JP7821738B2 - Method for producing polyhydroxybutyric acid copolymer and its use - Google Patents

Description

The present invention relates to a method for producing polyhydroxybutyric acid copolymers and their use.

Polyhydroxybutyric acid copolymer (hereinafter sometimes referred to as "PHB copolymer") is known to be biodegradable.

PHB copolymers produced by microorganisms accumulate within the microbial cells. Therefore, to use PHB copolymers as plastics, a process for separating and purifying the PHB copolymers from the microbial cells is required. In the process of separating and purifying the PHB copolymers, biologically derived components other than the PHB copolymer are solubilized, and then the PHB copolymer is extracted from the resulting aqueous suspension. This process involves separation procedures such as centrifugation, filtration, and drying. For drying, for example, a spray dryer, fluidized bed dryer, or drum dryer can be used. However, a spray dryer is preferred due to its ease of operation.

To date, the present inventors have developed a technique for preventing aggregation of PHB copolymers in aqueous suspensions at pH 7.0 or below by adding polyvinyl alcohol (PVA) as a dispersant before adjusting the pH of the aqueous suspension to 7.0 or below, and then spray-drying the resulting aqueous suspension at pH 7.0 or below (Patent Document 1). The present inventors have also developed a technique for producing PHB copolymers with a high composition ratio of 3-hydroxyhexanoate (hereinafter sometimes referred to as "3HH") units, which involves disrupting bacterial cells containing the PHB copolymer using high-pressure disruption (Patent Documents 2 and 3).

A known example of a polyhydroxybutyric acid copolymer is poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (hereinafter sometimes referred to as "PHBH").

International Publication No. 2018/070492 International Publication No. 2018/186278 Japanese Patent Application Laid-Open No. 2019-97518

While the above technology is excellent, there is room for further improvement in terms of ease of handling during the manufacturing process.

The object of the present invention is to provide a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles during the manufacturing process, particularly a PHB copolymer having a high bulk density and a specific composition ratio of 3-hydroxybutyrate (hereinafter sometimes referred to as "3HB") units to hydroxyalkanoate units other than 3HB units, and a method for manufacturing the same.

As a result of extensive research to solve the above-mentioned problems, the inventors discovered for the first time that a PHB copolymer with a high bulk density can be obtained while suppressing aggregation of PHB copolymer particles during the production process by including a step of enzymatically treating, with a specific enzyme, bacterial cells containing a polyhydroxybutyric acid copolymer having a specific composition ratio of 3HB units/hydroxyalkanoate units other than 3HB units, in which the composition ratio of hydroxyalkanoate units other than 3HB units is high. This led to the completion of the present invention.

Therefore, one aspect of the present invention is a method for producing a PHB copolymer, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol), and the method includes: (a) adding an alkaline protease to a culture solution containing bacterial cells containing the PHB copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing an aqueous suspension from the aqueous suspension obtained in step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa·s or more and 0.5 Pa·s or less; and (d) spray-drying the aqueous suspension prepared in step (c).

Another aspect of the present invention is a PHB copolymer powder comprising a PHB copolymer, peptidoglycan, and a dispersant, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol), a bulk density of 0.45 g/mL or more, and a median particle size of 80 to 200 μm.

Furthermore, one aspect of the present invention is a PHB copolymer powder comprising a PHB copolymer and a nitrogen compound, wherein the PHB copolymer has a composition ratio of 3-hydroxybutyrate units/hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol), a bulk density of 0.45 g/mL or more, and a median particle size of 80 to 200 μm.

According to one aspect of the present invention, it is possible to provide a PHB copolymer having a high bulk density while suppressing aggregation of PHB copolymer particles during the manufacturing process, particularly a PHB copolymer having a high bulk density and a specific composition ratio of 3HB units/hydroxyalkanoate units other than 3HB units.

One embodiment of the present invention is described in detail below. Unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B." Furthermore, all documents cited in this specification are incorporated herein by reference.

1. Overview of the present invention
A method for producing a PHB copolymer according to one embodiment of the present invention (hereinafter referred to as "this production method") is characterized in that the PHB copolymer has a composition ratio of 3HB units/hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol), and includes the steps of: (a) adding an alkaline protease to a culture solution containing bacterial cells containing the PHB copolymer to enzymatically treat the bacterial cells; (b) adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; (c) preparing an aqueous suspension from the aqueous suspension obtained in step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less; and (d) spray-drying the aqueous suspension prepared in step (c).

The inventors discovered a new problem in that, in the conventional method (Patent Document 1), a high-concentration slurry (PHB copolymer aqueous suspension) is used, but PHB copolymer particles with a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units aggregate, resulting in an increase in the viscosity of the PHB copolymer aqueous suspension, making it difficult to transport and potentially complicating spray drying. Furthermore, they found that reducing the concentration of the PHB copolymer aqueous suspension to solve this problem requires more energy for spray drying, which is undesirable in terms of energy efficiency, and also creates other problems, such as a low bulk density of the resulting powder and poor powder transportability.

On the other hand, the methods described in Patent Documents 2 and 3 involve physically disrupting the bacterial cells using high pressure, which can complicate the process when scaled up. Furthermore, the method described in Patent Document 3 also has the problem of requiring a long manufacturing process.

Therefore, the present inventors conducted extensive research to solve the above problems and discovered for the first time that by including a step of enzymatically treating, with a specific enzyme, bacterial cells containing a PHB copolymer having a specific 3HB unit/hydroxyalkanoate unit composition ratio, it is possible to obtain a PHB copolymer with high bulk density while suppressing aggregation of PHB copolymer particles during the production process, even when spray-drying is performed. The present inventors speculate that this is because the above step causes the aqueous PHB copolymer suspension to contain peptidoglycan derived from bacterial cells, thereby preventing aggregation of PHB copolymer particles.

Furthermore, the present inventors further investigated the PHB copolymer (PHB copolymer powder (intermediate)) obtained above from the perspective of increasing its purity, and as a result discovered for the first time that by washing the PHB copolymer (PHB copolymer powder (intermediate)) with a specific enzyme, a highly pure PHB copolymer can be obtained in a state of high bulk density (here, "high purity" means that the amount of nitrogen compounds, which are impurities, is low).

Therefore, this production method can produce a PHB copolymer (e.g., PHB copolymer powder) having a high bulk density and a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units. This production method can also produce a PHB copolymer (e.g., PHB copolymer powder) having a high bulk density and a specific composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units, with low levels of nitrogen compounds as impurities. Furthermore, this production method can produce the PHB copolymer without using a physical crushing step (e.g., high-pressure crushing), which is advantageous from the perspective of scale-up.

Furthermore, the above-mentioned configuration can reduce the amount of plastic waste generated, thereby contributing to the achievement of Sustainable Development Goals (SDGs), such as Goal 12 "Ensure sustainable consumption and production patterns" and Goal 14 "Conserve and sustainably use the oceans and marine resources for sustainable development." The configuration of this manufacturing method is described in detail below.

2. Method for producing PHB copolymer
This production method includes the following steps (a) to (d) as essential steps: The PHB copolymer in this production method has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol).
Step (a): adding an alkaline protease to a culture solution containing bacterial cells containing a PHB copolymer to enzymatically treat the bacterial cells; Step (b): adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment; Step (c): preparing an aqueous suspension from the aqueous suspension obtained in step (b), the aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less; Step (d): spray-drying the aqueous suspension prepared in step (c). In step (c) of this production method, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less is prepared from the aqueous suspension obtained in step (b). In this aqueous suspension, the PHB copolymer is present in a dispersed state in the aqueous medium. In this specification, an aqueous suspension containing at least a PHB copolymer may be abbreviated as "PHB copolymer aqueous suspension."

<Step (a)>
Step (a) is a step of adding an alkaline protease to a culture solution containing bacterial cells containing a PHB copolymer to enzymatically treat the bacterial cells.

