Process for preparing purified polyhydroxyalkanoates

Abstract

The method for preparing a purified polyhydroxyalkanoate according to the present invention can make the b * value of the final product lower than the conventional value through a decolorization step using two types of oxidants in sequence. Furthermore, in the present invention, by using sodium chlorite (NaClO2) as the first oxidant, the color improvement effect is significant, and the molecular weight is hardly reduced.

Description

Technical Field

[0001] The present disclosure relates to a method for preparing purified polyhydroxyalkanoate, and more particularly, to a method for preparing purified polyhydroxyalkanoate having excellent color through a decolorization process using an oxidizing agent. Background Art

[0002] Polyhydroxyalkanoates (PHAs) are biodegradable polymers composed of various hydroxycarboxylic acids produced by various microorganisms and used as intracellular storage materials. PHAs have similar physical properties to traditional petroleum-derived synthetic polymers such as polybutylene terephthalate (PBAT), polybutylene succinate (PBS), polybutylene succinate terephthalate (PBST), and polybutylene succinate adipate (PBSA), and exhibit complete biodegradability and excellent biocompatibility.

[0003] Specifically, polyhydroxyalkanoate is a natural thermoplastic polyester polymer that accumulates in microbial cells. Since it is a biodegradable material, it can be composted and eventually decomposed into carbon dioxide, water, and organic waste without generating toxic waste. In particular, polyhydroxyalkanoate is biodegradable under any environmental conditions (such as soil and ocean) and has environmentally friendly characteristics.

[0004] [Prior art literature]

[0005] (Patent Document 1) Korean Patent Publication No. 2001-0009719 Summary of the invention

[0006] Technical issues

[0007] In order to obtain polyhydroxyalkanoate (PHA), the fermentation broth obtained from the microorganism is subjected to cell crushing and separation to prepare a crude product. Then, it is necessary to remove impurities through a purification process such as decolorization to reduce the color of the final PHA. However, in the case where PHA is flexible and sticky, there are problems such as impurities attached to the PHA particles cannot be removed during the purification process and the particle size of the PHA is still at a low level, making it difficult to perform subsequent repeated solid-liquid separation and washing, so that the impurity content in the final PHA is still high. As mentioned above, the traditional PHA purification method cannot effectively reduce the impurity content in some PHAs, resulting in the b* value of the purified final PHA to increase to 15 or more, which makes its commercialization difficult. This problem is more serious for some PHAs with low or no crystallinity.

[0008] The present inventors have conducted research and found that in the purification process of polyhydroxyalkanoate, the color of the final product can be improved by using two types of oxidants for decolorization in sequence, thereby solving the problems of the conventional purification method.

[0009] Therefore, an object of the present disclosure is to provide a method for preparing purified polyhydroxyalkanoate having excellent color, which method includes a decolorization step using an oxidizing agent.

[0010] Technical Solution

[0011] According to one aspect of the present disclosure, there is provided a method for preparing purified polyhydroxyalkanoate, the method comprising preparing a crude product comprising polyhydroxyalkanoate (PHA); performing a first decolorization on the crude product to obtain a first product; and performing a second decolorization on the first product to obtain a second product, wherein the first decolorization is performed using a first oxidant comprising sodium chlorite (NaClO2).

[0012] In one embodiment, the second decolorization can be performed using a second oxidizing agent comprising hydrogen peroxide (H2O2).

[0013] In another embodiment, the first decolorization may be performed at a pH of 2 to 7, and the second decolorization may be performed at a pH of 8 to 13.

[0014] In another embodiment, in the first decolorization, the amount of sodium chlorite used may be 0.01 to 1 wt % based on the total weight of the crude product.

[0015] In another embodiment, in the second decolorization, the amount of hydrogen peroxide used may be 0.01 to 1 wt % based on the total weight of the first product.

[0016] In another embodiment, the lipid content of the first product may be less than 1 wt % based on the weight of the total solids content.

[0017] In another embodiment, the method may further comprise deproteinizing the second product using a protease.

[0018] In another embodiment, in the deproteinization, the amount of the protease used may be 0.001 to 0.1 wt % based on the total weight of the second product.

[0019] In another embodiment, deproteinization can be performed at a pH of 7 to 11.

[0020] In another embodiment, a crude product containing polyhydroxyalkanoate can be prepared by a method comprising: subjecting a fermentation broth containing polyhydroxyalkanoate (PHA) to solid-liquid separation to obtain biomass; mixing an additive containing a surfactant with the biomass to obtain a suspension; crushing the biomass in the suspension to obtain a slurry; and subjecting the slurry to solid-liquid separation.

[0021] According to another aspect of the present disclosure, there is provided a polyhydroxyalkanoate prepared by the above method and having a b* value of less than 15 in CIELAB color coordinates.

[0022] In one embodiment, the crystallization temperature (Tc) of the polyhydroxyalkanoate may not be measurable, or may be measured to be between 60°C and 120°C.

[0023] 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).

