Fine particles containing polyhydroxyalkanoic acid (PHA) and method for producing the same

Abstract

The present invention provides: microparticles each of which contains 3-hydroxybutyric acid (3-HB) as a repeating unit for a polyhydroxyalkanoic acid (PHA) that is a biodegradable polymer and has a particle diameter of 0.2 μm or more and less than 10 μm; and a method for producing the microparticles. The microparticles according to the present invention are biodegradable, have excellent processability and also have biocompatibility, and therefore can be used in wide varieties of use applications including medicinal use applications.

Description

Technical Field

[0001] The present invention relates to fine particles containing polyhydroxyalkanoic acid (PHA), which contain 3-hydroxybutanoic acid (3-HB) units as repeating units of polyhydroxyalkanoic acid (PHA) and have a particle diameter of 0.2 to less than 10 μm, and a method for producing the same.

Background Art

[0002] Fine particles made of synthetic resins have become indispensable materials in many industrial fields, because they have various applications, including use as modifiers such as plastic resin modifiers and cosmetic modifiers, use as additives such as paint additives, toner additives and cosmetic additives, use as fillers such as liquid crystal spacers, chromatographic packing materials and adhesive tape fillers, as well as various applications in the medical field such as drug delivery systems (DDS) and test particles for medical diagnosis. However, in addition to the problems that the price of petroleum, which is the raw material, fluctuates and the raw material supply is not stable, fine particles made of synthetic resins also produce greenhouse gases as a negative impact on the environment. After being used during or after the production process, they enter rivers, oceans and the like directly or through wastewater treatment plants, causing various problems. Thus, solutions to these problems on a global scale are urgently needed.

[0003] In particular, microplastic pollution in the ocean has become a problem in recent years. For example, when plastic waste is crushed by waves and ultraviolet rays, it becomes microplastics with a length of 5 millimeters or less, and such microplastics accumulate in the bodies of fish. It is predicted that the total weight of plastic waste in the ocean will exceed the total weight of fish in 50 years, and reducing plastic waste has become an urgent issue for humanity. In particular, since fine particles made of synthetic resins are themselves microplastics, their use is already subject to significant restrictions. Therefore, in industrial fields where the use of fine particles made of synthetic resins is indispensable, there is an urgent need to replace conventional synthetic resin raw materials for fine particles (Non-Patent Document 1).

[0004] Furthermore, as mentioned above, microparticles made of synthetic resins are highly anticipated in the medical field for application in drug delivery systems (DDS), etc. However, in the medical field, in addition to the challenges associated with microparticles made of synthetic resins, it is also necessary to satisfy conditions such as biocompatibility in order to enable safe use in vivo (Non-Patent Literature 2).

[0005] One proposed solution to these challenges is to use biodegradable biopolymers as raw materials for microparticles. While the development of microparticles using biopolymers such as polylactic acid (PLA), polyhydroxyalkanoic acid (PHA), and cellulose has already been proposed (Patent Documents 1 and 2), it has been pointed out that only those using polyhydroxyalkanoic acid (PHA) and cellulose as raw materials can be considered "biodegradable" in actual river and marine environments, rather than in high-temperature and high-humidity environments such as compost.

[0006] On the other hand, regarding the production of microparticles using polyhydroxyalkanoic acid (PHA) as a raw material for biodegradable biopolymers, development has mainly focused on the use of poly-3-hydroxybutanoic acid (3-PHB). Patent Document 3 describes microparticles that are injectable by syringe and consist of a biocompatible and biodegradable polymer, using a copolymer of 3-hydroxybutanoic acid and 4-hydroxybutanoic acid (poly(4-hydroxybutyrate-co-3-hydroxybutyrate)) as the polymer. Furthermore, Patent Document 4 describes a cosmetic composition in the form of microparticles containing polyhydroxyalkanoate (PHA), with poly-3-hydroxybutyrate (PHB), poly-3-hydroxyhexanoate (PHH), and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) given as examples of PHA. Furthermore, Patent Document 5 lists porous resin particles containing a polyhydroxyalkanoate, with poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), which is a copolymer of 3-hydroxybutyrate units and 3-hydroxyhexanoate units, as a preferred example of the polyhydroxyalkanoate. In addition, Patent Document 6 describes a nonwoven fabric containing a polyhydroxyalkanoate, more preferably poly-4-hydroxybutyrate and its copolymer, which is a nonwoven fabric produced by a dry spinning process and comprises fine fibers having a specific average diameter and bursting strength. However, poly-3-hydroxybutanoic acid has problems with its physical properties such as brittleness and hardness, and the production and purification costs are high. As a result, the practical application of fine particles with desired physical properties using poly-3-hydroxybutanoic acid as the main raw material has not yet been achieved. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Patent No. 5133478 [Patent Document 2] U.S. Patent Application Publication No. 2006 / 0177513 [Patent Document 3] U.S. Patent No. 10463619 [Patent Document 4] International Publication No. 2018 / 178899 [Patent Document 5] International Publication No. 2017 / 056908 [Patent Document 6] Special Publication No. 2013-534978 [Non-patent literature]

[0008] [Non-Patent Document 1] Report on Chemical Substance Safety Measures for FY2016 (Survey on Domestic Emissions of Microplastics) February 2017 JFE Techno Research Corporation FY2016 Commissioned Survey Report by the Ministry of Economy, Trade and Industry [Non-Patent Document 2] Microparticles, Microspheres, and Microcapsules for Advanced Drug Delivery, Sci. Pharm. 2019, 87,20 [Overview of the project] [Problems that the invention aims to solve]

[0009] Therefore, in order to provide fine particles that can be used in a wide range of applications, including medical applications, there was a need for suitable biodegradable polymers to produce fine particles that have the physical properties required for each application, in particular, important physical properties such as appropriate particle size distribution and retention of other substances. At the same time, there was a need to establish a simpler method for producing fine particles with physical properties that can be used in a wide range of applications from these biodegradable polymers. [Means for solving the problem]

[0010] As a result of their investigations to solve the above problems, the present inventors found that polyhydroxyalkanoic acid (PHA) with excellent melt-fluid properties can be produced by a method comprising the steps of preparing a microorganism that produces polyhydroxyalkanoic acid (PHA), growing the microorganism in a culture medium, having an animal ingest the grown microorganism, and recovering the polyhydroxyalkanoic acid (PHA) from the animal's excrement. Based on this finding, they discovered that fine particles containing 3-hydroxybutanoic acid (3-HB) as the repeating unit of such polyhydroxyalkanoic acid (PHA) and having a particle size of 0.2 to less than 10 μm have the desired physical properties, and thus completed the present invention (for the above findings, see Japanese Patent Application No. 2019-086889: Filing date April 26, 2019). In other words, we have confirmed that fine particles containing polyhydroxyalkanoic acid (PHA), which include 3-hydroxybutanoic acid (3-HB) units as repeating units, and having a particle size of 0.2 to less than 10 μm, possess the desired physical properties, and have thus completed the present invention. The present invention is defined by the following specific features.