(PHB copolymer)
The PHB copolymer in this production method is a copolymer of 3HB and a hydroxyalkanoate other than 3HB.

Examples of hydroxyalkanoates other than 3HB include 3-hydroxyhexanoate (3HH), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxyoctanoate (3HO), 3-hydroxyoctadecanoate (3HOD), and 3-hydroxydecanoate (3HD).

A preferred example of a PHB copolymer is PHBH, but this is not limiting. For ease of explanation, the following description will primarily focus on PHBH as a representative example.

By changing the composition ratio of the repeating units of 3HB and 3HH, the melting point and degree of crystallinity of PHBH can be changed, and as a result, physical properties such as Young's modulus and heat resistance can be changed, making it possible to give it physical properties between those of polypropylene and polyethylene.

The PHB copolymer produced by this manufacturing method has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol), preferably 81/19 to 87/13 (mol/mol), and more preferably 82/18 to 86/14 (mol/mol). When the composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units is 88/12 (mol/mol) or less, sufficient hardness is obtained, and when it is 80/20 (mol/mol) or more, sufficient flexibility is obtained.

In one embodiment of the present invention, the weight-average molecular weight (hereinafter sometimes referred to as "Mw") of the PHB copolymer is not particularly limited, but is preferably 150,000 to 800,000, more preferably 200,000 to 700,000, and even more preferably 250,000 to 600,000. A weight-average molecular weight of 150,000 or more provides sufficient mechanical properties, while a weight-average molecular weight of 800,000 or less provides a sufficient crystallization rate and achieves good moldability. The weight-average molecular weight of the P3HB resin can be determined as the polystyrene-equivalent molecular weight by gel permeation chromatography (GPC) (Shodex GPC-101 manufactured by Showa Denko) using a polystyrene gel (Shodex K-804 manufactured by Showa Denko) as the column and chloroform as the mobile phase.

(fungal body)
The bacterial cells used in step (a) are not particularly limited as long as they are microorganisms capable of producing PHB copolymers intracellularly. For example, microorganisms isolated from nature and deposited in a depository institution for strains (e.g., IFO, ATCC, etc.), or mutants and transformants prepared from them, can be used. For example, the first bacterial cell to produce P3HB, an example of a PHB copolymer, was Bacillus megaterium, discovered in 1925. Other examples include naturally occurring microorganisms such as Cupriavidus necator (formerly classified as Alcaligenes eutrophus and Ralstonia eutropha) and Alcaligenes latus. It is known that PHB copolymers accumulate intracellularly in these microorganisms.

Fungi that produce copolymers of hydroxybutyrate and other hydroxyalkanoates, which are an example of PHB copolymers, include Aeromonas caviae, which produces P3HB3HV and P3HB3HH, and Alcaligenes eutrophus, which produces P3HB4HB. In particular, with regard to P3HB3HH, Alcaligenes eutrophus AC32 (FERM BP-6038) (T. Fukui, Y. Doi, J. Bateriol., 179, pp. 4821-4830 (1997)), into which genes encoding PHB copolymer synthases have been introduced, is more preferred. In addition to the above, the bacterial cells may be genetically modified microorganisms into which various PHB copolymer synthesis-related genes have been introduced according to the PHB copolymer to be produced.

PHB copolymers can also be produced, for example, by the method described in International Publication No. 2010/013483.

(alkaline protease)
As used herein, the term "alkaline protease" refers to a protease that has the activity of decomposing proteins in an alkaline environment (for example, in a solution of pH 8.5).

In one embodiment of the present invention, the alkaline protease is not particularly limited as long as it has the activity to degrade proteins in an alkaline environment, and examples include serine-specific proteases (e.g., subtilisin, chymotrypsin), cysteine-specific proteases (e.g., papain, bromelain), etc. From the standpoint of versatility and economy, serine-specific proteases, particularly alcalase, which includes subtilisin, are preferred. These may be used alone or in combination of two or more.

Commercially available alkaline protease enzymes can be used, such as "Alcalase" and "Esperase" manufactured by Novozyme; "Protin SD-AY10" and "Protease P 'Amano' 3SD" manufactured by Amano Enzyme Inc.; "Multifect PR6L" and "Optimase PR89L" manufactured by Danisco Japan Co., Ltd.; "Sumiteam MP" manufactured by Shin-Nihon Chemical Industry Co., Ltd.; "Delvolase" manufactured by DSM Japan Co., Ltd.; "Bioprase OP," "Bioprase SP-20FG," and "Bioprase SP-4FG" manufactured by Nagase ChemteX Corporation; "Orientase 22BF" manufactured by HBI Corporation; and "Aroase XA-10" manufactured by Yakult Pharmaceutical Co., Ltd.

When enzymatically treating the bacterial cells with an alkaline protease in step (a), it is preferable to adjust the pH and temperature of the culture solution to match the optimal pH and temperature of the alkaline protease used. Furthermore, it is preferable that the pH in step (a) be lower than the pH adjusted by adding an alkaline aqueous solution in step (b). There are no particular limitations on the method for adjusting the pH and temperature of the culture solution, and known methods can be used.

In one embodiment of the present invention, the optimal pH of the alkaline protease is not particularly limited as long as the alkaline protease is active in an alkaline environment, but is, for example, 8.0 to 12.0, preferably 8.0 to 11.0, more preferably 8.0 to 10.0, even more preferably 8.0 to 9.0, and most preferably 8.5.

In one embodiment of the present invention, the optimum temperature for the alkaline protease is not particularly limited, but is preferably 70°C or lower, and more preferably 60°C or lower, from the viewpoint of not requiring excessive heating and being able to prevent thermal changes (thermal decomposition) of the PHB copolymer. The lower limit of the optimum temperature is not particularly limited, but is preferably room temperature (e.g., 25°C) or higher, from the viewpoint of not requiring excessive cooling and being economical.

In one embodiment of the present invention, the amount of alkaline protease added is not particularly limited, but is, for example, 0.05 to 1.0 phr, preferably 0.1 to 0.5 phr, and more preferably 0.15 to 0.3 phr. If the amount of alkaline protease added is within the above range, it can adequately decompose the bacterial cells.

In step (a), it is preferable to add substantially no lytic enzyme simultaneously with the alkaline protease. As used herein, "lytic enzyme" refers to an enzyme that has the activity of degrading (lysing) bacterial cell walls (e.g., peptidoglycan). Furthermore, as used herein, "substantially no lytic enzyme is added" refers to adding lytic enzymes at 0.0005 phr or less, which may be 0 phr. The lytic enzyme is not particularly limited as long as it falls within the above definition, but examples include lysozyme, rabia, β-N-acetylglucosaminidase, endolysin, and autolysin. By not substantially adding a lytic enzyme in step (a), peptidoglycan remains in the PHB copolymer aqueous suspension, thereby achieving the effects of the present invention.

In step (a), the PHB copolymer-containing bacterial cells are preferably inactivated. The inactivation method is not particularly limited, but an example is a method in which a culture solution containing the PHB copolymer-containing bacterial cells is heated and stirred at 60-70°C for 7 hours, as described in the Examples. After heating and stirring, the culture solution is preferably further cooled to a temperature suitable for step (a).

<Step (b)>
Step (b) is a step of adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment.

Step (b) includes the following steps (b1) and (b2).
Step (b1): Adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0. Step (b2): Adding a surfactant (step (b1)).
As described above, step (b1) is a step of adding an alkaline aqueous solution to the culture solution obtained in step (a) to adjust the pH to 10.0 to 12.0. This step disperses and dissolves bacterial cell-derived impurities (nucleic acids, proteins, etc.), thereby enabling separation of a highly pure PHB copolymer from the bacterial cells.