[0024] Effects of the Invention

[0025] According to the method disclosed herein, the b* value of the final product is reduced to less than 15 by sequentially using two types of oxidants in the decolorization step, thereby solving the color increase problem that may occur in the traditional water-based purification method of polyhydroxyalkanoate (PHA). In addition, the use of two types of oxidants in sequence as described above to solve the traditional color problem can reduce the amount of enzyme used, thereby reducing production costs and being economical.

[0026] In particular, sodium chlorite (NaClO2) used as the first oxidant in the present disclosure produces a significant color improvement effect, while, unlike other chlorine-based oxidants (such as NaClO), it hardly causes changes in polymer properties through a decrease in molecular weight. Therefore, compared with conventional polyhydroxyalkanoates, the purified polyhydroxyalkanoate obtained by the method of the present disclosure has improved color and purity, while also having an excellent effect in maintaining molecular weight.

[0027] In particular, the above effects of the present disclosure can be more significantly produced in the purification process of amorphous PHA. When the method of the present disclosure is applied to the purification process of other crystalline PHA and semi-crystalline PHA, the effects of improving color and purity and maintaining molecular weight can also be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A method of preparing purified polyhydroxyalkanoate according to an embodiment of the present disclosure is shown.

[0029] Figure 2 An example of a method for preparing a crude product of polyhydroxyalkanoate is shown. DETAILED DESCRIPTION

[0030] Best Mode for Carrying Out the Invention

[0031] The present disclosure will be described in more detail below with reference to various embodiments.

[0032] In the present specification, the terms relating to each component are used to distinguish them from each other, and are not intended to limit the scope of the present disclosure. In addition, in the present specification, a singular expression is interpreted as also covering the plural, unless otherwise indicated in the context.

[0033] In this specification, the term "comprising" is intended to specify specific features, regions, steps, processes, elements and / or components. It does not exclude the existence or addition of any other features, regions, steps, processes, elements and / or components, unless there is a specific description to the contrary.

[0034] In this specification, the terms first, second, etc. are used to describe various components. However, the components should not be limited by these terms. These terms are used to distinguish one element from another element.

[0035] In this specification, the molecular weight or weight average molecular weight is usually not described in units, but it is understood that it has units of g / mol or Da.

[0036] Method for preparing purified polyhydroxyalkanoate

[0037] According to one embodiment, a method for preparing purified polyhydroxyalkanoate includes preparing a crude product containing polyhydroxyalkanoate (PHA); performing a first bleaching on the crude product to obtain a first product; and performing a second bleaching on the first product to obtain a second product, wherein the first bleaching is performed using a first oxidant containing sodium chlorite (NaClO2).

[0038] The method may further comprise deproteinizing the second product using a protease.

[0039] Figure 1A method of preparing purified polyhydroxyalkanoate according to an embodiment of the present disclosure is shown.

[0040] refer to Figure 1 According to one embodiment, a crude product of polyhydroxyalkanoate is prepared (S100). Acid is added to the crude product to adjust the pH to about 3 to 5, and sodium chlorite (NaClO2) is added to perform a first decolorization at about 50 to 70°C (S210). An alkaline component is added to the product to adjust the pH to about 8 to 10, and hydrogen peroxide (H2O2) is added to perform a second decolorization at about 50 to 70°C (S220). An alkaline component is added to the decolorized product to adjust the pH to about 8 to 9.5, and deproteinization is performed using a protease (S310). After deproteinization, the PHA solid is recovered (410). Water washing and dehydration are performed (S430) to obtain the purified final polyhydroxyalkanoate (S500).

[0041] Meanwhile, a crude product of polyhydroxyalkanoate used as a starting material in the present disclosure may be prepared from a fermentation broth through processes such as filtration, crushing, and purification.

[0042] Figure 2 An example of a method for preparing a crude product of polyhydroxyalkanoate is shown.

[0043] refer to Figure 2 According to one embodiment, a fermentation broth of polyhydroxyalkanoate is prepared (S10). The bacterial cells are separated by centrifugation or the like (S20). A surfactant or the like is added (S30). The bacterial cells are crushed by a high-pressure homogenizer (S40). Solid-liquid separation is performed by centrifugation or the like (S50). A crude product of polyhydroxyalkanoate is obtained (S100).

[0044] Each step of the preparation method according to the present disclosure will be described in detail below.

[0045] Polyhydroxyalkanoate

[0046] Polyhydroxyalkanoates (PHA) can be classified into crystalline, semi-crystalline or amorphous polyhydroxyalkanoates according to their molecular structure.

[0047] According to one embodiment, the polyhydroxyalkanoate may include amorphous polyhydroxyalkanoate. In the case of amorphous polyhydroxyalkanoate, due to its unique flexibility and viscosity, repeated solid-liquid separation and washing in a water-based purification process are difficult, and the impurity content in the purified final PHA is still high, making its commercialization difficult. However, according to the present disclosure, when two types of oxidants are used sequentially for decolorization in the purification process of amorphous PHA, the color of the final product can be significantly improved.

[0048] 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.

[0049] The polyhydroxyalkanoate may comprise at least one repeating 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).