[0011] (1) Fine particles containing polyhydroxyalkanoic acid (PHA), characterized in that the polyhydroxyalkanoic acid (PHA) contains 3-hydroxybutanoic acid (3-HB) as a repeating unit, and the particle size is 0.2 to less than 10 μm. (2) Fine particles as described in (1), wherein the particle size is 7 μm or less. (3) The fine particles according to (1) or (2), wherein polyhydroxyalkanoic acid (PHA) further comprises 3-hydroxyhexanoic acid (3-HH) as a repeating unit. (4) The fine particles according to (3), wherein the proportion of 3-hydroxyhexanoic acid (3-HH) is 27% (by weight) or less of the total weight of the repeating units of polyhydroxyalkanoic acid (PHA). (5) The fine particles according to (3) or (4), wherein the polyhydroxyalkanoic acid (PHA) comprises a copolymer of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH). The fine particles according to any one of (1) to (5), wherein (6) the polyhydroxyalkanoic acid (PHA) further comprises a 4-hydroxybutanoic acid (4-HB) unit as a repeating unit thereof. The fine particles according to (6), wherein (7) the proportion of 4-hydroxybutanoic acid (4-HB) is 40 to 50% by weight relative to the total weight of the repeating units of the polyhydroxyalkanoic acid (PHA). The fine particles according to (6) or (7), wherein (8) the polyhydroxyalkanoic acid (PHA) comprises a copolymer of 3-hydroxybutanoic acid (3-HB) and 4-hydroxybutanoic acid (4-HB). The fine particles according to any one of (1) to (8), wherein (9) the average molecular weight (Mw) of the polyhydroxyalkanoic acid (PHA) is 100,000 to 1,300,000. The fine particles according to any one of (1) to (9), wherein (10) the melting point of the polyhydroxyalkanoic acid (PHA) is 55°C or higher and 170°C or lower. The fine particles according to any one of (1) to (10), wherein (11) the fine particles comprise a resin other than polyhydroxyalkanoic acid (PHA). The fine particles according to (11), wherein (12) the resin other than polyhydroxyalkanoic acid (PHA) is a biodegradable resin. The fine particles according to any one of (1) to (12), wherein (13) the fine particles are spherical. The fine particles according to any one of (1) to (13), wherein (14) the fine particles are porous. The fine particles according to any one of (1) to (14), wherein (15) the fine particles retain another substance on the surface and / or inside thereof. The fine particles according to any one of (1) to (15), wherein (16) the 10% compressive strength of the fine particles is 0.23 to 2.20 MPa. The fine particles according to any one of (1) to (16), wherein (17) the fine particles are dispersible in an aqueous solvent. (18) A method for producing the fine particles according to any one of (1) to (17), comprising the following steps. Step 1: a step of preparing a microorganism that produces polyhydroxyalkanoic acid (PHA), Step 2: a step of growing the microorganism of Step 1 in a medium, Step 3: a step of allowing an animal to ingest the proliferated microorganisms, Step 4: a step of recovering and purifying polyhydroxyalkanoate (PHA) from excreta of the animal of step 3, and Step 5: a step of micronizing the polyhydroxyalkanoate (PHA) obtained in step 4. (19) The method for producing fine particles according to (18), wherein step 5 is a step of micronizing a resin composition containing the polyhydroxyalkanoate (PHA) obtained in step 4. Effects of the Invention

[0012] According to the present invention, fine particles containing polyhydroxyalkanoate (PHA) include 3-hydroxybutanoic acid (3-HB) as a repeating unit of polyhydroxyalkanoate (PHA), whereby the fine particles containing polyhydroxyalkanoate (PHA) are excellent in biodegradability in natural environments and processability. Further, according to the present invention, when the particle diameter of the fine particles containing polyhydroxyalkanoate (PHA) is 0.2 or more and less than 10 µm, the fine particles can have physical properties such as melting point, particle diameter, porosity, compressive strength, and substance retention properties that enable use in a wide range of applications. Further, according to the present invention, a simple method for producing such fine particles containing polyhydroxyalkanoate (PHA) is provided. Therefore, according to the present invention, it is possible to provide fine particles containing polyhydroxyalkanoate (PHA) that is biodegradable, has excellent processability, has biocompatibility, and has physical properties usable in a wide range of applications. Since the fine particles according to the present invention can be provided as fine particles excellent in biodegradability under natural environments, they can contribute to solving problems such as marine pollution and microplastic pollution. Further, since biodegradation can be performed at the time of disposal, the effect of reducing incineration treatment and lowering the environmental load can also be expected. Furthermore, in addition to the biocompatibility and in vivo degradability inherent to polyhydroxyalkanoate (PHA), the fine particles according to the present invention can have physical properties usable in a wide range of applications, and thus are highly likely to be widely usable in medical applications in addition to conventional applications. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 is a conceptual diagram showing the basic configuration of an electrospray deposition apparatus. [Figure 2] Figure 2 shows SEM images of the fine particles produced from P(3-HB) in Example 1. [Figure 3] Figure 3 shows SEM images of porous microparticles produced from P(3-HB) in Example 2. [Figure 4] Figure 4 shows SEM images of the fine particles produced from P(3-HB-co-3-HH) in Example 3. [Figure 5] Figure 5 shows SEM images of the fine particles produced from P(3-HB-co-4-HB) in Example 4. [Figure 6] Figure 6 is an SEM image showing that the fine particles produced from P(3-HB) in Example 5 retained silica particles on their surface. [Figure 7] Figure 7 is an SEM image showing that the fine particles produced from P(3-HB) in Example 5 retained silica particles internally. [Figure 8] Figure 8 is a SEM BSE observation image showing that the fine particles produced from P(3-HB) in Example 5 retained silica particles. [Modes for carrying out the invention]

[0014] Next, specific embodiments will be described, including the best mode for carrying out the present invention.

[0015] [Polyhydroxyalkanoic acid (PHA)] Polyhydroxyalkanoic acid (PHA) is a polyester of hydroxyalkanoic acid, as exemplified by the following chemical formula (1), and is a biodegradable polymer.

[0016] [ka] (Chemical formula (1), R represents an alkyl group.)

[0017] The 3-hydroxyalkanoic acid unit (3-HA) and the 4-hydroxyalkanoic acid unit (4-HA) are described below as chemical formula (2) and chemical formula (3) respectively, as repeating units of polyhydroxyalkanoic acid (PHA) according to the present invention.

[0018] [ka] (Chemical formula (2), R represents an alkyl group.)

[0019] [ka] (Chemical formula (3))

[0020] The 3-hydroxyalkanoic acid unit (3-HA) can have a methyl group, ethyl group, propyl group, butyl group, pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, etc. as the alkyl group (R). However, the repeating unit of polyhydroxyalkanoic acid (PHA) according to the present invention is characterized by containing 3-hydroxybutanoic acid (3-HB: chemical formula (4) below), in which the alkyl group described below is a methyl group. Furthermore, in another embodiment of the present invention, the polyhydroxyalkanoic acid (PHA) is characterized by containing 3-hydroxyhexanoic acid (3-HH: chemical formula (5) below), in which the alkyl group is a propyl group, as a repeating unit. When the polyhydroxyalkanoic acid (PHA) according to the present invention contains 3-hydroxybutanoic acid units (3-HB) and 3-hydroxyheptanoic acid units (3-HH) as repeating units, it is preferable that it is included as a copolymer of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH) (P(3-HB-co-3-HH)) as exemplified below.

