid content may be 1.5 wt % or less, 1.3 wt % or less, 1 wt % or less, or 0.7 wt % or less, or 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more, based on the total solid weight in the product upon deproteinization.

[0094] Purified Polyhydroxyalkanoate According to the present disclosure, there is provided a purified polyhydroxyalkanoate obtained by the process described above.

[0095] According to one embodiment, the purified polyhydroxyalkanoate may be in a solid form, such as a solid resin, or according to another embodiment, the purified polyhydroxyalkanoate may be in a liquid form, such as a liquid resin or an aqueous suspension.

[0096] The polyhydroxyalkanoates prepared according to the present disclosure may have improved color. According to one embodiment, the polyhydroxyalkanoates may have a b* value of less than 15 on the CIELAB color coordinate system. For example, the b* value of the purified polyhydroxyalkanoates on the CIELAB color coordinate system may be 14 or less, 13.5 or less, or 13 or less, and may be 0 or more, 1 or more, 5 or more, or 10 or more.

[0097] The glass transition temperature (Tg) of the polyhydroxyalkanoate may be, for example, -45°C to -10°C, -35°C to -10°C, -35°C to -15°C, -35°C to -20°C, or -30°C to -20°C.

[0098] Furthermore, the melting point (Tm) of the polyhydroxyalkanoate does not have to be measurable, and may be, for example, 100°C to 170°C, 100°C to 160°C, 110°C to 160°C, or 120°C to 150°C.

[0099] The polyhydroxyalkanoate of the present disclosure may include amorphous polyhydroxyalkanoates, semi-crystalline polyhydroxyalkanoates, and mixtures thereof. For example, the crystallization temperature (Tc) of the polyhydroxyalkanoate may not be measurable, and the crystallization temperature (Tc) may be measured to be 60°C to 120°C, specifically 60°C to 110°C, 70°C to 120°C, or 75°C to 115°C.

[0100] The repeating units constituting the polyhydroxyalkanoate may include at least one selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxybutyrate (4-HB), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH).

[0101] According to one embodiment, the polyhydroxyalkanoate may contain 4-hydroxybutyrate (4-HB) repeat units. Specifically, it may include copolymers containing 4-HB repeat units. For example, the content of hydroxybutyrate (4-HB) repeat units in the polyhydroxyalkanoate may be 0.1 wt% or more, 1 wt% or more, 5 wt% or more, 10 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, or 60 wt% or more, and 100 wt% or less, 99 wt% or less, 90 wt% or less, 80 wt% or less, 70 wt% or less, or 60 wt% or less, based on the total weight of the polyhydroxyalkanoate. As a specific example, the polyhydroxyalkanoate may contain 4-hydroxybutyrate (4-HB) repeat units in an amount of 25 wt% or more.

[0102] The weight average molecular weight (Mw) of the polyhydroxyalkanoate may be, for example, 50,000 or more, 100,000 or more, 200,000 or more, 300,000 or more, 400,000 or more, or 500,000 or more, and may be 1,200,000 or less, 900,000 or less, 800,000 or less, 700,000 or less, or 600,000 or less.

[0103] The purity of the polyhydroxyalkanoate may be 95% or more, 97% or more, 98% or more, 98.5% or more, 98.55% or more, 98.6% or more, or 99.0% or more.

[0104] The present disclosure will now be described in more detail by the following examples, which, however, are provided for illustrative purposes only and are not intended to limit the present disclosure.

[0105] Preparation Example 1: Preparation of PHA fermentation broth A PHA fermentation broth was prepared using a genetically engineered E. coli strain. First, the PHA-producing strain was cultured as a seed culture, and after securing a certain amount, the main fermentation was carried out. For the fermentation of the PHA-producing strain, glucose or raw sugar was used as the carbon source, and various inorganic components were added as nutrients. During fermentation, the pH was adjusted using sodium hydroxide solution or ammonia gas. The fermentation process using the PHA-producing strain was carried out at 30-40°C and pH 6-9.