[0050] In addition, the polyhydroxyalkanoate may contain isomers. For example, the polyhydroxyalkanoate may contain structural isomers, enantiomers or geometric isomers. Specifically, the polyhydroxyalkanoate may contain structural isomers.

[0051] The polyhydroxyalkanoate may be a homopolymer or a copolymer. According to one embodiment, the polyhydroxyalkanoate may comprise a copolymer, in particular, a copolymer comprising two or more different repeating units randomly distributed in the polymer chain.

[0052] As an example, the polyhydroxyalkanoate may include 4-hydroxybutyrate (4-HB) repeating units. For example, the content of 4-hydroxybutyrate (4-HB) repeating units in the polyhydroxyalkanoate may be 0.1% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, or 60% by weight or more, and may be 100% by weight or less, 99% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less. Specifically, the polyhydroxyalkanoate may include 25% by weight or more of 4-hydroxybutyrate (4-HB) repeating units.

[0053] Specifically, the polyhydroxyalkanoate may be a copolymer comprising a 4-HB repeating unit. Examples of repeating units that may be included in the polyhydroxyalkanoate together with 4-HB may 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).

[0054] More specifically, in addition to the 4-HB repeating unit, the polyhydroxyalkanoate may include 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. For example, the polyhydroxyalkanoate may include a copolymer containing a 4-HB repeating unit and a 3-HB repeating unit. Specifically, it may include poly-3-hydroxybutyrate-co-4-hydroxybutyrate (P3HB-co-4HB).

[0055] The PHA may comprise one or more types of PHA. For example, it may comprise 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 comprise, 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 of 4-HB repeating units. The second PHA may include, for example, 0.1 wt % to 30 wt %, 0.5 wt % to 30 wt %, 1 wt % to 30 wt %, 3 wt % to 30 wt %, 1 wt % to 28 wt %, 1 wt % to 25 wt %, 1 wt % to 24 wt %, 1 wt % to 20 wt %, 1 wt % to 15 wt %, 2 wt % to 25 wt %, 3 wt % to 25 wt %, 3 wt % to 24 wt %, 5 wt % to 24 wt %, 5 wt % to 20 wt %, greater than 5 wt % to less than 20 wt %, 7 wt % to 20 wt %, 10 wt % to 20 wt %, 15 wt % to 25 wt %, or 15 wt % to 24 wt % of 4-HB repeating units. In addition, the PHA may be a mixture of the first PHA and the second PHA in a weight ratio of 20:80 to 80:20 or 30:70 to 70:30.

[0056] Crude product of polyhydroxyalkanoate

[0057] In this step, a crude product of polyhydroxyalkanoate is prepared.

[0058] Generally, polyhydroxyalkanoates can be produced by a fermentation process. A crude product of polyhydroxyalkanoates can also be obtained by fermentation.

[0059] The method of producing polyhydroxyalkanoate (PHA) by fermentation can be performed by a known method. For example, polyhydroxyalkanoate can be produced by fermentation using a wild-type or transgenic microorganism cultured on a specific substrate, and is not particularly limited.

[0060] During fermentation, the polyhydroxyalkanoates thus produced accumulate within the microbial cells; therefore, a process for isolating and purifying them is absolutely necessary.

[0061] For example, biomass containing polyhydroxyalkanoate is separated from a fermentation broth, a surfactant is added to the biomass, and microbial cells are disrupted to separate only polyhydroxyalkanoate.

[0062] As an example, a crude product containing polyhydroxyalkanoate can be prepared by a method comprising: subjecting a fermentation broth containing polyhydroxyalkanoate (PHA) to solid-liquid separation to obtain biomass; mixing an additive containing a surfactant with the biomass to obtain a suspension; crushing the biomass in the suspension to obtain a slurry; and subjecting the slurry to solid-liquid separation.

[0063] First, a fermentation broth formed by a microorganism is subjected to solid-liquid separation using a mechanical separation method (such as centrifugation and membrane separation) or other known methods to separate a biomass containing cells in which polyhydroxyalkanoate (PHA) is accumulated.

[0064] The biomass thus separated can be mixed with a solvent to adjust the solid concentration. The biomass can be mixed with a surfactant, etc. For example, an anionic surfactant or a nonionic surfactant can be used as a surfactant. Specifically, sodium laureth sulfate, sodium lauryl sulfate, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate can be used. As a result, a suspension comprising biomass, a surfactant, etc. can be obtained.

[0065] After this, the biomass in the suspension is broken, and the breaking can be carried out by mechanical breaking (physical breaking) or chemical breaking (non-mechanical breaking). For example, the breaking can be carried out by ultrasonic breaking, high pressure breaking or milling breaking. Ultrasonic breaking can be carried out for example at 20Hz or higher energy levels for 10 minutes to 60 minutes. In addition, high pressure breaking can be carried out for example at 10 bar or higher pressure for 1 minute to 60 minutes. In addition, milling breaking can be carried out for 1 minute to 60 minutes by colloid mill, bead mill or ball mill.