[0021] [ka]

[0022] Another embodiment of the polyhydroxyalkanoic acid (PHA) according to the present invention is such that the proportion of 3-hydroxyhexanoic acid (3-HH) in the repeating units of the polyhydroxyalkanoic acid (PHA) according to the present invention is 30% or less (by weight) of the total amount of repeating units of the polyhydroxyalkanoic acid (PHA), preferably 27% or less. When the proportion of 3-hydroxyhexanoic acid (3-HH) is 27% or less of the total amount of repeating units of the polyhydroxyalkanoic acid, it is possible to produce porous fine particles, but if it exceeds 27%, it may become difficult to produce porous fine particles.

[0023] Furthermore, another embodiment of the polyhydroxyalkanoic acid (PHA) according to the present invention is characterized in that the repeating unit of the polyhydroxyalkanoic acid (PHA) according to the present invention includes 4-hydroxybutanoic acid (4-HB) as described in the above chemical formula (3). When the polyhydroxyalkanoic acid (PHA) according to the present invention includes 4-hydroxybutanoic acid units (4-HB) as its repeating unit, it is preferable that it is included as a copolymer of 3-hydroxybutanoic acid (3-HB) and 4-hydroxybutanoic acid (4-HB) (P(3-HB-co-4-HB)), as exemplified below.

[0024] [ka]

[0025] The proportion of 4-hydroxybutanoic acid (4-HB) in the repeating units of polyhydroxyalkanoic acid (PHA) according to the present invention is 40-50% (by weight) of the total amount of repeating units of polyhydroxyalkanoic acid (PHA), preferably 40-45%, and more preferably 40-42%. When 4-hydroxybutanoic acid (4-HB) is included within the above proportion range relative to the total amount of repeating units of polyhydroxyalkanoic acid (PHA), good biocompatibility and biodegradability can be expected, and it becomes possible to manufacture fine particles that can be widely used in medical applications. However, if the proportion is included outside the above proportion range, good biocompatibility and biodegradability may not be expected, and it may become difficult to manufacture fine particles that can be widely used in medical applications.

[0026] Another embodiment of the polyhydroxyalkanoic acid (PHA) according to the present invention is one in which the weight-average molecular weight of the polyhydroxyalkanoic acid (PHA) is 1.0 × 10⁶. 5 ~13.0×10 5 The concentration is g / mol, preferably 3.0 × 10⁻⁶. 5 ~10.0×10 5 The concentration is g / mol, and more preferably 3.0 × 10⁻⁶. 5 ~8.0×10 5 The value is g / mol. When the weight-average molecular weight of polyhydroxyalkanoic acid (PHA) is within the above range, it is possible to provide polyhydroxyalkanoic acid (PHA) in which solubility in solvents, hardness / softness when formed into fine particles, heat resistance, and durability can be controlled. However, if it deviates from the above range, these effects may not be obtained.

[0027] Another embodiment of the polyhydroxyalkanoic acid (PHA) according to the present invention is one in which the melting point of the polyhydroxyalkanoic acid (PHA) is 55°C to 170°C, preferably 60°C to 160°C, and more preferably 80°C to 120°C. By setting the melting point of the polyhydroxyalkanoic acid (PHA) within the above range, it is possible to provide a polyhydroxyalkanoic acid (PHA) that can be used to produce fine particles suitable for various application conditions. The melting point of the polyhydroxyalkanoic acid (PHA) can be measured by any method, but for example, it can be measured by DSC analysis.

[0028] [Method for producing polyhydroxyalkanoic acid (PHA)] The method for producing polyhydroxyalkanoic acid (PHA) according to the present invention is not particularly limited, as long as it yields polyhydroxyalkanoic acid (PHA) possessing the characteristics of polyhydroxyalkanoic acid according to the present invention.

[0029] For example, one embodiment of the method for producing polyhydroxyalkanoic acid (PHA) according to the present invention may include the following steps. Step 1: A step to prepare microorganisms that produce polyhydroxyalkanoic acid (PHA). Step 2: A step in which the microorganisms from Step 1 are grown in a culture medium. Step 3: The process of having animals ingest the proliferated microorganisms, and Step 4: A process to recover and purify polyhydroxyalkanoates (PHAs) from the animal excrement produced in Step 3.

[0030] The polyhydroxyalkanoic acid (PHA) according to the present invention is preferably produced using microorganisms. Examples of microorganisms that have the ability to produce polyhydroxyalkanoic acid include Bacillus megaterium, Cupriavidus necator, Ralstonia eutropha, and Alcaligenes latus. Among these, Cupriavidus necator is particularly preferred.

[0031] Preferably, the microorganisms used are those in which genes involved in the synthesis of polyhydroxyalkanoic acid (PHA) are deleted or introduced. For example, it is preferable to use microorganisms in which the acetoacetyl-CoA reductase gene is deleted. It is also preferable to introduce the hydroxyalkanoic acid synthase gene or the enoyl-CoA hydratase gene. This makes it possible to increase the content of 3-hydroxyhexanoic acid units (3-HB) contained in the polyhydroxyalkanoic acid. Furthermore, it is possible to produce copolymer P (3HB-co-3HH) consisting of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH) which has high melt fluidity and excellent processability.

[0032] The culture medium used for culturing microorganisms is not particularly limited as long as it allows the microorganisms to grow. For example, the medium may contain alcohols such as methanol, ethanol, and butanol as a carbon source; fatty acids such as saturated and unsaturated fatty acids such as acetic acid, propionic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, oleic acid, palmitic acid, linoleic acid, linolenic acid, and myristic acid; sugars such as glucose and fructose; organic acids such as lactic acid; and oils and fats containing a large amount of saturated and unsaturated fatty acids with 10 or more carbon atoms. Examples of oils and fats include vegetable oils such as coconut oil, palm kernel oil, palm oil, palm olein, rapeseed oil, soybean oil, rice oil, and sesame oil; animal oils such as lard and beef tallow; and fish oil. Unrefined oils and waste cooking oils can also be used. Palm kernel oil or coconut oil containing lauric acid is preferred as an oil and fat to be added to the culture medium as a carbon source. By including palm kernel oil or coconut oil, the polyhydroxyalkanoic acid (PHA) content can be increased.

[0033] For the production of polyhydroxyalkanoic acid (PHA) according to the present invention, aerobic conditions are preferred for culturing the microorganisms. If necessary, a nitrogen source or inorganic substances may be added. Examples of nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium phosphate, and other ammonium salts. Examples of inorganic substances include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, and sodium chloride. The culture temperature is preferably 20°C to 40°C, and more preferably 25°C to 35°C. The culture time is not particularly limited, but is preferably 48 to 72 hours.

[0034] In the method for producing polyhydroxyalkanoic acid (PHA) according to the present invention, the content of 3-hydroxyhexanoic acid (3-HH) in the copolymer of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH) can be controlled by controlling the expression levels of the acetoacetyl-CoA reductase gene and the enoyl-CoA hydratase gene. Furthermore, the content of 3-hydroxyhexanoic acid (3-HH) in the copolymer of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH) can be controlled by controlling the remaining amount of carbon source, adjusting the concentration of inorganic components in the culture medium, and adjusting the amount of oxygen permeation and culture time.

