JP7652378B2 - Particle analysis method and particle manufacturing method - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies ▲1▼ Publication date: August 28, 2019 Publication: Web version of abstracts from the 68th Annual Meeting of the Japan Society for Analytical Chemistry Disclosed by: Ayako Tatematsu, Ryusuke Abe, Makoto Kono, and Yoshiya Fushimi ▲2▼ Date held: September 11, 2019 Meeting name and location: 68th Annual Meeting of the Japan Society for Analytical Chemistry Chiba University Nishi-Chiba Campus (1-33 Yayoi-cho, Inage-ku, Chiba City, Chiba Prefecture) Disclosed by: Ayako Tatematsu, Ryusuke Abe, Makoto Kono, and Yoshiya Fushimi

The present invention relates to a particle analysis method and a particle manufacturing method.

Microcapsules are particles that encapsulate liquid or solid ingredients. The particle diameter of microcapsules is less than 1 mm. Microcapsules have a membrane (capsule base material) that covers the encapsulated ingredients. For example, various techniques have been proposed for industrially producing microcapsules that encapsulate active ingredients such as medicines, pesticides, fragrances, or food ingredients. Methods for manufacturing microcapsules include, for example, chemical methods, physical methods, and biological methods. Chemical methods include, for example, interfacial polymerization, liquid-based effective coating, and molecular inclusion methods using cyclodextrin. Physical methods include, for example, spray drying, spray cooling, and air-suspended coating. Biological methods include, for example, a method of encapsulating ingredients within the cytoskeleton of yeast, etc.

In biological techniques, yeast-derived capsule substrates are mainly used. Yeast-derived capsule substrates are made of the yeast cytoskeleton remaining after soluble contents are removed from yeast. The cytoskeleton includes cell walls, cell membranes, and other solid components. Specifically, yeast-derived capsule substrates are made of the solid components of yeast remaining after soluble components within the yeast cell are removed from the yeast cell by washing and enzyme treatments. The cytoskeleton of the yeast cell wall, etc. is used as the capsule substrate. For example, Patent Document 1 discloses a technology for releasing intracellular components outside the yeast cell by enzyme treatment of the yeast.

Patent Document 1 discloses that a hydrophobic or hydrophilic substance is encapsulated in a capsule base material derived from yeast. Patent Document 1 discloses that the hydrophobic substance is at least one type of derived lipid selected from the group consisting of long-chain hydrocarbons, long-chain alcohols, long-chain amino alcohols, long-chain aldehydes, long-chain ketones, long-chain acids and their salts, terpenoids, steroids, and carotenoids. It also discloses that the hydrophilic substance is at least one type of substance selected from the group consisting of amino acids, water-soluble vitamins, nucleotides, and sugars.

Patent Document 1 also discloses that the yeast is at least one type of microorganism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces rouxii, Saccharomyces carlsbergensis, Candida utilis, Candida tropicalis, Candida lipolytica, and Candida flavori.

Incidentally, to quantitatively analyze whether or not the component to be encapsulated is encapsulated in the microcapsule, for example, high performance liquid chromatography (HPLC) is generally used. Specifically, the encapsulated component is extracted from the microcapsule, the encapsulated component is quantified using high performance liquid chromatography, and the amount of the encapsulated component per gram of microcapsule is calculated based on the measurement results, thereby analyzing (evaluating) whether or not the component to be encapsulated is encapsulated in the microcapsule.

Japanese Patent Application Publication No. 8-243378

However, quantitative analysis using high performance liquid chromatography only estimates whether or not the target component is contained in the microcapsules (the particles being analyzed).

The present invention has been made in consideration of the above problems, and its purpose is to provide a particle analysis method and a particle manufacturing method that can analyze for each analysis target whether or not the analysis target particle contains a target component.