[0066] As a result of the disruption, a slurry containing fine particles of polyhydroxyalkanoate and cell-derived impurities (such as cell walls) can be obtained. The slurry is subjected to solid-liquid separation using a mechanical separation method (such as centrifugation and membrane separation) or other known methods to obtain a crude product of polyhydroxyalkanoate.

[0067] Since the crude product of polyhydroxyalkanoate contains trace amounts of cell-derived impurities (proteins, lipids, etc.), further purification is required.

[0068] For example, the protein content in the crude product can be 3.5 wt % or less, 3 wt % or less, 2.7 wt % or less, or 2.5 wt % or less, and can be 0.1 wt % or more, 1 wt % or more, or 1.5 wt % or more, based on the total solid weight in the crude product.

[0069] In addition, the lipid content in the crude product can be 1.5 wt % or less, 1.3 wt % or less, 1 wt % or less, or 0.7 wt % or less, and can be 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more, based on the total solid weight in the crude product.

[0070] In addition, the solid content in the crude product can be 1 wt % or more, 3 wt % or more, 5 wt % or more, 7 wt % or more, or 10 wt % or more, and can be 80 wt % or less, 50 wt % or less, 30 wt % or less, 20 wt % or less, or 15 wt % or less, based on the total weight of the crude product.

[0071] First decolorization

[0072] In this step, a crude product containing polyhydroxyalkanoate is first decolorized to obtain a first product.

[0073] The first decolorization is performed by a first oxidant comprising sodium chlorite (NaClO2). When sodium chlorite is used as an oxidant for decolorization, the color improvement effect is significant, and there is almost no decrease in molecular weight, unlike other chlorine-based oxidants (such as NaClO). This is because cell-derived impurities (proteins, lipids, etc.) in the crude product are decomposed by reaction with sodium chlorite, while PHA is hardly decomposed.

[0074] The amount of sodium chlorite used in the first decolorization can be 0.01% by weight or more, 0.02% by weight or more, 0.03% by weight or more, 0.04% by weight or more, 0.05% by weight or more, 0.06% by weight or more, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, 0.4% by weight or more, or 0.5% by weight or more, and can be 1.3% by weight or less, 1.2% by weight or less, 1.1% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, or 0.6% by weight or less. As a specific example, in the first decolorization, the amount of sodium chlorite can be 0.01 to 1% by weight or 0.05 to 1% by weight based on the gross weight of the crude product. When the amount of sodium chlorite is within the above-mentioned preferred range, the decolorization effect can be fully achieved while minimizing the reduction in molecular weight.

[0075] In addition, an additional oxidant can be used together with sodium chlorite (NaClO2) as the first oxidant in the first decolorization. For example, the amount of chlorine dioxide (ClO2) can be 0.05 to 1 weight % based on the total weight of the crude product.

[0076] The first decolorization can be carried out under acidic pH conditions. For example, the pH range in the first decolorization can 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, and can 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 decolorization can be carried out at a pH of 1 to 7 or a pH of 2 to 7. As a more specific example, the first decolorization can be carried out at a pH of 3 to 6 or a pH of 3 to 5.

[0077] The acid component can be used to adjust the acidic pH. Examples of acid components used for pH adjustment in the first decolorization include phosphoric acid, sulfuric acid, hydrochloric acid and nitric acid, but are not limited thereto. Because the crude product contains an appropriate amount of this acid component, it can be adjusted to the desired acidic pH.

[0078] In addition, the temperature of 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° C. to 90° C. or 50° C. to 70° C.

[0079] In addition, the first decolorization required time can be 10 minutes or more, 30 minutes or more or 50 minutes or more, and can be 3 hours or less, 2 hours or less, or 1 hour and 30 minutes or less. As a specific example, it can be 30 minutes to 1 hour and 30 minutes.

[0080] The impurity content in the first product obtained by the first bleaching can be reduced relative to the impurity content in the previous step.

[0081] For example, the protein content in the first product can be 3.5 wt % or less, 3 wt % or less, or 2.7 wt % or less, and can be 0.1 wt % or more, 1 wt % or more, or 1.5 wt % or more, based on the total solid weight in the first product.

[0082] In addition, the lipid content in the first product can be 1.5 % by weight or less, 1.3 % by weight or less, 1 % by weight or less, or 0.7 % by weight or less based on the total solid weight in the first product, and can be 0.1 % by weight or more, 0.2 % by weight or more, or 0.3 % by weight or more. Specifically, the lipid content of the first product can be less than 1 % by weight based on the weight of the total solid content, which is significantly reduced relative to the lipid content in the previous step.

[0083] In addition, the solid content in the first product can be 1 wt % or more, 3 wt % or more, 5 wt % or more, 7 wt % or more, or 10 wt % or more, based on the total weight of the first product, and can be 80 wt % or less, 50 wt % or less, 30 wt % or less, 20 wt % or less, or 15 wt % or less.

[0084] Second decolorization

[0085] In this step, the first product obtained by the first decolorization is subjected to second decolorization to obtain a second product.