[0035] The method for recovering and purifying polyhydroxyalkanoic acid (PHA) is not particularly limited, but examples include recovering it from a culture medium by centrifugation and extracting it with a solvent, or digesting and absorbing the microorganisms with animals and recovering it as excrement. From the viewpoint of being able to easily concentrate the concentration of polyhydroxyalkanoic acid (PHA), the method of digesting and absorbing microorganisms with animals and recovering it as granular polyhydroxyalkanoic acid (PHA) contained in the excrement is preferred. The animals mentioned above include rodents, goats, sheep, cattle, birds, aquatic organisms, beetles, and insects. Among these, beetle larvae such as mealworms are preferred, and 35-day-old houseflies that have been eaten by insects (larvae of the mite beetle, Tenebrio molitor) are even more preferred. After feeding mealworm larvae or similar organisms with the above-mentioned microorganisms, the fecal pellets are collected, sieved using a mesh, and then washed with water and a base such as sodium hydroxide, followed by drying to recover polyhydroxyalkanoic acid (PHA).

[0036] Specific examples of methods for producing polyhydroxyalkanoic acid (PHA) are given below. (1) Method for producing P(3-HB) Using the Cupriavidus necator H16 strain, pre-culture was performed, followed by a 10g / L solution. The cells were transferred to a 500 mL conical flask containing palm oil, 0.54 g / L urea, and 100 μL of MM composition solution (composition described in (3) below), and cultured with shaking at 200 rpm for 24 hours at 30°C. After culture, the cells were freeze-dried, and approximately 5 g was dissolved in 500 mL of chloroform and stirred at room temperature for 5 days. Cellular residue was separated from the solution by filtration. This solution was concentrated using a rotary evaporator, added dropwise to cold methanol, and stirred for approximately 2 hours until a precipitate formed, thereby purifying polyhydroxyalkanoic acid (PHA) as poly-3-hydroxybutanoic acid P (3-HB) that does not contain 3-hydroxyhexanoic acid (3-HH). The resulting precipitate was vacuum-filtered through a 0.2 μm PTFE filter and dried.

[0037] (2) Method for producing P(3-HB-co-3-HH) (a) Preparation of mineral medium for P(3HB-co-3HH) production The mineral medium for P(3HB-co-3HH) production consisted of 4.0 g / L NaH2PO4, 4.6 g / L Na2HPO4, 0.45 g / L K2SO4, 0.39 g / L MgSO4, 62 mg / L CaCl2, and 1 mL / L trace element solution (the trace element solution contained 15 g / L FeSO4·7H2O, 2.4 g / L MnSO4·H2O, 2.4 g / L ZnSO4·7H2O, and 0.48 g / L CuSO4·5H2O dissolved in 0.1 M HCl). The pH of the medium was adjusted to 7.0 before sterilization by autoclaving. (i) Biosynthesis of P(3HB-co-3HH) using a 13L fermenter P(3HB-co-3HH) biosynthesis is inherited by polyhydroxyalkanoate synthase. The procedure was performed using Capriavidas necatol, into which offspring had been introduced. First, Capriavidus nekator, into which a gene encoding polyhydroxyalkanoate synthase was introduced, was streaked onto an agar plate and incubated at 30°C for 24 hours. Next, as a pre-culture, Capriavidus nekator was inoculated twice into 50 mL of culture medium using a platinum loop, and the mixture was shaken in a 30°C incubator shaker for 8 hours until the OD600 nm of the culture medium reached 4. Approximately 3 mL of the culture medium was inoculated into 100 mL of mineral medium containing 0.54 g / L urea, 0.39 g / L MgSO4, 62 mg / L CaCl2, 1 mL / L trace element solution, and 1% by mass crude palm kernel oil. The crude palm kernel oil was autoclaved before being added to the mineral medium. Furthermore, this mineral medium was incubated for 18 hours and inoculated into a 6 L fermenter. The morphology of the inoculated Capriavidus nekator was checked before transferring it to the fermenter (10% v / v). The culture medium temperature was maintained at 30°C, and the pH of the medium was set to 7.0 ± 0.1 by adding 3M NaOH and 3M H3PO4. Agitation was performed using a Rushton turbine at a stirring speed of 200 to 900 rpm. Air was supplied through a filter cartridge (Sartorius stedim, Germany) at a rate of 1 vvm (air volume / working volume of fermenter / min) to maintain a dissolved oxygen concentration of 40% or higher. MgSO4·7H2O was added at 18 hours after incubation, and urea was added every 6 hours. Trace elements were added at a concentration of 1 mL during inoculation and at 18 hours of incubation. Crude palm kernel oil was supplied at a concentration of 10 g / L to 20 g / L every 6 hours, depending on the oil consumption by the microorganisms. Sampling was performed every 6 hours to determine the residual oil content, wet cell weight, and optical density of the bacterial culture. The culture time ranged from 48 to 72 hours, depending on bacterial growth. (c) Biological recovery of P(3HB-co-3HH) 35-day-old mealworms (larvae of the mite insect, Tenebrio molitor) were reared in plastic containers at ambient temperature (approximately 25°C). 100g of the reared mealworms were fed dried microorganisms containing the above-mentioned P(3HB-co-3HHx). The amount of microorganisms supplied was based on the mealworm's body weight (5% of body weight per day). Before supplying a new batch of microorganisms, mealworm fecal pellets were collected and sieved using meshes of 0.50 mm and 0.25 mm. Double sieving helped remove other impurities and facilitated the subsequent washing process. (e) Purification of P(3HB-co-3HH) using distilled water Approximately 10% (w / v) fecal pellets were added to tap water to a concentration of 100 g / L. The fecal pellet suspension was rinsed several times, and the supernatant was allowed to settle before discarding. After removing the supernatant, the recovered P(3HB-co-3HH) was dried in a 50°C oven until it reached a certain mass. Furthermore, the dried P(3HB-co-3HH) was rinsed in 0.25 M NaOH for 1 hour to allow the mixture to settle, the supernatant was removed, and the recovered pellets were stirred in tap water for another 1 hour until the pH dropped to less than 9.5. Then, the recovered P(3HB-co-3HH) granules were dried in a 50°C oven until a certain mass was reached, and the target P(3HB-co-3HH) was recovered.

[0038] (3) Method for producing P(3HB-co-4HB) Using Cupriavidus necator Re2058 / pHT1phaCCs strain Next, the cells were cultured on NR agar medium (with 50 μg / mL kanamycin added) at 30°C to allow them to grow. The grown cells were collected and transferred to 50 ml of MM medium and cultured at 48°C and 200 rpm for 48 hours. After culturing, the cells were collected by centrifugation at 8000 rpm for 10 minutes and freeze-dried for 2 days. Extraction of P(3HB-co-4HB) was performed using chloroform in the same manner as in (1) above. Composition of MM medium Fructose 10 g / L 4-Sodium hydroxybutanoate 9g / L Sodium dihydrogen phosphate 4.0 g / L Disodium hydrogen phosphate 4.6 g / L Potassium sulfate 0.45 g / L Magnesium sulfate 0.39 g / L Calcium chloride 62 mg / L Urea 0.54g / L TE solution 1.0g / L Composition of TE solution Ferrous sulfate 15g / L Manganese sulfate 2.4g / L Zinc sulfate 2.4g / L Copper sulfate 0.48g / L

[0039] [Resin composition containing polyhydroxyalkanoic acid (PHA)] The polyhydroxyalkanoic acid (PHA) of the present invention can be mixed with other additives to form a resin composition, provided that its physical properties are not impaired. Other additives that can be used include other resins besides the polyhydroxyalkanoic acid (PHA) of the present invention, antioxidants, ultraviolet absorbers, plasticizers, flame retardants, inorganic fillers, crystal nucleating agents, and the like.