The particle analysis method of the present invention includes a first observation step of magnetically migrating a first particle and observing it; a first migration velocity measurement step of measuring the magnetic migration velocity of the first particle from the result of the observation by the first observation step; a first magnetic susceptibility measurement step of measuring the volume magnetic susceptibility of the first particle based on the result of the measurement by the first migration velocity measurement step; an inclusion processing step of performing an inclusion processing to make the first particle include a component to be included that is smaller than the first particle; a second observation step of magnetically migrating a second particle to be analyzed, which is the first particle after the inclusion processing, and observing it; a second migration velocity measurement step of measuring the magnetic migration velocity of the second particle from the result of the observation by the second observation step; a second magnetic susceptibility measurement step of measuring the volume magnetic susceptibility of the second particle based on the result of the measurement by the second migration velocity measurement step; and a step of analyzing whether the component to be included is included in the second particle based on the result of the measurement by the first magnetic susceptibility measurement step and the result of the measurement by the second magnetic susceptibility measurement step.

In one embodiment, the particle analysis method further includes a step of spectroscopically observing the second particles to analyze whether the component to be included is included in the second particles.

In one embodiment, the particle analysis method further includes a first zeta potential measurement step of measuring the zeta potential of the first particle, a second zeta potential measurement step of measuring the zeta potential of the second particle, and a step of analyzing whether the component to be encapsulated is encapsulated in the second particle based on the measurement results of the first zeta potential measurement step and the measurement results of the second zeta potential measurement step.

The particle manufacturing method of the present invention includes the particle analysis method described above.

In one embodiment, the first particles contain solid components of the yeast that remain after intracellular components are released from the yeast.

In one embodiment, the particle production method further includes a first particle production step of producing the first particles. The first particle production step includes a step of releasing the intracellular components from the yeast.

In one embodiment, the encapsulation process includes mixing the first particles and the component to be encapsulated in a solvent.

The particle analysis method and particle production method of the present invention make it possible to analyze whether or not the particles to be analyzed contain the components to be contained therein for each of the particles to be analyzed.

1 is a flowchart showing a particle manufacturing method according to a first embodiment of the present invention. FIG. 1 is a schematic diagram of an analysis device according to a first embodiment of the present invention. 1A and 1B are diagrams illustrating the movement of particles. 1 is a diagram showing a configuration of an analysis device according to a first embodiment of the present invention. 10 is a flowchart showing a particle manufacturing method according to a second embodiment of the present invention. 10 is a flowchart showing a particle manufacturing method according to a third embodiment of the present invention. FIG. 1 is a diagram showing measurement results according to Example 1 of the present invention. FIG. 11 is a diagram showing measurement results according to Example 2 of the present invention. FIG. 13 is a diagram showing measurement results according to Example 3 of the present invention. FIG. 11 is a diagram showing a first measurement result according to Example 4 of the present invention. FIG. 11 is a diagram showing a second measurement result according to Example 4 of the present invention. FIG. 13 is a diagram showing a third measurement result according to Example 4 of the present invention. FIG. 13 is a diagram showing a fourth measurement result according to Example 4 of the present invention. FIG. 13 is a diagram showing a first measurement result according to Example 5 of the present invention. FIG. 13 is a diagram showing a second measurement result according to Example 5 of the present invention. FIG. 13 is a diagram showing measurement results according to Example 6 of the present invention.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following embodiment. Note that where the explanation overlaps, the explanation may be omitted as appropriate. In addition, in the drawings, the same or equivalent parts are given the same reference symbols and the explanation will not be repeated.

[Embodiment 1]
First, the particle analysis method and the particle manufacturing method of this embodiment will be described with reference to Fig. 1. Fig. 1 is a flow chart showing the particle manufacturing method according to this embodiment. As shown in Fig. 1, the particle manufacturing method of this embodiment includes a first particle preparation step S1, a first observation step S2, a first migration speed measurement step S3, a first magnetic susceptibility measurement step S4, an encapsulation process step S5, a second observation step S6, a second migration speed measurement step S7, a second magnetic susceptibility measurement step S8, and a magnetic susceptibility analysis step S9. Among these steps S1 to S9, the first observation step S2, the first migration speed measurement step S3, the first magnetic susceptibility measurement step S4, the encapsulation process step S5, the second observation step S6, the second migration speed measurement step S7, the second magnetic susceptibility measurement step S8, and the magnetic susceptibility analysis step S9 represent the particle analysis method of this embodiment.