[0086] The second decolorization can be performed using a second oxidant including hydrogen peroxide (H2O2). When hydrogen peroxide is used as an oxidant in the second decolorization step after sodium chlorite is used as an oxidant in the first decolorization step, the content of impurities (protein and lipid) after the second decolorization can be significantly reduced, and the efficiency of dissolving impurities can be improved when recovering PHA solids, thereby also reducing the re-recovery rate of impurities. Therefore, the amount of enzyme used for protein decomposition can be reduced in the subsequent deproteinization step, thereby reducing production costs.

[0087] The amount of hydrogen peroxide used in the second decolorization can be 0.01% by weight or more, 0.02% by weight or more, 0.03% by weight or more, 0.04% by weight or more, 0.05% by weight or more, 0.06% by weight or more, 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight or more, 0.4% by weight or more, or 0.5% by weight or more, and can be 1.3% by weight or less, 1.2% by weight or less, 1.1% by weight or less, 1% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, or 0.6% by weight or less. As a specific example, in the second decolorization, the amount of hydrogen peroxide can be 0.01 to 1% by weight or 0.05 to 1% by weight 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 the prior art, which is made possible by the advantages of sequential use with sodium chlorite (the first oxidant).

[0088] The second decolorization can be carried out under alkaline pH conditions. For example, the pH range in the second decolorization can be 7 or more, 7.5 or more, 8 or more, or 8.5 or more, and can 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 decolorization can be carried out at a pH of 7 to 13 or a pH of 8 to 13. As a more specific example, the second decolorization can be carried out at a pH of 8 to 11 or a pH of 8 to 10.

[0089] The alkaline component can be used to adjust the alkaline pH value. Examples of alkaline components used for pH adjustment in the second decolorization include sodium hydroxide, potassium hydroxide, lithium hydroxide and sodium carbonate, but are not limited thereto. Because the first product contains an appropriate amount of such alkaline components, it can be adjusted to the desired alkaline pH.

[0090] In addition, the temperature of the second decolorization 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 decolorization may be performed at a temperature of 40° C. to 90° C. or 50° C. to 70° C.

[0091] In addition, the required time for the second decolorization can be 10 minutes or more, 30 minutes or more, or 50 minutes or more, and can be 3 hours or less, 2 hours or less, or 1 hour and 30 minutes or less. As a specific example, it can be 30 minutes to 1 hour and 30 minutes.

[0092] The impurity content in the second product obtained by the second bleaching can be reduced relative to the impurity content in the previous step.

[0093] For example, the protein content in the second product can be 2.5 wt % or less, 2 wt % or less, or 1.5 wt % or less, and can be 0.1 wt % or more, 0.5 wt % or more, or 1 wt % or more, based on the total solid weight in the second product.

[0094] In addition, the lipid content in the second product can be 1 wt % or less, 0.8 wt % or less, 0.6 wt % or less, or 0.4 wt % or less, based on the total solid weight in the second product, and can be 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more.

[0095] In addition, the solid content in the second product can be 1 wt % or more, 3 wt % or more, 5 wt % or more, 7 wt % or more, or 10 wt % or more, based on the total weight of the second product, and can be 80 wt % or less, 50 wt % or less, 30 wt % or less, 20 wt % or less, or 15 wt % or less.

[0096] Deproteinization

[0097] Thereafter, a step of deproteinizing the second product using a protease may be performed.

[0098] Examples of the protease include alkaline protease, pepsin, trypsin, papain, chymotrypsin, aminopeptidase, and carboxypeptidase, and one or two or more of them may be used.

[0099] The amount of the protease used in the deproteinization can be 0.001% by weight or more, 0.002% by weight or more, 0.003% by weight or more, 0.004% by weight or more, 0.005% by weight or more, 0.006% by weight or more, 0.01% by weight or more, 0.015% by weight or more, 0.02% by weight or more, or 0.025% by weight or more, and can be 0.2% by weight or less, 0.15% by weight or less, 0.1% by weight or less, 0.05% by weight or less, 0.045% by weight or less, or 0.04% by weight or less. As an example, in the deproteinization, the amount of the protease can be 0.001 to 0.1% by weight or 0.005 to 0.1% by weight based on the total weight of the second product.

[0100] In addition to proteases, lipolytic enzymes, cell wall decomposing enzymes, DNA decomposing enzymes, etc. can be further added to the second product. Examples of lipolytic enzymes include lipases, phospholipases, cholinesterases, and phosphatases. Examples of cell wall decomposing enzymes include lysozymes, amylases, cellulases, maltase, sucrase, α-glycosidase, β-glycosidase, and N-glycosidase. Examples of DNA decomposing enzymes include ribonucleases.

[0101] Deproteinization can be carried out under alkaline pH conditions. For example, the pH range in the deproteinization can be 7 or more, 7.5 or more, 8 or more, or 8.5 or more, and can be 12 or less, 11 or less, 10.5 or less, 10 or less, 9.5 or less, or 9 or less.