[0040] Other resins besides polyhydroxyalkanoic acid (PHA) of the present invention include thermoplastic resins and thermosetting resins, such as polyethylene, polypropylene and other polyolefin resins, polyimide, polyamide, polyphenylene ether, polyether ketone, polyether ketone ketone, polybutadiene, polystyrene, polyester, polylactic acid, phenolic resin, poly(meth)acrylic acid, norbornene resin, etc. Among these, biodegradable resins are preferred.

[0041] [Fine particles containing polyhydroxyalkanoic acid (PHA)] The polyhydroxyalkanoic acid (PHA)-containing fine particles of the present invention can take on various shapes, such as spherical, plate-like, spindle-like, and needle-like, but a spherical shape is preferred. Furthermore, considering its application to medical uses, the particle size of the polyhydroxyalkanoic acid (PHA)-containing fine particles of the present invention is 0.2 to less than 10 μm, preferably 7 μm or less. The method for measuring the particle size of the polyhydroxyalkanoic acid (PHA)-containing fine particles of the present invention is described in detail below, and involved processing of SEM observation images with software (ImageJ) and dynamic light scattering. Furthermore, the fine particles containing polyhydroxyalkanoic acid (PHA) of the present invention can take on a porous form in order to increase their surface area.

[0042] Furthermore, the polyhydroxyalkanoic acid (PHA)-containing microparticles of the present invention can retain other substances not only on the surface of the microparticles but also internally. These other substances are not particularly limited as long as they do not impair the characteristics of the polyhydroxyalkanoic acid-containing microparticles of the present invention, but examples include inorganic powders such as calcium carbonate, aluminum oxide, magnesium oxide, magnesium carbonate, mica, talc, and silica, or organic powders such as magnesium stearate and zinc stearate, as well as solvent-soluble substances.

[0043] Furthermore, the fine particles containing polyhydroxyalkanoic acid (PHA) according to the present invention can be configured to have a 10% compressive strength of 0.23 to 2.20 MPa. The 10% compressive strength of the fine particles according to the present invention can be adjusted, for example, by mixing in a resin such as cellulose.

[0044] Furthermore, the fine particles containing polyhydroxyalkanoic acid (PHA) according to the present invention include a configuration that allows for dispersion in an aqueous solvent. While water is an example of an aqueous solvent, it is not limited to water; a mixed solvent of water and a hydrophilic solvent such as alcohol is also possible. Because the fine particles containing polyhydroxyalkanoic acid (PHA) according to the present invention can be dispersed in an aqueous solvent, it is expected that they will be applicable to a variety of uses.

[0045] [Manufacturing of polyhydroxyalkanoic acid (PHA) microparticles] While various methods such as spray drying and dispersion can be applied to produce fine particles from polyhydroxyalkanoic acid (PHA) according to the present invention, the electrospray deposition method is preferred because it can be applied to many types of polyhydroxyalkanoic acid (PHA), allows for a wide range of changes in particle size and strength of the fine particles, and has a simple manufacturing process. The electrospray deposition method (ESD method) will be described below, but the method for producing fine particles from polyhydroxyalkanoic acid (PHA) according to the present invention is not limited to the electrospray deposition method.

[0046] [Electrospray Deposition Method] This paper describes the principle of the electrospray deposition method used as a specific embodiment of the present invention, and an electrospray deposition apparatus (ESD: electrostatic spraying apparatus) used to implement the electrospray deposition method.

[0047] Figure 1 shows a conceptual diagram illustrating the basic configuration of an electrospray deposition apparatus. As shown in the figure, the container CNT contains the sample solution SL. The sample solution SL is, for example, an organic polymer solution or a polymer solution. In this embodiment, the sample solution is a polyhydroxyalkanoic acid (PHA) solution dissolved in a solvent, or a silica microparticle dispersion.

[0048] ESD is a highly complex physical phenomenon, and its entire process is not fully understood, but it is generally thought to involve the following: The sample solution is contained in a narrow capillary nozzle NZL, and a voltage of several thousand to tens of thousands of volts is applied to the target substrate TS (counter electrode) facing it. At the tip of the capillary, a strong electric field is generated due to the effect of electric field concentration, causing charged microdroplets to gather on the liquid surface and form a cone (called a Taylor cone). From this tip, the sample solution breaks the surface tension and becomes a jet. The jet is strongly charged, and due to electrostatic repulsion, it becomes a spray (Coulomb explosion). The droplets formed by the spray are very small, and the solvent evaporates and dries in a short time, becoming fine nanoparticles or nanofibers. Of course, it is also possible to deposit the sample in a wet state without evaporation or drying. These charged microparticles and narrow nanofibers are attracted to the target substrate TS, which functions as the counter electrode, by electrostatic force. The deposition pattern can be controlled by insulating masks and auxiliary electrodes (not shown). The sample can be any liquid, not just a solution, but also a dispersion.

[0049] Preferably, the sample solution in the container CNT is subjected to extrusion pressure toward the nozzle NZL side by an air pressure syringe pump or plunger (discharge means, not shown). The extrusion pressure is provided, for example, by a stepping motor and a screw feed mechanism (not shown). The sample solution SL, subjected to the extrusion pressure, has its internal pressure increased within the container CNT and is discharged from the tip of the nozzle NZL. As described above, by providing an adjustment mechanism (stepping motor and screw feed mechanism) to adjust the speed at which the sample solution is discharged, it is possible to adjust to an appropriate discharge speed.

[0050] The nozzle NZL is made of metal, and a positive voltage is supplied from the high-voltage power supply HPS via a conductive wire WL. The negative side of the high-voltage power supply HPS is connected to the target substrate TS (the substrate that acts as the counter electrode). By applying voltage from the high-voltage power supply HPS, a positive voltage is applied to the sample solution SL via the nozzle NZL, and the solution becomes positively charged. Note that the polarity of the voltage applied to the sample solution SL may be negative. To implement the present invention, it is necessary to suppress the formation of nanofibers and produce only fine particles, and to control their particle size and physical properties. This requires adjusting the selection of the sample and solvent, the concentration of the sample solution, the voltage, the spray distance, and environmental conditions such as temperature and humidity.

[0051] The sprayed material forms fibers or droplets, which repeatedly split during flight due to repulsion caused by electrostatic charge, forming nanofibers and nanoparticles. Because the sprayed material is nano-sized and has a large surface area, it is almost dry by the time it reaches the substrate or receiving tank. The shape and size can be changed by the spraying conditions; for example, when using a polymer solution, a higher molecular weight and higher concentration will form thicker nanofibers, while a lower molecular weight and lower concentration will form thinner nanofibers or nanoparticles. In addition, various conditions such as the voltage and distance between the nozzle and the substrate, ambient temperature, and humidity also have an effect. In this embodiment, polyhydroxylalkanoates soluble in various solvents were used as samples, and fine particles were produced under various conditions. The particle size, shape, and surface shape of the fine particles were confirmed using the method described in the examples. As an electrospray deposition apparatus, other types of ESD apparatus can be used in addition to the apparatus described above, and especially when the purpose is mass production, the method using the airflow described in Table 2009 / 060898 is preferred.