[0106] Example 1: Preparation of purified polyhydroxyalkanoates Step (1): Solid-liquid separation and cell disruption The PHA fermentation broth was subjected to solid-liquid separation using a mechanical separator to separate the PHA-containing medium impurities and biomass. The separated biomass was adjusted to a total solid content of 10-15 wt%. A surfactant (sodium laureth sulfate, 30%, Miwon Specialty Chemical Co.) was added at 0.13 wt% based on the total weight of the reaction solution. After thorough stirring, physical cell disruption was performed using a high-pressure homogenizer (Bertoli) (400-800 bar, 1 pass). The resulting PHA solution was subjected to a second solid-liquid separation to separate the PHA-containing solids and cell-derived impurities.

[0107] Process (2): Bleaching Acid (10% H3PO4, DUKSAN PURE CHEMICALS) was added to the separated PHA solution to obtain a reaction solution with a pH adjusted to approximately 3.5 to 4. Sodium chlorite (NaClO2) was added in an amount of 0.55 wt% (approximately 5 wt% in terms of solid content) based on the total weight of the reaction solution, and the first decolorization was carried out at 60°C for 1 hour.

[0108] An alkali (40% NaOH, DUKSAN PURE CHEMICALS) was added to the first decolorized slurry to obtain a reaction solution with a pH adjusted to approximately 9 to 9.5. Hydrogen peroxide (HO) was added in an amount of 0.22 wt% (approximately 2 wt% in terms of solid content) based on the total weight of the reaction solution, and the second decolorization was carried out at 60°C for 1 hour.

[0109] Step (3): Deproteinization To the reaction solution obtained in the decolorization step, alkaline protease (Alcalase, Novozymes) was added in an amount of 0.044 wt% (approximately 0.4 wt% in terms of solid content) based on the total weight of the reaction solution, and deproteinization was carried out at a pH of approximately 9.5 for 2 hours. After deproteinization, the recovered solid (PHA) was washed with water and then dried to obtain the final purified polyhydroxyalkanoate resin.

[0110] Example 2 A purified polyhydroxyalkanoate resin was obtained by repeating the same process as in Example 1, except that the amounts of the first oxidizing agent (NaClO2) and the second oxidizing agent (H2O2) were changed as shown in Table 1 below.

[0111] Comparative Example 1 A purified polyhydroxyalkanoate resin was obtained by repeating the same process as in Example 1, except that decolorization was carried out using only hydrogen peroxide (H2O2) as the oxidizing agent under the conditions shown in Table 1 below.

[0112] Comparative Example 2 A purified polyhydroxyalkanoate resin was obtained by repeating the same process as in Example 1, except that decolorization was carried out using only sodium chlorite (NaClO2) as the oxidizing agent under the conditions shown in Table 1 below.

[0113] Comparative Example 3 A purified polyhydroxyalkanoate resin was obtained by repeating the same process as in Example 1, except that decolorization was carried out using only sodium hypochlorite (NaClO) as the oxidizing agent under the conditions shown in Table 1 below.

[0114] Comparative Example 4 A purified polyhydroxyalkanoate resin was obtained by repeating the same process as in Example 1, except that decolorization was carried out using sodium hypochlorite (NaClO) as the first oxidizing agent and hydrogen peroxide (HO) as the second oxidizing agent under the conditions shown in Table 1 below.

[0115] The oxidizing agents and reaction conditions used in the examples and comparative examples are summarized in the table below.

[0116] [Table 1]

[0117] Test Example 1: Color and Molecular Weight The polyhydroxyalkanoate resins obtained in the deproteinization in the Examples and Comparative Examples were subjected to the following tests.

[0118] (1) Color (b* value) The b* value of the PHA solid was measured using a spectrophotometer (Konica Minolta). Approximately 8 to 10 g of the PHA solid was placed in a petri dish for the spectrophotometer, and the color was measured.