[0102] The alkaline component can be used to adjust the alkaline pH. Examples of alkaline components used for pH adjustment in deproteinization include sodium hydroxide, potassium hydroxide, lithium hydroxide and sodium carbonate, but are not limited thereto. Because the second product contains an appropriate amount of such alkaline components, it can be adjusted to the desired alkaline pH.

[0103] In addition, the deproteinization temperature 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.

[0104] As a specific example, deproteinization may be performed at a pH of 7 to 11, more specifically at a pH of 8 to 10, and a temperature of 50°C to 70°C.

[0105] The protein content can be significantly reduced by deproteinization. For example, the protein content can be 1 wt % or less, 0.8 wt % or less, 0.6 wt % or less, or 0.4 wt % or less based on the total solid weight in the product after deproteinization, and can be 0.1 wt % or more, 0.15 wt % or more, 0.2 wt % or more, or 0.25 wt % or more.

[0106] In addition, the lipid content can be 1.5 wt % or less, 1.3 wt % or less, 1 wt % or less, or 0.7 wt % or less, based on the total solid weight in the deproteinized product, and can be 0.1 wt % or more, 0.2 wt % or more, or 0.3 wt % or more.

[0107] Purified polyhydroxyalkanoate

[0108] According to the present disclosure, a purified polyhydroxyalkanoate obtained by the above method is provided.

[0109] According to one embodiment, the purified polyhydroxyalkanoate may have a solid form, and may be, for example, a solid resin. According to another embodiment, the purified polyhydroxyalkanoate may have a liquid form, and may be, for example, a liquid resin or an aqueous suspension.

[0110] The polyhydroxyalkanoates prepared according to the present disclosure may have improved color. According to one embodiment, the polyhydroxyalkanoates may have a b* value in CIELAB color coordinates of less than 15. For example, the b* value of the purified polyhydroxyalkanoates in CIELAB color coordinates may be 14 or less, 13.5 or less, 13 or less, and may be 0 or more, 1 or more, 5 or more, or 10 or more.

[0111] In addition, 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.

[0112] Furthermore, the melting temperature (Tm) of the polyhydroxyalkanoate may not be measurable, or 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.

[0113] The polyhydroxyalkanoate of the present disclosure may include amorphous polyhydroxyalkanoate, semi-crystalline polyhydroxyalkanoate and mixtures thereof. For example, the crystallization temperature (Tc) of the polyhydroxyalkanoate may not be measured, or the crystallization temperature (Tc) may be measured as 60°C to 120°C, specifically, 60°C to 110°C, 70°C to 120°C, or 75°C to 115°C.

[0114] 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).

[0115] According to one embodiment, the polyhydroxyalkanoate may include 4-hydroxybutyrate (4-HB) repeating units. Specifically, it may include a copolymer containing 4-HB repeating units. For example, the content of 4-hydroxybutyrate (4-HB) repeating units in the polyhydroxyalkanoate may be 0.1% by weight or more, 1% by weight or more, 5% by weight or more, 10% by weight or more, 20% by weight or more, 30% by weight or more, 40% by weight or more, 50% by weight or more, or 60% by weight or more, and may be 100% by weight or less, 99% by weight or less, 90% by weight or less, 80% by weight or less, 70% by weight or less, or 60% by weight or less. As a specific example, the polyhydroxyalkanoate may include 25% by weight or more of 4-hydroxybutyrate (4-HB) repeating units.

[0116] In addition, 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.

[0117] Additionally, the polyhydroxyalkanoate may have a purity of 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.

[0118] Mode for Carrying Out the Invention

[0119] The present disclosure will be described in more detail below through the following examples. However, these examples are provided for illustrative purposes only, and the present disclosure is not limited thereto.

[0120] Preparation Example 1: Preparation of PHA fermentation broth

[0121] PHA fermentation broth is prepared using genetically engineered Escherichia coli (E. coli) strains. First, the PHA-producing strain is seed cultured to ensure a certain amount, and then the main fermentation is carried out. For the fermentation of the PHA-producing strain, glucose or raw sugar is used as a carbon source, and various mineral components are added as nutrients. Sodium hydroxide solution or ammonia gas is used to adjust the pH during the fermentation process. The fermentation process using the PHA-producing strain is carried out at 30 to 40°C and a pH of 6 to 9.

[0122] Example 1: Preparation of purified polyhydroxyalkanoate

[0123] Step (1): solid-liquid separation and cell disruption

[0124] The PHA fermentation broth was subjected to solid-liquid separation using a mechanical separator to separate the culture medium impurities and the biomass containing PHA. The separated biomass was adjusted to a total solid content of 10 to 15% by weight. A surfactant (sodium laureth sulfate, 30%, Miwon Corporation) was added in an amount of 0.13% by weight based on the total weight of the reaction solution. After sufficient stirring, physical cell disruption was performed using a high-pressure homogenizer (Bertoli) (400 to 800 bar, 1 pass). The PHA solution thus obtained was subjected to a second solid-liquid separation to separate the solids containing PHA and cell-derived impurities.