[0052] To produce the polyhydroxyalkanoic acid (PHA) fine particles of the present invention, it is important to select a suitable solvent. The solvent is not particularly limited as long as it sufficiently dissolves the PHA polymer, more strongly suppresses the formation of nanofibers, promotes the formation of fine particles, and also has a useful effect in changing the particle size. In the following examples, chloroform and dimethyl carbonate were used as suitable solvents for these reasons.

[0053] [Method for recovering fine particles containing polyhydroxyalkanoic acid (PHA)] Regarding the recovery method for the manufactured microparticles, if the material manufactured from polyhydroxyalkanoic acid (PHA) is a nanofiber, it is possible to allow it to form a nanofiber structure at the landing site. However, if the manufactured material is microparticles, it is necessary to recover these microparticles from the landing site without causing them to adhere. Therefore, depending on the environmental conditions of the landing site, as well as the physical properties of the polyhydroxyalkanoic acid (PHA) and the solvent used, it may be necessary to recover them in a liquid rather than on a solid surface.

[0054] Regarding the specific method for recovering the fine particles, it is necessary to recover them without them binding together due to surface potential or other factors. When the manufactured fine particles were recovered using tap water, it was observed that the particles bound together due to the influence of surface potential, etc. Therefore, to recover the fine particles in a dispersed state without them binding together, recovery using ethanol is preferable. Anhydrous ethanol can be used as the ethanol, but depending on the fine particles produced, it may also be possible to use it diluted with water at a concentration of 10-30%. Furthermore, even with tap water, it is possible to recover the fine particles in a dispersed state without them binding together by appropriately adding anionic surfactants, etc. To recover the dried particles, vacuum drying is preferable when using ethanol for recovery, but natural drying is also possible. When using tap water containing a surfactant for recovery, vacuum drying is preferable.

[0055] [Method for measuring the particle size of fine particles containing polyhydroxyalkanoic acid (PHA)] (1) SEM observation images and processing with software (ImageJ) This measurement method obtains particle size by processing images obtained by SEM with ImageJ. ImageJ is open-source, public-domain image processing software widely used for image analysis in scientific research and has become the de facto standard analysis tool in biology. Processing with ImageJ makes it possible to calculate particle size from SEM images. The specific procedure is as follows: 1. Image input, 2. Automatic binarization, 3. Removal of unwanted particles, 4. Filling in the binary image, and 5. Measurement results. After investigation, it was found that this method is particularly suitable for fine particles of several μm or larger. The accuracy of the measurement results obtained by this method was confirmed by measuring the particle size using fine particles on the order of several hundred nm obtained from P(3-HB-co-4-HB) by both this method and dynamic light scattering. As a result, as described in Example 4 below, the measurement results obtained by both methods were almost identical. In particular, considering that the particle size is extremely small, less than 500 nm, the accuracy of the measurement results obtained by this method can be judged to be very high. (2) Dynamic light scattering method There are many methods for measuring the particle size (diameter) of fine particles, in addition to those mentioned above. Methods such as the electrical sensor method, centrifugal sedimentation method, laser diffraction method, and FFF method are used, each with its own characteristics. However, dynamic light scattering is said to be the only method capable of measuring particles at the electron microscope level, and is therefore utilized for ultrafine particle measurement. In this development, dynamic light scattering was used to measure the particle size of submicron-level fine particles. The device used was the HORIBA NANO PARTICLE ANALYZER SZ-100, and the measurement conditions were as follows: Detection angle: 90 Holder temperature: 25.0℃ Sample refractive index: 1.500-0.000i Distributed medium file: water Refractive index of dispersion medium: 1.333 Dispersion medium viscosity: 0.896mPa·s Molecular form (dispersity): polydispersity Particle size standard: scattered light intensity Count rate: 1285kCPS

[0056] The present invention will be described in more detail below with reference to examples. However, these examples do not limit the present invention in any way. [Examples]

[0057] [Example 1] Production of fine particles from P(3-HB) 1.5 g of P(3-HB) resin, which consists of 3-HB repeating units without 3-HH, was dissolved in chloroform to prepare 100 g of a 1.5% sample solution by weight. 1 mL of this sample solution was placed in a glass syringe (Tsubasa Kogyo white hard syringe 1 ml) equipped with a metal double nozzle NZL (Musashi Engineering Co., Ltd. DN-24G) with an inner diameter of 0.29 mm, as shown in Figure 1, and mounted on an electrospray deposition apparatus (Fuence Co., Ltd. Esprayer ES-2000). An 8.9 g iron container with a thickness of 0.25 mm, a diameter of 5 cm, and a height of 1 cm was placed on the target substrate TS (collector substrate), and 15 ml of anhydrous methanol was injected into it. The electrospray conditions at this time were a nozzle-collector (target substrate TS) voltage of 25KV, a nozzle-collector distance of 4cm, and a liquid flow rate of 20μl / min. The spray was evenly scanned across the substrate in all directions (forward, backward, left, and right) to disperse the particles and obtain fine particles of P(3-HB). The solution concentration was set to 0.7-3.0% by weight, and other conditions were the same to obtain a liquid containing fine particles of P(3-HB). By drying this liquid, the fine particles shown in Figure 2 were obtained. The average particle size was determined from the SEM observation image using ImageJ particle size analysis, and the average particle size was found to be 6.70μm.

[0058] [Example 2] Production of porous microparticles from P(3-HB) 1.5 g of P(3-HB) resin, which consists of 3-HB repeating units without 3-HH, was dissolved in chloroform to prepare 100 g of a 1.5% concentration sample solution. 1 mL of this sample solution was placed in a glass syringe (Tsubasa Kogyo white hard syringe 1 ml) equipped with a metal nozzle NZL (Musashi Engineering Co., Ltd. 27G) with an inner diameter of 0.21 mm, as shown in Figure 1, and mounted in an electrospray deposition apparatus (Fuence Co., Ltd. Esprayer ES-2000). On the target substrate TS (collector substrate), a 0.5 mm thick, 8 cm long x 10 cm wide sample was placed. A 11.5g aluminum container, approximately 5mm in height, was placed on the substrate, and 15ml of anhydrous ethanol was injected into it for microparticle collection. The electrospray conditions at this time were a nozzle-collector (target substrate TS) voltage of 25KV, a nozzle-collector distance of 2.5cm, and a liquid flow rate of 10μl / min. The substrate was evenly scanned in all directions (forward, backward, left, and right) and sprayed to obtain dispersed 3-PHB (PHA 0%) microparticles. Liquids containing P(3-HB) microparticles were also obtained under other nozzle diameter and flow rate conditions with solution concentrations of 0.7-3.0% by weight. These liquids were dried to obtain the microparticles shown in Figure 3. SEM observation images confirmed that the microparticles were spherical in shape and had a porous surface. Furthermore, similar to Example 1, the particle size was measured using SEM observation images and software (ImageJ), and the average particle size was approximately 6.4μm. It is believed that the physical properties of the resin itself are related to the factors that cause porosity. When the content of 3-hydroxyhexanoic acid (3-HH) as a repeating unit constituting the polyhydroxyalkanoic acid (PHA) according to the present invention is high, the generation of porous fine particles is not observed. For example, in P(3-HB-co-3-HH) which contains 27% 3-hydroxyhexanoic acid (3-HH), the generation of porous fine particles was hardly observed.