<First particle preparation step S1>
In the first particle preparation step S1, the first particle p1 is prepared. The first particle p1 has a particle diameter of, for example, 1 nm or more and several hundred μm or less. The first particle p1 is, for example, a base material of a microcapsule (capsule base material). The capsule base material has a membrane component that separates the outside from the inside. However, the first particle p1 is not limited to a capsule base material. The first particle p1 may be any particle that can encapsulate the component pt to be encapsulated.

The first particle p1 may be, for example, a capsule substrate derived from yeast. For example, the yeast-derived capsule substrate is made of the solid components of yeast that remain after the intracellular components are released from the yeast. In this case, the yeast-derived capsule substrate is mainly made of the skeletal portion of the yeast. More specifically, the yeast-derived capsule substrate has a yeast cell wall and cell membrane. The intracellular components mainly include amino acids, nucleic acid-related substances, minerals, and vitamins.

The yeast-derived capsule substrate is produced, for example, from torula yeast, baker's yeast, brewer's yeast, or sake yeast. The form of the yeast-derived capsule substrate is not particularly limited, and may be, for example, in the form of compressed yeast, dried yeast, active dry yeast, dead yeast, or pasteurized dry yeast.

The yeast-derived capsule base material may be a substance derived from the solid components of yeast. The substance derived from the solid components of yeast has substantially the same composition as the solid components of yeast. Specifically, the yeast-derived capsule base material may be a pulverized or powdered form of the solid components of yeast.

The yeast used in the production of the yeast-derived capsule base material is not particularly limited, and may be, for example, a yeast belonging to the genus Saccharomyces or a yeast belonging to the genus Candida. For example, the yeast may be Saccharomyces cerevisiae. Alternatively, the yeast may be Candida utilis.

The first particles p1 are not limited to capsule substrates derived from yeast. The first particles p1 may be, for example, yeast, algae such as Euglena, lactic acid bacteria, acetic bacteria, Bacillus subtilis, or cell membrane fragments. The yeast, Euglena, lactic acid bacteria, acetic bacteria, and Bacillus subtilis may be alive or dead. For example, the yeast, algae such as Euglena, lactic acid bacteria, acetic bacteria, and Bacillus subtilis may be dried before or after sterilization. Furthermore, the first particles p1 are not limited to microorganisms, and may be animal cells or plant cells.

Alternatively, the first particle p1 may be a vesicle. More specifically, the first particle p1 may be, for example, a liposome. The surface membrane of the vesicle is made of lipids. More specifically, the surface membrane of the vesicle has a lipid bilayer. A liposome is a vesicle whose surface membrane is made of phospholipids. Liposomes are used as encapsulating materials in DDS (drug delivery systems). The encapsulating materials used in DDS encapsulate, for example, a physiologically active substance as the component pt to be encapsulated.

<First particle manufacturing process>
The first particle preparation step S1 may include a first particle production step of producing the first particles p1. When the first particles p1 are capsule base materials derived from yeast, the first particle production step includes a step of releasing intracellular components from the yeast. The treatment for releasing intracellular components from the yeast is not particularly limited, and may be, for example, any of a hot water treatment method, an autolysis method, and an enzymatic decomposition method.

For example, the hot water treatment method is a process in which a high-temperature solution is stirred for a predetermined period of time. Yeast is added to the solution as a solute. The solvent is, for example, distilled water. However, the solvent is not limited to distilled water. The solvent may be any liquid that can release intracellular components from the yeast. For example, a buffer solution or an emulsifier may be used as the solvent.

When intracellular components are released from yeast by hot water treatment, the solid components remaining after the intracellular components are released from the yeast may be subjected to an enzyme treatment in which protease and/or cellulase is added. Furthermore, an emulsifier may be added to the solid components. The timing of adding the emulsifier may be before or after the enzyme treatment. Alternatively, the timing of adding the emulsifier may be simultaneous with the enzyme treatment.