[0125] Step (2): Decolorization

[0126] Acid (10% H3PO4, Deoksan Scientific) was added to the separated PHA solution to obtain a reaction solution having a pH adjusted to about 3.5 to 4. Sodium chlorite (NaClO2) was added in an amount of 0.55 wt% (about 5 wt%, based on solid content) based on the total weight of the reaction solution, and then first decolorization was performed at 60°C for 1 hour.

[0127] Alkali (40% NaOH, Deoksan Scientific) was added to the first decolorized slurry to obtain a reaction solution with a pH adjusted to about 9 to 9.5. Hydrogen peroxide (H2O2) was added in an amount of 0.22 wt% (about 2 wt%, based on the solid content) based on the total weight of the reaction solution, and then the second decolorization was performed at 60°C for 1 hour.

[0128] Step (3): Deproteinization

[0129] Alcalase (Novozyme) was added to the reaction solution obtained in the decolorization step in an amount of 0.044 wt % (about 0.4 wt %, based on the solid content) based on the total weight of the reaction solution, and deproteinization was performed for 2 hours at a pH of about 9.5. After deproteinization, the recovered solid (PHA) was washed with water and then dried to obtain the final purified polyhydroxyalkanoate resin.

[0130] Example 2

[0131] The same procedure as in Example 1 was repeated to obtain a purified polyhydroxyalkanoate resin, except that the amounts of the first oxidizing agent (NaClO 2 ) and the second oxidizing agent (H 2 O 2 ) were each changed as shown in Table 1 below.

[0132] Comparative Example 1

[0133] The same procedure as in Example 1 was repeated to obtain a purified polyhydroxyalkanoate resin, except that decolorization was performed using only hydrogen peroxide (H2O2) as an oxidizing agent under the conditions shown in Table 1 below.

[0134] Comparative Example 2

[0135] The same procedure as in Example 1 was repeated to obtain a purified polyhydroxyalkanoate resin, except that only sodium chlorite (NaClO 2 ) was used as an oxidizing agent for decolorization under the conditions shown in Table 1 below.

[0136] Comparative Example 3

[0137] The same procedure as in Example 1 was repeated to obtain a purified polyhydroxyalkanoate resin, except that decolorization was performed using only sodium hypochlorite (NaClO) as an oxidizing agent under the conditions shown in Table 1 below.

[0138] Comparative Example 4

[0139] The same steps as in Example 1 were repeated to obtain a purified polyhydroxyalkanoate resin, except that sodium hypochlorite (NaClO) was used as the first oxidant and hydrogen peroxide (H2O2) was used as the second oxidant, and decolorization was performed under the conditions shown in Table 1 below.

[0140] The oxidants and reaction conditions used in the Examples and Comparative Examples are summarized in the following table.

[0141] [Table 1]

[0142]

[0143]

[0144] Test Example 1: Color and Molecular Weight

[0145] The polyhydroxyalkanoate resins obtained after deproteinization in Examples and Comparative Examples were each subjected to the following tests.

[0146] (1) Color (b* value)

[0147] The b* value of the PHA solid was measured using a spectrophotometer (Konica Minolta). About 8 to 10 g of the PHA solid was placed on a petri dish for the spectrophotometer, and the color was measured.

[0148] (2) Molecular weight

[0149] The molecular weight of PHA was measured by gel permeation chromatography (GPC). Each PHA was dissolved by adding chloroform equivalent to 50 times the solid content of PHA. Impurities were removed using a 0.45 μm syringe filter, and GPC analysis was performed to calculate the weight average molecular weight (Mw).

[0150] The results are shown in the table below.

[0151] [Table 2]

[0152] b* value M Summarized results Comparative Example 1 16.2 541,408 b* is 15 or higher Comparative Example 2 15.6 560,771 b* is 15 or higher Example 1 10.0 535,546 Improved color without loss of molecular weight Example 2 12.4 563,062 Improved color without loss of molecular weight Example 3 7.4 384,926 Molecular weight reduction Example 4 7.4 474,711 Molecular weight reduction

[0153] As can be seen from the above table, 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 no color improvement effect.

[0154] 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, resulting in a significant improvement in color. 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 color was improved without a decrease in molecular weight.

[0155] In contrast, when sodium hypochlorite (NaClO) was used alone as an oxidant as in Comparative Example 3, a color-improving effect was confirmed, but the molecular weight was significantly reduced (about 29% lower than in Comparative Example 1). Even when it was used sequentially with hydrogen peroxide as in Comparative Example 4, a color-improving effect was observed, but the molecular weight was significantly reduced.

[0156] Test Example 2: Analysis of impurity content

[0157] The content of cell-derived impurities (protein and lipid) in the products prepared in each step of the examples and comparative examples was analyzed. Specifically, the PHA slurry obtained in each purification step was centrifuged to separate solid and liquid components. Then, protein analysis was performed using the Dumas analysis method, and lipid extraction was performed using ether. The results are shown in Tables 3 and 4 below.

[0158] [Table 3]

[0159]

[0160] [Table 4]

[0161]

[0162] As can be seen from Table 3 above, in Comparative Example 1 using hydrogen peroxide as an oxidant, protein was decomposed, while lipid was hardly decomposed. In addition, it can be understood that the increase in the b* value of the final PHA resin is affected by the increase in the content of impurities (protein and lipid) during the solid recovery process after purification.