[0059] [Example 3] Production of fine particles from P(3-HB-co-3-HH) 1.5 g of P(3-HB-co-3-HH) resin containing 27% 3-hydroxyhexanoic acid (3-HH) as a repeating unit was dissolved in chloroform to prepare 100 g of a 1.5% concentration sample solution by weight. 1 mL of this sample solution was placed in a glass syringe (Tsubasa Kogyo white hard syringe 1 ml) equipped with a metal nozzle NZL (Musashi Engineering Co., Ltd. SNA-22G) with an inner diameter of 0.42 mm, as shown in Figure 1, and mounted on an electrospray deposition apparatus (Fuence Co., Ltd. EsprayerES-2000). An 8.9 g iron container with a thickness of 0.25 mm, a diameter of 5.5 cm, and a height of 1 cm was placed on the target substrate TS (collector substrate), and 15 ml of 90% concentration ethanol was injected into it. The electrospray conditions in this case were a nozzle-collector (target substrate TS) voltage of 25KV, a nozzle-collector distance of 5cm, and a liquid flow rate of 20μl / min. The spray was evenly scanned across the substrate in all directions (forward, backward, left, and right) to disperse the particles and obtain fine particles of P(3-HB-co-3-HH). Similarly, a solution containing fine particles of P(3-HB-co-3-HH) was obtained under the same conditions (metal nozzles 24G, 21G, DN-24) with a solution concentration of 0.7-3.0 wt%. By drying this solution, the fine particles shown in Figure 4 were obtained. SEM observation images showed that the shape of these fine particles was almost spherical, with almost no porosity observed. Similar to Example 1, the particle size was measured using SEM observation images and software (ImageJ), and the average particle size was approximately 6.6μm.

[0060] [Example 4] Production of fine particles from P(3-HB-co-4-HB) 1.5 g of P(3-HB-co-4-HB) resin containing 42% 4-hydroquibutanoic acid (4-HB) as a repeating unit was dissolved in dimethyl carbonate to prepare 150 g of a 1.0% concentration sample solution by weight. 1 mL of this sample solution was placed in a glass syringe (Tsubasa Kogyo white hard syringe 1 ml) equipped with a metal nozzle NZL (Musashi Engineering Co., Ltd. DN-24G) with an inner diameter of 0.29 mm, as shown in Figure 1, and mounted on an electrospray deposition apparatus (Fuence Co., Ltd. Esprayer ES-2000). An 8.9 g iron container with a thickness of 0.25 mm, a diameter of 5 cm, and a height of 1 cm was placed on the target substrate TS (collector substrate), and 15 ml of tap water (5 drops of surfactant in 50 ml) was injected into it. The electrospray conditions at this time were a nozzle-collector (target substrate TS) voltage of 25KV, a nozzle-collector distance of 5cm, and a liquid flow rate of 20μl / min. The spray was evenly scanned across the substrate in all directions (forward, backward, left, and right) to disperse the material and obtain fine particles of P(3-HB-co-4-HB). Similarly, a solution containing fine particles of P(3-HB-co-4-HB) was obtained under the same conditions but with a solution concentration of 0.7-3.0% by weight. The fine particles shown in Figure 5 were obtained by drying this solution.

[0061] The particle size of the obtained fine particles was measured using a method combining SEM observation images and software (ImageJ) processing, as well as the dynamic scattered light intensity method. The measurement conditions were as described above. As a result, the average particle diameter measured using SEM observation images and software (ImageJ) processing was 0.42 μm. On the other hand, the mode diameter measured using dynamic scattered light intensity spectroscopy was 0.34 μm (335.1 nm). Therefore, the particle diameter of 420 nm (0.42 μm) measured using SEM observation images and software (ImageJ) processing and the mode diameter of 335.1 nm measured using dynamic scattered light intensity spectroscopy are extremely similar results, confirming that measuring particle diameter using SEM observation images and software (ImageJ) processing is a highly reliable measurement method.

[0062] [Example 5] Production of fine particles retaining other compounds 1.5 g of P(3-HB) resin, consisting solely of 3-HB as the repeating unit of polyhydroxyalkanoic acid (PHA), was mixed with 0.5% silica particles AdmaFine SC2500-SPJ (manufactured by Admatex Co., Ltd.) by weight. This mixture was dissolved in chloroform to prepare approximately 100 g of a 1.5 wt percent P(3-HB) solution. 1 mL of this sample solution was placed in a glass syringe (Tsubasa Kogyo white hard syringe 1 ml) fitted with a metal nozzle NZL (Musashi Engineering Co., Ltd. DN-24G) with an inner diameter of 0.29 mm, as shown in Figure 1, and mounted on an electrospray deposition apparatus (EsprayerES-2000, manufactured by Fuence Co., Ltd.). An 8.9 g iron container with a thickness of 0.25 mm, a diameter of 5 cm, and a height of 1 cm was placed on the target substrate TS (collector substrate), and 15 ml of anhydrous alcohol was injected into it. The electrospray conditions at this time were: nozzle NZL-collector (target substrate TS) voltage 25KV, nozzle-collector distance 4cm, and liquid flow rate 20μl / min (10μl / min per nozzle due to double nozzles). The substrate was evenly scanned in all directions (forward, backward, left, and right) and sprayed to obtain a dispersion of P(3-HB) fine particles. These fine particles were air-dried to obtain the fine particles shown in Figure 6. The particle size of these fine particles was approximately 6-10μm, and they retained silica particles on their surface.

[0063] Regarding the P(3-HB) microparticles that retain the silica particles described above, it was confirmed that these silica particles are also retained within the P(3-HB) microparticles by the following method. The following procedure was used to prepare the surface using the ultrathin sectioning method, and then observed with a scanning electron microscope (SEM). 1) Place the sample on a microscope slide. 2) Place one drop of embedding resin onto the powder and allow it to harden. This was done using EPON812 (epoxy resin) at 60°C for 48 hours. 3) Place the beam capsule filled with embedding resin over the hardened resin. 4) Allow the embedding resin to harden (60°C for 48 hours). 5) Warm the slide glass and peel off the beam capsule, whose embedding resin has hardened. 6) Remove the hardened resin from the beam capsule. 7) Use an optical microscope to locate and mark the positions of the powder particles. 8) Trim around the powder. 9) Place the sample in the ultramicrotome and prepare the surface with a diamond knife. 10) Cut the sample to a height that will fit on the sample stand. 11) Attach the sample to the copper plate using conductive double-sided tape. 12) Deposit carbon. Figure 7 shows the SEM observation image, which confirms the presence of particles equivalent to silica particles within the P(3-HB) nanoparticles. Furthermore, Figure 8 shows the SEM BSE image. The SEM BSE image is a backscattered electron image, a technique that allows for confirmation of the compositional distribution within the sample. From this observation image, it was confirmed that the P(3-HB) nanoparticles contain particles with a different chemical composition from the P(3-HB) nanoparticles.