[0119] (2)Molecular weight The molecular weight of the PHA was measured by gel permeation chromatography (GPC). Each PHA was dissolved in chloroform in an amount equivalent to 50 times the solid content of the PHA. Impurities were removed using a 0.45 μm syringe filter, and GPC analysis was performed to calculate the weight-average molecular weight (Mw). The results are shown in the table below.

[0120] [Table 2]

[0121] As can be seen from the table above, when only one of hydrogen peroxide (H2O2) and sodium chlorite (NaClO2) was used, as in Comparative Examples 1 and 2, the b* value of the final PHA resin was measured to be 15 or more, indicating that there was no effect in improving coloration.

[0122] In contrast, when sodium chlorite (NaClO2) and hydrogen peroxide (H2O2) were used sequentially as in Examples 1 and 2, the b* value of the final PHA resin was less than 15, and discoloration was significantly improved. In particular, the molecular weight (Mw) of the PHA resin obtained in Example 1 showed almost no difference from those of Comparative Examples 1 and 2, indicating that discoloration was improved without a decrease in molecular weight.

[0123] In contrast, when sodium hypochlorite (NaClO) alone was used as an oxidizing agent as in Comparative Example 3, the effect of improving coloring was confirmed, but the molecular weight was significantly reduced (about 29% lower than in Comparative Example 1). Even when sodium hypochlorite and hydrogen peroxide were used in combination as in Comparative Example 4, the effect of improving coloring was observed while the molecular weight was significantly reduced.

[0124] Test Example 2: Analysis of impurity content The contents of cell-derived impurities (proteins and lipids) in the products prepared in each step of the Examples and Comparative Examples were analyzed. Specifically, the PHA slurry solutions obtained in each purification step were centrifuged to separate solid and liquid components. Protein analysis was then performed using the Dumas analysis method, and lipid extraction was performed using diethyl ether. The results are shown in Tables 3 and 4 below.

[0125] [Table 3]

[0126] [Table 4]

[0127] As can be seen from Table 3 above, in Comparative Example 1, in which hydrogen peroxide was used as an oxidizing agent, proteins were decomposed while lipids were hardly decomposed. It is also understood that the increase in the b* value of the final PHA resin was influenced by the increase in the content of impurities (proteins and lipids) during solid recovery after purification.

[0128] In contrast, as can be seen from Table 4 above, in Example 2, in which the first oxidizing agent (NaClO2) and the second oxidizing agent (HO2), were used sequentially, the protein and lipid contents were significantly reduced during the second decolorization. Furthermore, the proportion of impurities re-recovered during solid recovery was lower than in Comparative Example 1. It is understood that decolorization with the first oxidizing agent (NaClO2) caused the collapse and decomposition of protein and lipid structures, while decolorization with the second oxidizing agent (HO2) caused a rapid increase in the decomposition rate of proteins and lipids, resulting in the rapid decomposition of cell-derived impurities. As a result, the content of impurities remaining in the final PHA resin was reduced, leading to a decrease in the b* value.

[0129] Test Example 3: Analysis of purified PHA The polyhydroxyalkanoate (PHA) resins finally obtained in the examples and comparative examples were tested as follows.

[0130] (1) b* value The b* value of the PHA solid was measured using a spectrophotometer (Konica Minolta). Approximately 8 g of the PHA solid was placed in a petri dish for the spectrophotometer, and the color was measured.

[0131] (2)4-HB content The 4-HB content in PHA was measured by gas chromatography (GC) after butanolysis. γ-Butyrolactone (GBL) was used as a standard reagent for 4-HB content analysis. The same butanolysis reaction was performed for conversion to butyl esters, and quantification was performed using concentration-specific standards. A 20 mg PHA sample was added to the butanolysis solution (n-butanol and 4 M hydrochloric acid in dioxane) and an internal standard solution (diphenylmethane) and thoroughly dissolved by ultrasonication at 70 °C for 1 hour. The solution was then incubated in a hot water bath at 95 °C for 6 hours. Water was added to induce layer separation, and the upper (organic) layer was collected and analyzed by GC to calculate the 4-HB content.