In one embodiment of the present invention, the alkaline aqueous solution is an aqueous solution containing a basic compound. Examples of basic compounds contained in the alkaline aqueous solution include, but are not limited to, alkali metal or alkaline earth metal hydroxides such as sodium hydroxide and potassium hydroxide; metal carbonates such as sodium carbonate and potassium carbonate; and metal phosphates or metal hydrogen phosphates such as sodium phosphate, potassium phosphate, sodium hydrogen phosphate, and potassium hydrogen phosphate.

In one embodiment of the present invention, the basic compound contained in the alkaline aqueous solution is preferably an alkali metal hydroxide or an alkaline earth metal hydroxide, and more preferably sodium hydroxide. One basic compound may be used alone, or two or more basic compounds may be used in combination.

In step (b1), the pH is preferably adjusted to 10.0 to 12.0 by adding an alkaline aqueous solution, more preferably to 10.2 to 11.8, and even more preferably to 10.4 to 11.6. Adjusting the pH to 10.0 or higher has the advantage of enabling the decomposition and dissolution of bacterial components. Furthermore, adjusting the pH to 12.0 or lower can prevent unintended damage to the bacterial cells.

The temperature in step (b1) is preferably less than 100°C, and more preferably less than 80°C. There is no particular lower limit, but it is preferably, for example, 40°C or higher.

(Step (b2))
Step (b2) is a step of adding a surfactant to the culture solution obtained in step (a). This step can efficiently treat cell membranes in particular and remove a larger amount of impurities derived from the bacterial cells, allowing a more highly pure PHB copolymer to be separated from the bacterial cells.

In one embodiment of the present invention, the surfactant is not particularly limited, but examples include anionic surfactants, cationic surfactants, amphoteric surfactants, and nonionic surfactants. Of these, anionic surfactants are preferred from the standpoint of their high ability to remove cell membranes. These surfactants may be used alone or in combination of two or more.

Examples of anionic surfactants include alkyl sulfates, alkylbenzene sulfonates, alkyl sulfate ester salts, alkenyl sulfate ester salts, alkyl ether sulfate ester salts, alkenyl ether sulfate ester salts, α-olefin sulfonates, α-sulfofatty acid salts, esters of α-sulfofatty acid salts, alkyl ether carboxylates, alkenyl ether carboxylates, amino acid surfactants, and N-acylamino acid surfactants. Among these, alkyl sulfate ester salts are preferred, and sodium dodecyl sulfate (SDS) is particularly preferred due to its high cell membrane removal ability and low cost. These surfactants may be used alone or in combination of two or more.

In step (b2), the amount of surfactant added is not particularly limited and is, for example, 0.1 to 5.0% by weight, preferably 0.3 to 2.5% by weight, relative to the culture medium.

Step (b2) may be performed before, simultaneously with, or after step (b1). Preferably, step (b2) is performed after step (b1).

<Step (b')>
In one embodiment of the present invention, the production method may further comprise step (b').

Step (b') is a step of centrifuging the culture medium obtained in step (b) and removing the supernatant to obtain an aqueous suspension of concentrated PHB copolymer. In other words, this is a step of removing impurities from the PHB copolymer separated from the bacterial cells, concentrating, and purifying the PHB copolymer.

In step (b'), the method for centrifuging the culture medium is not particularly limited, and any known method can be used.

In step (b'), it is preferable to centrifuge the culture medium, remove the supernatant, add a solution to the precipitate, and then repeat the steps of centrifuging again and removing the supernatant. This procedure allows for the production of a more concentrated and purified PHB copolymer aqueous suspension. Here, it is preferable that the solution added after removing the supernatant is an alkaline aqueous solution adjusted to the same pH as the culture medium.

Since the amount of impurities remaining in the final product is largely determined by step (b'), it is preferable to reduce these impurities as much as possible. Naturally, depending on the application, impurities may be present as long as they do not impair the physical properties of the final product. However, when a highly pure PHB copolymer is required, such as for medical applications, it is preferable to reduce the amount of impurities as much as possible. An example of an indicator of the degree of purity is the amount of protein in the aqueous PHB copolymer suspension. The amount of protein in the aqueous PHB copolymer suspension is not particularly limited, as long as it achieves the residual protein amount in the PHB copolymer powder described below. The amount of protein is preferably 10,000 ppm or less, more preferably 5,000 ppm or less, and even more preferably 3,000 ppm or less, per weight of PHB copolymer in the aqueous PHB copolymer suspension.

In step (b'), the solvent ("solvent" is also referred to as "aqueous medium") constituting the PHB copolymer aqueous suspension is not particularly limited and may be water or a mixed solvent of water and an organic solvent. Furthermore, in the mixed solvent, the concentration of the organic solvent is not particularly limited as long as it is equal to or lower than the solubility of the organic solvent used in water. Furthermore, the organic solvent is not particularly limited, and examples thereof include alcohols such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, pentanol, hexanol, and heptanol; ketones such as acetone and methyl ethyl ketone; ethers such as tetrahydrofuran and dioxane; nitriles such as acetonitrile and propionitrile; amides such as dimethylformamide and acetamide; dimethyl sulfoxide, pyridine, and piperidine. Among these, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, isobutanol, acetone, methyl ethyl ketone, tetrahydrofuran, dioxane, acetonitrile, propionitrile, etc. are preferred because they are easily removed. Furthermore, methanol, ethanol, 1-propanol, 2-propanol, butanol, acetone, etc. are more preferred because they are easily available. Furthermore, methanol, ethanol, and acetone are particularly preferred.

The water content in the aqueous medium constituting the PHB copolymer aqueous suspension is preferably 5% by weight or more, more preferably 10% by weight or more, even more preferably 30% by weight or more, and particularly preferably 50% by weight or more.

The aqueous suspension of PHB copolymer in step (b') may contain other solvents, components derived from bacterial cells, compounds generated during purification, etc., as long as this does not impair the essence of the present invention.

<Step (c)>
In step (c) of this production method, an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa·s or more and 0.5 Pa·s or less is prepared from the aqueous suspension obtained in step (b).

Before being subjected to step (c) of the present production method, the PHB copolymer aqueous suspension typically has a pH greater than 7.0 due to step (b). Therefore, in step (c) of the present production method, the pH of the PHB copolymer aqueous suspension is adjusted to 7.0 or less. The adjustment method is not particularly limited, and examples include adding an acid. The acid is not particularly limited, and may be either an organic acid or an inorganic acid, regardless of whether it is volatile. More specifically, examples of acids that can be used include sulfuric acid, hydrochloric acid, phosphoric acid, and acetic acid.

The upper limit of the pH of the PHB copolymer aqueous suspension adjusted in the above adjustment step is 7.0 or less, preferably 5.0 or less, and more preferably 4.0 or less, from the viewpoint of reducing discoloration when the PHB copolymer is heated and melted, and ensuring molecular weight stability during heating and/or drying. The lower limit of the pH is preferably 1 or more, more preferably 2.0 or more, and even more preferably 3.0 or more, from the viewpoint of acid resistance of the container. By adjusting the pH of the PHB copolymer aqueous suspension to 7.0 or less, discoloration during heating and melting is reduced, and a PHB copolymer with suppressed molecular weight reduction during heating and/or drying is obtained.

The concentration of the PHB copolymer in the aqueous suspension prepared in step (c) of this production method is preferably 30% by weight or more, more preferably 40% by weight or more, and even more preferably 50% by weight or more. A PHB copolymer concentration of 30% by weight or more is economically advantageous in terms of drying utility, improves productivity, and increases the bulk density of the powder obtained in the subsequent step (d), thereby improving transportability. Furthermore, the upper limit of the PHB copolymer concentration is preferably 65% by weight or less, more preferably 60% by weight or less, because close packing may occur and sufficient fluidity may not be ensured. The method for adjusting the PHB copolymer concentration is not particularly limited, and examples include adding an aqueous medium and removing a portion of the aqueous medium (e.g., by centrifuging and then removing the supernatant). The PHB copolymer concentration may be adjusted in either step (c) or step (b).