[0064] From the above results, although the strength of these microparticles in retaining silica particles is not clear, it was confirmed that silica particles were retained on the surface and inside the P(3-HB) particles after the electrospray deposition operation. This indicates that not only is polyhydroxyalkanoic acid (PHA) microparticleized by the electrospray deposition operation, but other substances are also dissolved or dispersed in the spray solution during the process, causing the polyhydroxyalkanoic acid (PHA) microparticles according to the present invention to retain, mix with, or integrate other substances. This suggests that the polyhydroxyalkanoic acid (PHA) microparticles according to the present invention may be useful as carriers for other materials.

[0065] [Example 6] Measurement of compressive strength of fine particles containing polyhydroxyalkanoic acid (PHA) Since fracture strength and deformation strength, as physical properties of fine particles, are practically important factors when considering their application to various uses, a compression test was conducted on the fine particles containing polyhydroxyalkanoic acid (PHA) according to the present invention to confirm the 10% compressive strength. The following samples were used. Sample (1) Fine particles produced from P(3-HB) (fine particles produced in Example 1) Sample (2) Fine particles produced from P(3-HB-co-3-HH) (fine particles produced in Example 3) Compression tests were conducted using a Shimadzu microcompression tester MCT-510 under the following conditions. A minute amount of the sample was scattered onto a glass plate, and each individual particle was compressed. The test results were evaluated using the average value.

[0066] [Table 1]

[0067] The results of the compression test are shown in the table below.

[0068] [Table 2]

[0069] From the above results, the samples of the polyhydroxyalkanoic acid (PHA)-containing fine particles according to the present invention showed a 10% compressive strength of 0.23 to 2.20 MPa, confirming that they possess strength capable of withstanding corresponding compressive strengths. Furthermore, the fine particles produced from P(3-HB) showed a higher 10% compressive strength than the fine particles produced from P(3-HB-co-3-HH) containing 27% 3-hydroxyhexanoic acid (3-HH) as repeating units, confirming that they possess higher strength. Therefore, the fine particles containing polyhydroxyalkanoic acid (PHA) according to the present invention can be adapted to various usage environments and applications by adjusting the blending ratio of 3-hydroxyhexanoic acid (3-HH) as repeating units, thereby adjusting their strength.

[0070] [Example 7] Thermal properties of polyhydroxyalkanoate (PHA) polymers Among the physical properties of polyhydroxyalkanoic acid (PHA), which is the raw material for the fine particles according to the present invention, the melting point is one of the important properties in terms of ease of processing for applying the fine particles to actual uses, and if it is possible to change the melting point, it can be an advantageous property in processing, etc. While the melting point of P(3-HB), which has been studied relatively well so far, is often reported to be 170~180°C, the melting point of P(3-HB-co-3-HH), which contains 27% 3-hydroxyhexanoic acid (3-HH) as a repeating unit, is unknown, and the melting point of P(3-HB-co-4-HB) varies considerably depending on the literature, so we confirmed it using the following method. Measurements were performed using a PerkinElmer differential scanning calorimeter DSC8500. The measurement conditions involved using approximately 6 mg of sample, heating from 5.00°C to 200.00°C at a rate of 5.00°C / min in a nitrogen gas atmosphere. As a result, it was confirmed that the melting point of P(3-HB-co-3-HH) is 79.8°C and the melting point of P(3-HB-co-4-HB) is 56°C. [Industrial applicability]

[0071] The polyhydroxyalkanoic acid (PHA)-containing fine particles according to the present invention have excellent processability, biodegradability in the natural environment, biocompatibility, and biodegradability. They also possess a broad melting point, particle size, and appropriate compressive strength, making them suitable for a wide range of industrial and medical applications, while completely eliminating environmental problems such as microplastics. [Explanation of Symbols]

[0072] CNT container HPS High Voltage Power Supply NZL Nozzle SL sample solution TS target substrate ESD Electrospray Deposition System WL wire

Claims

1. Fine particles containing polyhydroxyalkanoic acid (PHA), wherein the repeating units of the polyhydroxyalkanoic acid (PHA) include 3-hydroxybutanoic acid (3-HB), and the particle size is 0.2 to less than 10 μm. The polyhydroxyalkanoic acid (PHA) further contains 3-hydroxyhexanoic acid (3-HH) as a repeating unit, and the proportion of the 3-hydroxyhexanoic acid (3-HH) is 27% (by weight) or less of the total weight of the repeating units of the polyhydroxyalkanoic acid (PHA). The fine particles are characterized by being porous.

2. The fine particles according to claim 1, characterized in that the particle size is 7 μm or less.

3. The fine particles according to claim 1 or 2, characterized in that the polyhydroxyalkanoic acid (PHA) comprises a copolymer of 3-hydroxybutanoic acid (3-HB) and 3-hydroxyhexanoic acid (3-HH).

4. The fine particles according to any one of claims 1 to 3, characterized in that the polyhydroxyalkanoic acid (PHA) further comprises 4-hydroxybutanoic acid (4-HB) units as repeating units.

5. The fine particles according to claim 4, characterized in that the proportion of 4-hydroxybutanoic acid (4-HB) is 40 to 50% (by weight) of the total weight of the repeating units of polyhydroxyalkanoic acid (PHA).

6. The fine particles according to claim 4 or 5, characterized in that the polyhydroxyalkanoic acid (PHA) comprises a copolymer of 3-hydroxybutanoic acid (3-HB) and 4-hydroxybutanoic acid (4-HB).

7. The fine particles according to any one of claims 1 to 6, characterized in that the average molecular weight (Mw) of the polyhydroxyalkanoic acid (PHA) is between 100,000 and 1,300,000.

8. The fine particles according to any one of claims 1 to 7, characterized in that the melting point of the polyhydroxyalkanoic acid (PHA) is 55°C or higher and 170°C or lower.

9. The fine particles according to any one of claims 1 to 8, characterized in that the fine particles contain a resin other than polyhydroxyalkanoic acid (PHA).

10. The fine particles according to claim 9, characterized in that the resin other than polyhydroxyalkanoic acid (PHA) is a biodegradable resin.

11. The fine particles according to any one of claims 1 to 10, characterized in that the fine particles are spherical.

12. The fine particles according to any one of claims 1 to 11, characterized in that the fine particles retain other substances on their surface and / or inside.

13. The fine particles according to any one of claims 1 to 12, characterized in that the 10% compressive strength of the fine particles is 0.23 to 2.20 (MPa).

14. The fine particles according to any one of claims 1 to 13, characterized in that the fine particles can be dispersed in an aqueous solvent.

15. A method for producing fine particles according to any one of claims 1 to 14, comprising the following steps. Step 1: A step to prepare microorganisms that produce polyhydroxyalkanoic acid (PHA), Step 2: A step of growing the microorganisms from Step 1 in a culture medium. Step 3: The process of having animals ingest the proliferated microorganisms. Step 4: A step of recovering and purifying polyhydroxyalkanoic acid (PHA) from the animal excrement of Step 3, and Step 5: A step to atomize the polyhydroxyalkanoic acid (PHA) obtained in Step 4.

16. The method for producing fine particles according to claim 15, wherein step 5 is a step of micronizing the resin composition containing polyhydroxyalkanoic acid (PHA) obtained in step 4.

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