[0163] On the contrary, it can be seen from Table 4 above that in Example 2 in which the first oxidant (NaClO2) and the second oxidant (H2O2) were used sequentially, the contents of protein and lipid were each significantly reduced after the second decolorization. In addition, the recovery rate of impurities during the solid recovery process was lower than that of Comparative Example 1. It can be understood that the decolorization of the first oxidant (NaClO2) causes structural destruction and decomposition of proteins and lipids, and the decolorization of the second oxidant (H2O2) causes a rapid increase in the decomposition rate of proteins and lipids, thereby causing rapid decomposition of cell-derived impurities. As a result, the impurity content remaining in the final PHA resin is reduced, resulting in a reduction in the b* value.

[0164] Test Example 3: Analysis of purified PHA

[0165] The polyhydroxyalkanoate (PHA) resins finally obtained in Examples and Comparative Examples were each subjected to the following tests.

[0166] (1) b* value

[0167] The b* value of the PHA solid was measured using a spectrophotometer (Konica Minolta). About 8 g of the PHA solid was placed on a petri dish for the spectrophotometer, and the color was measured.

[0168] (2) 4-HB content

[0169] After the butanolysis reaction, the content of 4-HB in PHA was measured by gas chromatography (GC). γ-Butyrolactone (GBL) was used as a standard reagent for analyzing the content of 4-HB. The same butanolysis reaction was performed to convert into butyl esters, and a standard of a specific concentration was used to quantify it. The butanolysis solution (a dioxane solution of n-butanol and 4M HCl) and the internal standard solution (diphenylmethane) were added to 20 mg of PHA sample, and the PHA was fully dissolved by ultrasound at 70 ° C for 1 hour. The dissolved sample was reacted in a hot water bath at 95 ° C for 6 hours. Water was added to induce layer separation, the upper layer (organic layer) was separated and analyzed by GC to calculate the 4-HB content.

[0170] (3) Purity of PHA

[0171] The impurity content in the dried PHA solid component was measured, and the PHA purity was calculated using the following equation. The content of crude protein, crude lipids and other impurities (ash) as impurities was measured. The content of impurities was measured based on the total dry weight.

[0172] PHA purity (%) = 100 – (crude protein (%) + crude lipid (%) + other impurities (ash) (%))

[0173] The results are shown in the following table.

[0174] [Table 5]

[0175]

[0176] As can be seen from the above table, the final PHA resin obtained in Example 2 according to the present disclosure is an amorphous resin having a 4-HB content of more than 40 wt%, a high PHA purity of 98.5% or more, and a b* value (an indicator of yellowness index) of less than 15, which is significantly lower than that of Comparative Example 1, indicating excellent color.

Claims

1. A method for preparing purified polyhydroxyalkanoate, comprising: preparing a crude product comprising polyhydroxyalkanoate (PHA); Performing a first decolorization on the crude product to obtain a first product; and performing a second decolorization on the first product to obtain a second product, The first decolorization is carried out using a first oxidizing agent comprising sodium chlorite (NaClO2).

2. The method for preparing purified polyhydroxyalkanoate according to claim 1, wherein the second decolorization is performed using a second oxidizing agent comprising hydrogen peroxide (H2O2). 3 . The method for preparing purified polyhydroxyalkanoate according to claim 1 , wherein the first decolorization is performed at a pH of 2 to 7, and the second decolorization is performed at a pH of 8 to 13.

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

5. The method for preparing purified polyhydroxyalkanoate according to claim 2, wherein: In the second decolorization, hydrogen peroxide is used in an amount of 0.01 to 1 wt % based on the total weight of the first product.

6. The method for preparing purified polyhydroxyalkanoate according to claim 1, wherein the lipid content of the first product is less than 1 wt% based on the weight of the total solid content. 7 . The method for preparing purified polyhydroxyalkanoate according to claim 1 , further comprising deproteinizing the second product using a protease.

8. The method for preparing purified polyhydroxyalkanoate according to claim 7, wherein: In the deproteinization, the amount of protease used is 0.001 to 0.1 wt % based on the total weight of the second product. 9 . The method for preparing purified polyhydroxyalkanoate according to claim 7 , wherein the deproteinization is performed at a pH of 7 to 11.

10. The method for preparing purified polyhydroxyalkanoate according to claim 1, wherein the crude product containing polyhydroxyalkanoate is prepared by a method comprising: performing solid-liquid separation on a fermentation broth containing polyhydroxyalkanoate (PHA) to obtain a 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 The slurry is subjected to solid-liquid separation.

11. A polyhydroxyalkanoate prepared by the method of claim 1 and having a b* value of less than 15 in CIELAB color coordinates.

12. The polyhydroxyalkanoate according to claim 11, wherein the crystallization temperature (Tc) of the polyhydroxyalkanoate cannot be measured or is measured to be 60°C to 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).

Images