In one embodiment of the present invention, the concentration of PHB copolymer in the aqueous suspension prepared by step (c) is preferably 30 to 65% by weight.

In step (c), the shear viscosity of the PHB copolymer aqueous suspension is 0.005 to 0.5 Pa·s, preferably 0.007 to 0.4 Pa·s, and more preferably 0.009 to 0.3 Pa·s, from the viewpoint of ease of feeding into a spray dryer. The shear viscosity of the PHB copolymer aqueous suspension refers to the shear viscosity (Pa·s) at 20°C when a shear rate of 10 (1/s) is applied. The shear viscosity of the PHB copolymer aqueous suspension is measured by the method described in the Examples.

<Dispersant>
The aqueous suspension in one embodiment of the present invention may contain a dispersant. That is, the step (c) in one embodiment of the present invention may be a step of preparing an aqueous suspension further containing a dispersant in addition to the PHB copolymer. In one embodiment of the present invention, when the dispersant is used, it is preferable to add the dispersant to the PHB copolymer aqueous suspension before adjusting the pH to 7.0 or less. By including a dispersant in the PHB copolymer aqueous suspension in one embodiment of the present invention, the productivity and thermal stability of the PHB copolymer can be suitably improved.

Dispersants are not particularly limited, but examples include alkylene oxide-based dispersants, cellulose-based dispersants, polyvinyl alcohol (PVA), and sorbitan alkylate-based dispersants. By using these dispersants, PHB copolymers with large particle sizes can be obtained at lower hot air temperatures. The dispersant may be one type or two or more types.

In one embodiment of the present invention, the dispersant is at least one selected from the group consisting of alkylene oxide-based dispersants, cellulose-based dispersants, and polyvinyl alcohol.

In one embodiment of the present invention, the dispersant is preferably an alkylene oxide-based dispersant, from the perspective of effectively preventing aggregation of the PHB copolymer when adjusting the pH of the PHB copolymer aqueous suspension to 7.0 or less and effectively suppressing adhesion to the extruder shaft during powder processing. Furthermore, using an alkylene oxide-based dispersant in combination with a cellulose-based dispersant is even more preferable, as it further suppresses aggregation of the PHB copolymer and is also expected to act as a binder that makes the powder less likely to break after drying.

In one embodiment of the present invention, the alkylene oxide dispersant is not particularly limited as long as it achieves the above-mentioned effects, but it is preferable that it is composed of poly(ethylene oxide) (PEO) blocks and poly(propylene oxide) (PPO) blocks, and is in the form of PEO-PPO-PEO.

In this specification, "poly(ethylene oxide) (PEO) block" refers to the polymer portion formed by polymerization of ethylene oxide (EO) in the structure of an alkylene oxide-based dispersant.

In this specification, "poly(propylene oxide) (PPO) block" refers to the polymer portion formed by polymerization of propylene oxide (PO) in the structure of an alkylene oxide-based dispersant.

In one embodiment of the present invention, by setting the PEO molecular weight and PEO molecular weight/PPO molecular weight in the alkylene oxide-based dispersant to a specific range, the viscosity of the aqueous suspension can be kept low, and PHB copolymer (e.g., PHB copolymer powder) can be produced with high productivity.

In one embodiment of the present invention, the PEO molecular weight and PEO molecular weight/PPO molecular weight range in the alkylene oxide-based dispersant are preferably the following combinations:

In this specification, "PEO molecular weight" may be referred to as "EO amount" and "PPO molecular weight" may be referred to as "PO amount."

That is, in one embodiment of the present invention, the molecular weight of PEO in the alkylene oxide-based dispersant may be 1,500 or more, preferably 1,750 or more, and more preferably 2,000 or more. Furthermore, in one embodiment of the present invention, the upper limit of the molecular weight of PEO in the alkylene oxide-based dispersant is, for example, 30,000 or less, preferably 25,000 or less, and more preferably 20,000 or less.

In one embodiment of the present invention, the PEO molecular weight/PPO molecular weight ratio in the alkylene oxide dispersant is preferably 0.5 or greater, more preferably 0.6 or greater, and even more preferably 0.7 or greater. The upper limit of the PEO molecular weight/PPO molecular weight ratio is preferably 5.0 or less, more preferably 4.8 or less, and even more preferably 4.5 or less.

In one embodiment of the present invention, if the PEO molecular weight and PEO molecular weight/PPO molecular weight in the alkylene oxide-based dispersant are within the above ranges, the alkylene oxide-based dispersant will be hydrophilic and the number of molecules relative to the added weight of the alkylene oxide-based dispersant will be large, making it easier to maintain the dispersibility of the aqueous suspension.

In one embodiment of the present invention, the alkylene oxide dispersant has a PEO molecular weight of 1,500 or more and a PEO molecular weight/PPO molecular weight ratio of 0.5 to 5.0.

The alkylene oxide dispersant used in step (c) of this manufacturing method is not particularly limited, and commercially available products can be used. Examples of commercially available products include Pluronic® 10400 (manufactured by BASF), Pluronic® 10500 (manufactured by BASF), Genapol® PF80 (manufactured by Clariant), Unilube® 70DP-600B (manufactured by NOF Corporation), Unilube® 70DP-950B (manufactured by NOF Corporation), Pronon® 208 (manufactured by NOF Corporation), Epan® U105 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.), Epan® U108 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.), and Epan® 750 (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd.).

The amount of dispersant added to the aqueous suspension in step (c) of this production method is not particularly limited, but is preferably 0.1 to 20 parts by weight, more preferably 0.5 to 10 parts by weight, and even more preferably 0.75 to 5 parts by weight, per 100 parts by weight of the total amount of PHB copolymer contained in the aqueous suspension. Setting the amount of dispersant added within the above range further improves the dispersion stability of the PHB copolymer in the PHB copolymer aqueous suspension, allowing for efficient spray drying, which tends to result in more favorable improvements in the productivity and thermal stability of the PHB copolymer.

In one embodiment of the present invention, the cellulose-based dispersant is not particularly limited as long as it achieves the effects of the present invention. Examples include methyl cellulose (MC), ethyl cellulose, propyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC), carboxyethyl cellulose, carboxypropyl cellulose, carboxymethyl hydroxyethyl cellulose, acetyl cellulose, cyanoethyl cellulose, sodium cellulose sulfate, etc. Of these, methyl cellulose and hydroxypropyl methyl cellulose are preferred from the perspective of the wide range of substitution degrees over which they become water-soluble. Cellulose-based dispersants may be used alone or in combination.

The cellulose-based dispersant used in step (c) of this manufacturing method is not particularly limited, and commercially available products can be used. Examples of commercially available cellulose-based dispersants include MCE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-400 (manufactured by Shin-Etsu Chemical Co., Ltd.), MCE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SFE-4000 (manufactured by Shin-Etsu Chemical Co., Ltd.), SE-50 (manufactured by Shin-Etsu Chemical Co., Ltd.), and NE-100 (manufactured by Shin-Etsu Chemical Co., Ltd.).

The amount of cellulose-based dispersant added to the PHB copolymer aqueous suspension in step (c) of this production method is not particularly limited, but is preferably 0.01 to 10 parts by weight, more preferably 0.05 to 5 parts by weight, and even more preferably 0.08 to 3 parts by weight, per 100 parts by weight of the PHB copolymer contained in the aqueous suspension. By adding the cellulose-based dispersant in the amount within the above range, the effects of the present invention can be achieved.

In one embodiment of the present invention, it is preferable that the dispersant be a biodegradable substance from an environmental perspective.