[0132] (3) Purity of PHA The impurity content in the dried PHA solid component was measured, and the PHA purity was calculated using the following formula. The contents of crude protein, crude lipid, and other impurities (ash) were measured as impurities. The impurity content was measured based on the total dry weight. PHA purity (%) = 100 - (crude protein (%) + crude lipids (%) + other impurities (ash) (%)) The results are shown in the table below.

[0133] [Table 5]

[0134] As can be seen from the table above, the final PHA resin obtained in Example 2 according to the present disclosure was an amorphous resin, with a 4-HB content exceeding 40% by weight, a high PHA purity of 98.5% by weight or more, and a b* value, which is an index of yellowness, of less than 15, which was significantly lower than that of Comparative Example 1, indicating excellent coloration.

Claims

1. Preparing a crude product comprising polyhydroxyalkanoate (PHA); subjecting the crude product to a first decolorization to obtain a first product; and subjecting the first product to a second bleaching treatment to obtain a second product; The first bleaching step is performed using sodium chlorite (NaClO ２ 2. A process for preparing a purified polyhydroxyalkanoate, comprising:

2. The second bleaching is performed using hydrogen peroxide (H ２ O ２ 2. The process for preparing a purified polyhydroxyalkanoate according to claim 1, wherein the process is carried out with a second oxidizing agent comprising:

3. 2. The process for preparing a purified polyhydroxyalkanoate according to claim 1, wherein the first bleaching is carried out at a pH of 2 to 7, and the second bleaching is carried out at a pH of 8 to 13.

4. 2. The process for preparing a purified polyhydroxyalkanoate according to claim 1, wherein in the first decolorization, the sodium chlorite is used in an amount of 0.01 to 1% by weight, based on the total weight of the crude product.

5. 3. The process for preparing a purified polyhydroxyalkanoate according to claim 2, wherein in the second bleaching, the hydrogen peroxide is used in an amount of 0.01 to 1% by weight, based on the total weight of the first product.

6. 2. The process for preparing a purified polyhydroxyalkanoate according to claim 1, wherein the lipid content of the first product is less than 1% by weight, based on the weight of the total solids.

7. 2. The process for preparing a purified polyhydroxyalkanoate according to claim 1, further comprising deproteinizing the second product using a protease.

8. 8. The process for preparing a purified polyhydroxyalkanoate according to claim 7, wherein the protease is used in the deproteinization in an amount of 0.001 to 0.1% by weight, based on the total weight of the second product.

9. The process for preparing a purified polyhydroxyalkanoate according to claim 7, wherein the deproteinization is carried out at a pH of 7 to 11.

10. The crude product containing the polyhydroxyalkanoate is performing solid-liquid separation of the fermentation broth containing polyhydroxyalkanoates (PHAs) to obtain biomass; mixing an additive comprising a surfactant with the biomass to obtain a suspension; crushing the biomass in the suspension to obtain a slurry; and performing solid-liquid separation of the slurry.

11. 10. A polyhydroxyalkanoate prepared by the process of claim 1, having a b* value of less than 15 in the CIELAB color coordinate system.

12. 12. The polyhydroxyalkanoate of claim 11, wherein the crystallization temperature (Tc) of the polyhydroxyalkanoate is not measured or is measured to be between 60°C and 120°C.

13. The polyhydroxyalkanoate according to claim 11, wherein the repeating units constituting the polyhydroxyalkanoate include at least one selected from the group consisting of 2-hydroxybutyrate (2-HB), 3-hydroxybutyrate (3-HB), 3-hydroxypropionate (3-HP), 3-hydroxyvalerate (3-HV), 3-hydroxyhexanoate (3-HH), 3-hydroxyheptanoate (3-HHep), 3-hydroxyoctanoate (3-HO), 3-hydroxynonanoate (3-HN), 3-hydroxydecanoate (3-HD), 3-hydroxydodecanoate (3-HDd), 4-hydroxybutyrate (4-HB), 4-hydroxyvalerate (4-HV), 5-hydroxyvalerate (5-HV), and 6-hydroxyhexanoate (6-HH).

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