The volume median diameter of the PHB copolymer in the aqueous suspension of PHB copolymer obtained by step (c) of this production method (hereinafter sometimes simply referred to as the "volume median diameter of the PHB copolymer") is preferably 30 times or less, more preferably 20 times or less, and even more preferably 10 times or less, the volume median diameter of the primary particles of the PHB copolymer (hereinafter referred to as the "primary particle diameter"). When the volume median diameter of the PHB copolymer is 30 times or less the primary particle diameter, the aqueous suspension of the PHB copolymer exhibits superior fluidity, which allows the subsequent step (d) to be carried out with high efficiency and tends to further improve the productivity of the PHB copolymer. The volume median diameter of the PHB copolymer is measured, for example, using a HORIBA LA-950 laser diffraction/scattering particle size distribution analyzer.

The volume median diameter of the PHB copolymer can be used as an indicator of the dispersion state of the PHB copolymer in the aqueous PHB copolymer suspension. The method for adjusting the volume median diameter of the PHB copolymer is not particularly limited, and known means (e.g., stirring) can be applied. For example, a PHB copolymer aqueous suspension whose dispersion state has been disrupted due to exposure to acidic conditions can be subjected to physical, chemical, or biological treatments that would be considered by a person skilled in the art, thereby restoring the PHB copolymer in the aqueous PHB copolymer suspension to a dispersed state (e.g., a state having the above-mentioned PHB copolymer volume median diameter).

<Step (d)>
In step (d) of this production method, the PHB copolymer aqueous suspension prepared in step (c) is spray-dried. Examples of spray-drying methods include a method in which the PHB copolymer aqueous suspension is supplied as fine droplets into a dryer and dried while contacting hot air in the dryer. The method (atomizer) for supplying the PHB copolymer aqueous suspension in the form of fine droplets into the dryer is not particularly limited, and known methods such as a method using a rotating disk or a method using a nozzle are included. The method for contacting the droplets with hot air in the dryer is not particularly limited, and includes a co-current method, a counter-current method, and a combination of these.

The drying temperature during spray drying in step (d) may be any temperature that can remove most of the aqueous medium from the droplets of the PHB copolymer aqueous suspension, and can be appropriately set under conditions that allow drying to the desired moisture content and minimize the occurrence of quality deterioration (reduction in molecular weight, color tone, etc.), melting, etc. For example, the temperature of the hot air blown into the spray dryer can be selected appropriately from the range of 100 to 300°C. The volume of hot air blown into the dryer can also be appropriately set depending on, for example, the size of the dryer.

The present production method may include, after step (d), a step of further drying the obtained PHB copolymer (e.g., PHB copolymer powder) (e.g., a step of subjecting it to drying under reduced pressure). The present production method may also include other steps (e.g., a step of adding various additives to the PHB copolymer aqueous suspension).

This production method may further include the following steps (e) and (f) after step (d). These steps can reduce the nitrogen content derived from bacterial cell residues contained in the PHB copolymer, thereby producing a highly pure PHB copolymer.

<Step (e)>
Step (e) is a step of washing the PHB copolymer powder obtained in step (d) above. Step (e) preferably includes the following steps (e1) and (e2):
Step (e1): A step of dispersing the PHB copolymer powder obtained in the step (e) in pure water to a predetermined concentration to prepare a dispersion slurry, adding an alkaline aqueous solution to adjust the pH of the dispersion slurry, and stirring the slurry together with a washing liquid. Step (e2): A step of adding an alkaline aqueous solution to the dispersion slurry obtained in the step (e1), centrifuging the mixture, and removing the supernatant.

(Step (e1))
As described above, step (e1) is a step of preparing a dispersion slurry by dispersing the PHB copolymer powder obtained in step (d) in pure water to a predetermined concentration, adding an alkaline aqueous solution to adjust the pH of the dispersion slurry, and stirring the slurry together with a cleaning liquid. During stirring, the liquid may be heated.

The predetermined concentration of the dispersed slurry in step (e1) is not particularly limited, but is preferably 20 to 40%, more preferably 30 to 40%, from the viewpoint of minimizing the size of the dispersion tank and ensuring that the slurry has fluidity.

In step (e1), the alkaline aqueous solution is not particularly limited, but the alkaline aqueous solution described in step (b1) can be used. The pH of the dispersion slurry is preferably adjusted to, for example, 5.0 to 14.0.

The stirring time for washing in step (e1) is not particularly limited, but may be several hours. From the viewpoint of thoroughly washing the PHB copolymer, the stirring time may be 1 to 12 hours, or may be 2 to 10 hours.

The dispersed slurry in step (e1) may further contain sodium sulfate.

The washing solution in step (e1) may contain an alkaline protease and/or a lytic enzyme. The type of alkaline protease is not particularly limited, but may be one of those described in the "Alkaline Protease" section of this specification. The alkaline protease used in this step may be the same as or different from the alkaline protease used in step (a). The type of lytic enzyme is not particularly limited, but may be one of the lytic enzymes described in the explanation of step (a).

(Step (e2))
As described above, the step (e2) is a step of adding an alkaline aqueous solution to the dispersed slurry obtained in the step (e1), separating the PHB copolymer powder, and removing the supernatant.

In step (e2), the alkaline aqueous solution is not particularly limited, but may be the same as or different from the alkaline aqueous solution used in step (e1).

In step (e2), the method for separating the PHB copolymer powder is not particularly limited, and known methods can be used. For example, separation by centrifugation, filter dehydration, or hydrocyclone can be used.

In step (e2), it is preferable to repeatedly add an alkaline aqueous solution to the dispersed slurry, centrifuge the slurry, and remove the supernatant. This procedure allows for the production of a more concentrated and purified PHB copolymer.

<Step (f)>
Step (f) is a step of dehydrating and/or drying the polyhydroxyalkanoic acid powder obtained in step (e). For dehydration and/or drying, for example, a spray dryer, a fluidized bed dryer, a drum dryer, etc. are used, but a spray dryer is preferably used because of its simple operation. The spray drying method and drying temperature may be, for example, the method and temperature described in step (d).

[3. PHB copolymer powder]
(Intermediate)
A PHB copolymer powder according to one embodiment of the present invention (hereinafter referred to as "the first PHB copolymer powder") comprises a PHB copolymer, peptidoglycan, and a dispersant, wherein the PHB copolymer has a composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units of 80/20 to 88/12 (mol/mol), a bulk density of 0.45 g/mL or more, and a median particle size of 80 to 200 μm. The first PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units and has a high bulk density, making it extremely useful in a variety of fields.

In this embodiment, the terms "PHB copolymer," "peptidoglycan," and "dispersant" are defined above.

The first PHB copolymer powder contains a PHB copolymer. The content of the PHB copolymer in the first PHB copolymer powder is not particularly limited, but is, for example, 90 to 99% by weight, preferably 93 to 98% by weight, and more preferably 95 to 97% by weight. Setting the PHB copolymer content within this range has the advantage of not losing the physical properties of PHBH, such as fluidity.

The first PHB copolymer powder contains peptidoglycan. The content of peptidoglycan in the first PHB copolymer powder is not particularly limited, but is preferably 0.1 to 1.5 parts by weight (phr), and more preferably 0.3 to 1.3 parts by weight (phr), per 100 parts by weight of the PHB copolymer constituting the PPHB copolymer powder. By setting the peptidoglycan content within the above range, the bulk density of the PHB copolymer powder tends to be improved. The peptidoglycan content in the first PHB copolymer powder is measured by the method described in the Examples.

The first PHB copolymer powder contains a dispersant. The dispersant is preferably an alkylene oxide-based dispersant. The content of the dispersant in the PHB copolymer powder is not particularly limited, but is preferably 0.1 to 20 parts by weight, more preferably 0.5 to 10 parts by weight, and even more preferably 0.75 to 5 parts by weight, per 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. By adding the dispersant in the above range, the productivity of the PHB copolymer powder tends to improve.

From the viewpoint of improving powder transportability, the bulk density of the first PHB copolymer powder is 0.45 g/mL or more, preferably 0.48 g/mL or more, and more preferably 0.50 g/mL or more. While there are no particular upper limits, from the viewpoint of powder transportability, an example of the upper limit is 0.55 g/mL. The bulk density of the first PHB copolymer powder is measured by the method described in the examples.

From the viewpoint of achieving excellent fluidity, the median particle size of the first PHB copolymer powder is 80 to 200 μm, preferably 100 to 180 μm, and more preferably 105 to 160 μm. The median particle size of the first PHB copolymer powder is measured by the method described in the Examples.

The purity of the first PHB copolymer powder is not particularly limited to an upper limit, but is preferably less than 98%. The lower limit is 85% or more, preferably 90% or more, and more preferably 92% or more, from the viewpoint of not impairing the physical properties of the PHB copolymer. The purity of the PHB copolymer powder refers to the content (wt%) of the PHB copolymer in the PHB copolymer powder. The purity of the first PHB copolymer powder is measured by the method described in the Examples.

In one embodiment of the present invention, the residual protein content of the first PHB copolymer powder is preferably 0.1 to 1.0 phr, more preferably 0.12 to 0.5 phr, and even more preferably 0.15 to 0.3 phr, from the viewpoint of coloration. The residual protein content refers to the amount of protein remaining in the PHB copolymer powder and is expressed as the amount per 100 parts by weight of PHB copolymer in the PHB copolymer powder. The residual protein content of the first PHB copolymer powder is measured by the method described in the Examples.

Furthermore, this first PHB copolymer powder may contain various components that were generated or not removed during the manufacturing process, as long as the effects of the present invention are achieved.

In one embodiment of the present invention, the first PHB copolymer powder is an intermediate for the final product described below, and is produced by the above-described production method (e.g., steps (a) to (d)).

(Final product)
A PHB copolymer powder according to one embodiment of the present invention (hereinafter referred to as "the second PHB copolymer powder") comprises a PHB copolymer and a nitrogen compound, wherein the PHB copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol), a bulk density of 0.45 g/mL or more, and a median particle size of 80 to 200 μm. The second PHB copolymer powder is a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3-hydroxybutyrate units, and has a high bulk density and high purity, making it extremely useful in a variety of fields.

As used herein, "nitrogen compounds" refers to compounds that contain at least nitrogen. Nitrogen compounds are decomposition products of proteins and peptidoglycan, which are components of cell walls, remaining in the PHB copolymer powder.

In this embodiment, the terms "PHB copolymer" and "nitrogen compound" are defined above. Furthermore, the terms "bulk density" and "median particle size" are defined above in the (intermediate) section.

The second PHB copolymer powder contains a nitrogen compound. The total nitrogen content in the second PHB copolymer powder is not particularly limited, but is preferably 0.010 to 0.075 parts by weight (phr), and more preferably 0.01 to 0.06 parts by weight (phr), per 100 parts by weight of the PHB copolymer constituting the PHB copolymer powder. By keeping the total nitrogen content within the above range, the purity of the PHB copolymer powder tends to be improved. The total nitrogen content in the second PHB copolymer powder is measured by the method described in the Examples.

Furthermore, this second PHB copolymer powder may contain various components that were generated or not removed during the manufacturing process, as long as the effects of the present invention are achieved.

In one embodiment of the present invention, the second PHB copolymer powder is the final product and is produced by the above-described production method (e.g., steps (a) to (f)).

This PHB copolymer powder can be used for a variety of purposes, including paper, films, sheets, tubes, plates, rods, containers (e.g., bottle containers), bags, and parts.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

That is, one embodiment of the present invention is as follows.
<1> A method for producing a polyhydroxybutyric acid copolymer,
The polyhydroxybutyric acid copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol),
(a) adding an alkaline protease to a culture solution containing the bacterial cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the bacterial cells;
(b) adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment;
(c) preparing an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less from the aqueous suspension obtained in the step (b); and (d) spray-drying the aqueous suspension prepared in the step (c).
A method for producing a polyhydroxybutyric acid copolymer, comprising:
<2> After the step (d),
The method for producing a polyhydroxybutyric acid copolymer according to <1>, further comprising: (e) a step of washing the polyhydroxybutyric acid copolymer powder obtained in the step (d); and (f) a step of dehydrating and/or drying the polyhydroxybutyric acid copolymer powder obtained in the step (e).
<3> The method for producing a polyhydroxybutyric acid copolymer according to <1> or <2>, wherein the hydroxyalkanoate units other than 3-hydroxybutyrate units are 3-hydroxyhexanoate units.
<4> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <3>, wherein the surfactant in the step (b) is sodium dodecyl sulfate.
<5> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <4>, wherein the aqueous suspension further contains a dispersant.
<6> The method for producing a polyhydroxybutyric acid copolymer according to <5>, wherein the dispersant is at least one selected from the group consisting of alkylene oxide-based dispersants, cellulose-based dispersants, and polyvinyl alcohol.
<7> The method for producing a polyhydroxybutyric acid copolymer according to any one of <1> to <6>, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in the step (c) is 30 to 65% by weight.
<8> The method for producing a polyhydroxybutyric acid copolymer according to <2>, wherein the washing liquid in the step (e) contains an alkaline protease and/or a lytic enzyme.
<9> A composition comprising a polyhydroxybutyric acid copolymer, a peptidoglycan, and a dispersant,
The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol),
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g/mL or more and a median particle size of 80 to 200 μm.
<10> The polyhydroxybutyric acid copolymer powder according to <9>, wherein the peptidoglycan content is 0.1 to 1.5 phr.
<11> The polyhydroxybutyric acid copolymer powder according to <8> or <9>, wherein the residual protein content is 0.1 to 1.0 phr.
<12> A composition comprising a polyhydroxybutyric acid copolymer and a nitrogen compound,
The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol),
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g/mL or more and a median particle size of 80 to 200 μm.
<13> The polyhydroxybutyric acid copolymer powder according to <12>, wherein the total nitrogen content in the polyhydroxybutyric acid copolymer powder is 0.010 to 0.075 phr.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. In the examples, "PHBH" is used as "PHB copolymer," and "PHB copolymer" can be read as "PHBH."

[Measurement and evaluation methods]
Measurements and evaluations in the examples and comparative examples were carried out by the following methods.

(Volume median diameter)
The volume median diameter in the PHB copolymer aqueous suspension was measured using a laser diffraction/scattering particle size distribution measuring device LA-950 manufactured by HORIBA.

(Shear viscosity of PHB copolymer aqueous suspension)
The shear viscosity of the PHB copolymer aqueous suspension was measured by the following method. Specifically, the shear viscosity was measured in a coaxial double cylinder using an MCR302 manufactured by Anton Paar. The PHB copolymer aqueous suspension was charged into a 20 mL cylinder and cooled at a shear rate of 10 (1/s) until the liquid temperature reached 15°C. The liquid temperature was then raised to 20°C, and after reaching the target liquid temperature, the viscosity was measured when the change in torque with time became less than 1%.

(Amount of peptidoglycan)
The amount of peptidoglycan remaining in the PHB copolymer powder was measured using the following method. Specifically, first, 50 μL of peptidoglycan standard was placed in a microplate. Next, 50 μL of reagent from the SLP-HS Single Reagent Set (Wako LAL System) was placed in the microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the absorbance 3 hours after the start of measurement was defined as the final absorbance. The time until the absorbance reached 0.4 times the final absorbance was measured. Similarly, the peptidoglycan standard was diluted 1000-10,000 times with distilled water, and the same procedure as above was repeated to create a calibration curve. Then, the PHB copolymer aqueous suspension prepared in step (c) was diluted 1000-10,000 times with distilled water, and 50 μL of the diluted solution was placed in a microplate. Next, 50 μL of reagent from the SLP-HS Single Reagent Set (Wako LAL System) was placed in the microplate. The absorbance was measured every 15 seconds at a wavelength of 650 nm, and the absorbance 3 hours after the start of measurement was defined as the final absorbance. The amount of peptidoglycan was calculated from the time it took for the absorbance to reach 0.4 times the final absorbance.

(Residual protein amount)
The residual protein content of the PHB copolymer powder was measured using a BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific). Specifically, 10 mg of PHBH powder was placed in a 14 mL Falcon tube, 2 mL of the above reagent was added, and the tube was shaken at 60°C for 30 minutes. After 30 minutes, the tube was cooled, and the absorbance at a wavelength of 562 nm was measured.

(Bulk density)
The bulk density of the PHB copolymer powder was measured using a bulk density measuring device (manufactured by Kuramochi Scientific Instruments) in accordance with JIS K 7365:1999.

(median particle size)
The median particle size of the PHB copolymer powder was measured by the following method. Specifically, the median particle size was measured using a laser diffraction/scattering particle size distribution analyzer LA-950 (HORIBA). 0.05 g of sodium dodecyl sulfate as a surfactant was added to 20 mL of ion-exchanged water to obtain a surfactant aqueous solution. Next, 0.2 g of the PHB copolymer powder to be measured was added to the surfactant aqueous solution, and the PHB copolymer powder was dispersed in the surfactant aqueous solution to obtain a dispersion for measurement. The prepared dispersion was introduced into the laser diffraction/scattering particle size distribution analyzer, and measurement was performed.

(purity)
The purity of the PHB copolymer powder was measured by the following method. Specifically, using a TG-DTA (2000SE, manufactured by Nietzsch), approximately 10 mg of PHBH dry powder (weight: W) was heated at a rate of 10°C/min from 50°C to 500°C under a nitrogen atmosphere, and a graph of temperature vs. sample weight was created. The weights at point A, where a linear line representing the weight loss at 280-285°C intersects with a linear line representing the weight loss at 400-450°C, and point B, where a linear line representing the weight loss at 280-285°C intersects with a linear line representing the weight loss at 150-200°C, were calculated from the graph. The difference in weight between point A and point B was used to calculate the amount of PHBH, W', and W'/W was used to represent the purity of PHBH.

(Total nitrogen measurement)
The total nitrogen content of the PHB copolymer powder was measured using a trace total nitrogen analyzer TN-2100H (Nitto Seiko Analytech Co., Ltd.).

Example 1
(Preparation of bacterial cell culture solution)
Ralstonia eutropha, as described in International Publication No. WO 2019/142717, was cultured using the method described in paragraphs [0041] to [0048] of the same document to obtain a fungal cell culture solution containing fungal cells containing a PHB copolymer. Ralstonia eutropha is currently classified as Capriavidus necator. The composition ratio of the repeating units of the PHB copolymer (composition ratio of 3HB units to hydroxyalkanoate units other than 3HB units) was 80/20 to 88/12 (mol/mol).

(inactivation)
The bacterial cell culture solution obtained above was heated and stirred at an internal temperature of 60 to 70°C for 7 hours, sterilized, and then cooled to 55°C to obtain an inactivated culture solution.

(Enzyme treatment)
The pH of the inactivated culture solution was adjusted to 8.5±0.2 using 30% sodium hydroxide, followed by the addition of 0.2 phr of Alcalase (Novozymes), a protease, and the mixture was kept at 50°C for 2 hours or more while controlling the pH at 8.5 with 30% sodium hydroxide.

(lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above to adjust the pH to 11.5 or higher, and the solution was maintained at 45°C or higher for 4 hours or more. Subsequently, 0.6 to 1.0 wt% sodium dodecyl sulfate (Kao Corporation) was added, and 30% sodium hydroxide was added to adjust the pH to approximately 12. The solution was then diluted 2-fold with an alkaline aqueous solution (pH 10 to 11.5). After centrifugation, the supernatant was removed and the solution was concentrated 2-fold. To this concentrated aqueous suspension of PHB copolymer, an equal amount of aqueous sodium hydroxide solution (pH 11) to the removed supernatant was added, followed by centrifugation and removal of the supernatant. The procedure of adding the aqueous sodium hydroxide solution, centrifugation, and removing the supernatant was repeated 7 times. The aqueous PHB copolymer suspension was concentrated 4-fold to adjust the PHB copolymer concentration to 52 wt% or higher. The volume median diameter of this aqueous PHB copolymer suspension was measured using a HORIBA LA-950 laser diffraction/scattering particle size distribution analyzer and found to be 1.9 μm.

(granulation)
To the PHB copolymer aqueous suspension obtained above, 0.95 phr (0.95 parts by weight per 100 parts by weight of the PHB copolymer present in the aqueous suspension) of an ethylene oxide/propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added. Next, 0.3 phr of methylcellulose (trade name SM-15) was added. The resulting solution was stirred, the temperature was raised to 60°C, 10% sulfuric acid was added to adjust the pH to 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. The solids concentration of the mixture was then adjusted to 50% by mass by adding water. The volume median diameter of this PHB copolymer aqueous suspension was measured using a HORIBA laser diffraction/scattering particle size distribution analyzer LA-950 and found to be 1.9 μm. Furthermore, the shear viscosity of the PHB copolymer aqueous suspension was measured using an MCR302 manufactured by Anton Paar and was found to be 0.1 Pa s. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a pre-washed PHB copolymer powder. The amount of peptidoglycan contained in the resulting pre-washed PHB copolymer powder was 1.04 phr, the amount of residual protein was 0.18 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g/mL, the median particle size was 110 μm, the purity was 97%, and the total nitrogen content was 0.099 phr.

(Washing 1)
The unwashed PHB copolymer powder obtained above was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 1 phr of sodium sulfate was added to the dispersion slurry. The pH of the dispersion slurry was then adjusted to 6 with 30% sodium hydroxide. The dispersion slurry was heated to 50°C, and 0.0050 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.) was added, followed by stirring for 2 hours.

(Washing 2)
30% sodium hydroxide was added to the dispersion slurry obtained above to adjust the pH of the dispersion slurry to 11. Thereafter, an aqueous sodium hydroxide solution having a pH of 11 was added to the dispersion slurry in an amount equal to the weight of the dispersion slurry, followed by centrifugation and removal of the supernatant. The procedure of adding the aqueous sodium hydroxide solution, centrifugation, and removal of the supernatant was repeated three times.

(Drying)
10% sulfuric acid was added to the dispersion slurry obtained above, and the pH of the dispersion slurry was adjusted to 4. Water was then added so that the PHB copolymer concentration in the dispersion slurry was 35%. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 160°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a PHB copolymer powder. The total nitrogen content of the resulting PHB copolymer powder was 0.059 phr, and the bulk density of the PHB copolymer powder before washing was 0.50 g/mL and the median particle size was 102 μm.

Example 2
A pre-washed PHB copolymer powder was obtained in the same manner as in Example 1 up to (granulation). The pre-washed PHB copolymer powder was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 1 phr of sodium sulfate was added to the resulting dispersion slurry. The pH of the dispersion slurry was then adjusted to 10.5 with 30% sodium hydroxide. 0.05 phr of Esperase (Novozyme) was added and the mixture was stirred for 2 hours. The operations from (Washing 2) onward described in Example 1 were then carried out, and the total nitrogen content of the resulting PHB copolymer powder was 0.070 phr, the bulk density of the PHB copolymer powder was 0.50 g/mL, and the median particle size was 108 μm.

Example 3
A dispersion slurry of the PHB copolymer powder before washing was obtained in the same manner as in Example 1 up to (Washing 1). 30% sodium hydroxide was added to the obtained dispersion slurry to adjust the pH of the dispersion slurry to 10.5. 0.05 phr of Esperase (Novozyme) was added and the mixture was stirred for 2 hours. The operations from (Washing 2) onward described in Example 1 were then carried out. The total nitrogen content of the obtained PHB copolymer powder was 0.033 phr, the bulk density of the PHB copolymer powder was 0.50 g/mL, and the median particle size was 115 μm.

Example 4
A pre-washed PHB copolymer powder was obtained in the same manner as in Example 1 up to (granulation). The pre-washed PHB copolymer powder was dispersed in pure water to a PHB copolymer concentration of 37.5%, and 30% sodium hydroxide was added to adjust the pH to 11, followed by stirring for 2 hours. The operations from (Washing 2) onward described in Example 1 were then carried out, and the total nitrogen content of the obtained PHB copolymer powder was 0.098 phr, the bulk density of the pre-washed PHB copolymer powder was 0.51 g/mL, and the median particle size was 110 μm.

Comparative Example 1
An inactivated culture solution was obtained in the same manner as in Example 1 up to (inactivation).

(Enzyme treatment)
To the inactivated culture medium obtained above, 0.01 phr of egg white lysozyme (egg white lysozyme FG, manufactured by Nagase & Co., Ltd.), a peptidoglycan degrading enzyme, was added, and the mixture was kept at 50°C for 2 hours. Next, the pH was adjusted to 8.5±0.2 using 30% sodium hydroxide. Thereafter, Alcalase (manufactured by Novozymes), a protease, was added, and the mixture was kept at 50°C for 2 hours or more while controlling the pH to 8.5 with 30% sodium hydroxide.

(lysis, concentration)
An alkaline aqueous solution was added to the enzyme-treated solution obtained above to adjust the pH to 11.5 or higher, and the solution was maintained at 45°C or higher for 4 hours or more. Subsequently, 0.6 to 1.0 wt% sodium dodecyl sulfate (Kao Corporation) was added, and 30% sodium hydroxide was added to adjust the pH to approximately 12. The solution was then diluted 3-fold with an alkaline aqueous solution (pH 10 to 11.5). After centrifugation, the supernatant was removed and the solution was concentrated 2-fold. To this concentrated aqueous suspension of PHB copolymer, an equal amount of aqueous sodium hydroxide solution (pH 11) was added, followed by centrifugation and removal of the supernatant. The addition of aqueous sodium hydroxide solution, centrifugation, and supernatant removal were repeated. The volume median diameter of this aqueous PHB copolymer suspension was measured using a HORIBA LA-950 laser diffraction/scattering particle size distribution analyzer, and found to be 62 μm. Thereafter, the aqueous suspension of PHB copolymer was concentrated to a solids concentration of 50% by filtration using a Buchner funnel, but it was in the form of a wet powder with no fluidity and was in a state where it could not be spray-dried.

Comparative Example 2
An aqueous suspension of PHB copolymer was obtained in the same manner as in Comparative Example 1 up to the centrifugation step (lysing and concentration). The volume median diameter of this aqueous suspension of PHB copolymer was measured using a laser diffraction/scattering particle size distribution analyzer LA-950 manufactured by HORIBA, and was found to be 62 μm.

(granulation)
To the PHB copolymer aqueous suspension (solids concentration 20%) obtained above, 0.95 phr of an ethylene oxide/propylene oxide copolymer nonionic dispersant (polyethylene oxide molecular weight 8000, polypropylene oxide molecular weight 2000, trade name Pronon 208) was added. Next, 0.3 phr of methylcellulose (trade name SM-15) was added. The resulting solution was stirred, the liquid temperature was raised to 60°C, 10% sulfuric acid was added to adjust the pH to 4.0, and stirring was continued for 120 minutes to obtain a PHB copolymer aqueous suspension. Thereafter, the solids concentration of the mixture was adjusted to 15% by mass by adding water. The volume median diameter of this PHB copolymer aqueous suspension was measured using a HORIBA laser diffraction/scattering particle size distribution analyzer LA-950 and found to be 62 μm. Furthermore, the shear viscosity of the PHB copolymer aqueous suspension was measured using an Anton Paar MCR302 and found to be 0.1 Pa s. The resulting PHB copolymer aqueous suspension was spray-dried using a rotary atomizer-type spray dryer (OC-16, manufactured by Okawara Co., Ltd.) (hot air temperature: 115°C, exhaust air temperature: 75°C, rotary atomizer rotation speed: 11,000 rpm) to obtain a PHB copolymer powder. The resulting PHB copolymer powder contained 0.01 phr of peptidoglycan, 0.5 phr of residual protein, a bulk density of 0.20 g/mL, a median particle size of 71 μm, and a purity of 98%.

〔result〕
From Table 1, it is clear that the present production method makes it possible to produce a PHB copolymer having a high bulk density and a high ratio of hydroxyalkanoate units other than 3HB units.

According to the present invention, it is possible to produce a PHB copolymer having a high composition ratio of hydroxyalkanoate units other than 3HB units. Furthermore, because the PHB copolymer obtained by the production method of the present invention has a high bulk density, it can be suitably used in agriculture, fisheries, forestry, horticulture, medicine, hygiene products, clothing, non-clothing products, packaging, automobiles, building materials, and other fields.

Claims (8)

1. A method for producing a polyhydroxybutyric acid copolymer powder , comprising:
the polyhydroxybutyrate copolymer contained in the polyhydroxybutyrate copolymer powder has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol);
(a) adding an alkaline protease to a culture solution containing the bacterial cells containing the polyhydroxybutyric acid copolymer to enzymatically treat the bacterial cells;
(b) adding an alkaline aqueous solution to the culture solution obtained in the step (a) to adjust the pH to 10.0 to 12.0, and adding a surfactant either before, simultaneously with, or after the adjustment;
(c) preparing an aqueous suspension having a pH of 7.0 or less and a shear viscosity of 0.005 Pa s or more and 0.5 Pa s or less from the aqueous suspension obtained in the step (b); and (d) spray-drying the aqueous suspension prepared in the step (c).
Including,
After the step (d),
(e) a step of washing the polyhydroxybutyric acid copolymer powder obtained in the step (d);
further comprising
A method for producing a polyhydroxybutyric acid copolymer powder , wherein the washing liquid in the step (e) contains an alkaline protease and/or a bacteriolytic enzyme . After the step ( e ) ,
2. The method for producing a polyhydroxybutyric acid copolymer powder according to claim 1, further comprising the step of: (f) dehydrating and/or drying the polyhydroxybutyric acid copolymer powder obtained in the step (e). 3. The method for producing polyhydroxybutyric acid copolymer powder according to claim 1, wherein the hydroxyalkanoate units other than 3-hydroxybutyrate units are 3-hydroxyhexanoate units. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 3, wherein the surfactant in step (b) is sodium dodecyl sulfate. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 4, wherein the aqueous suspension further contains a dispersant. 6. The method for producing polyhydroxybutyric acid copolymer powder according to claim 5, wherein the dispersant is at least one selected from the group consisting of alkylene oxide-based dispersants, cellulose-based dispersants, and polyvinyl alcohol. The method for producing a polyhydroxybutyric acid copolymer powder according to any one of claims 1 to 6, wherein the concentration of the polyhydroxybutyric acid copolymer in the aqueous suspension prepared in step (c) is 30 to 65% by weight. A polyhydroxybutyric acid copolymer and a nitrogen compound are included,
The polyhydroxybutyrate copolymer has a composition ratio of 3-hydroxybutyrate units to hydroxyalkanoate units other than 3-hydroxybutyrate units of 80/20 to 88/12 (mol/mol),
A polyhydroxybutyric acid copolymer powder having a bulk density of 0.45 g/mL or more and a median particle size of 80 to 200 μm ,
The polyhydroxybutyric acid copolymer powder has a total nitrogen content of 0.010 to 0.075 phr .