The solid components remaining after the intracellular components are released from the yeast may be sterilized. That is, the yeast-derived capsule base material may be sterilized. In addition, the solid components may be dried in the final stage of the first particle manufacturing process. That is, the yeast-derived capsule base material may be in a dry state. However, the first particles p1 are not limited to being in a dry state. The first particles p1 may be in a paste form containing an appropriate amount of moisture.

<First observation step S2>
In the first observation step S2, the first particles p1 are magnetophoretically moved and observed. Specifically, the magnetophoretically moving first particles p1 are observed using an analysis device 10 described with reference to FIG.

<First migration speed measurement step S3>
In the first migration velocity measurement step S3, the magnetophoretic velocity v1 of the first particle p1 is measured from the results of the observation in the first observation step S2. Specifically, the magnetophoretic velocity v1 of the first particle p1 is measured using an analysis device 10 described with reference to Fig. 2. In this embodiment, the particle diameter d1 of the first particle p1 is measured from the results of the observation in the first observation step S2 together with the magnetophoretic velocity v1 of the first particle p1.

<First magnetic susceptibility measurement step S4>
In the first magnetic susceptibility measurement step S4, the volume magnetic susceptibility χs1 of the first particle p1 is measured based on the result of the measurement in the first migration velocity measurement step S3. Specifically, the volume magnetic susceptibility χs1 of the first particle p1 is measured using an analysis device 10 described with reference to FIG.

<Encapsulating processing step S5>
In the encapsulation process S5, an encapsulation process is performed to encapsulate a component pt to be encapsulated, which is smaller than the first particle p1, in the first particle p1, to obtain a second particle p2. The second particle p2 is the first particle p1 after the encapsulation process. More specifically, in the encapsulation process S5, the first particle p1 and the component pt to be encapsulated are mixed to contact the first particle p1 and the component pt to be encapsulated. Here, the first particle p1 to be encapsulated may be a first particle p1 whose volume magnetic susceptibility χs1 has been measured, or a first particle p1 whose volume magnetic susceptibility χs1 has not been measured. The second particle p2 may be, for example, a microcapsule. For example, when the first particle p1 is a capsule base material derived from yeast, the second particle p2 is a microcapsule.

The component pt to be encapsulated is not particularly limited as long as it can be encapsulated in the first particle p1. The component pt to be encapsulated may be a liquid or a solid. The component pt to be encapsulated may be, for example, a pharmaceutical, a pesticide, a flavoring, or a food ingredient.

Specifically, the component pt to be encapsulated may be a physiologically active substance, a mineral, a flavoring, a spice extract, a flavor oil, an animal fat, or a vegetable fat. For example, flavorings include d-limonene, carvone, ethyl caproate, chili flavor, vanilla flavor, grill flavor, wasabi flavor, coffee flavor, pepper, black pepper, mustard, curry spice, and meat flavor.

More specifically, the component pt to be encapsulated may be a hydrophobic substance or a hydrophilic substance. Furthermore, the component pt to be encapsulated may be a fat-soluble substance or a non-fat-soluble substance. When the first particle p1 has a hydrophobic region therein, the hydrophobic substance is encapsulated in the first particle p1 more stably than the hydrophilic substance. Similarly, when the first particle p1 has a hydrophilic region therein, the hydrophilic substance is encapsulated in the first particle p1 more stably than the hydrophobic substance.

The hydrophobic substance may be, for example, at least one derived lipid selected from the group consisting of long-chain hydrocarbons, long-chain alcohols, long-chain amino alcohols, long-chain aldehydes, long-chain ketones, long-chain acids and their salts, terpenoids, steroids, and carotenoids. Alternatively, the hydrophobic substance may be, for example, at least one simple lipid selected from the group consisting of waxes, glycerides, ether glycerides, and ceramides. Alternatively, the hydrophobic substance may be, for example, at least one complex lipid selected from the group consisting of phospholipids, glycolipids, phosphoglycolipids, sulfolipids, and amino acid lipids. The hydrophilic substance may be, for example, at least one substance selected from the group consisting of amino acids, water-soluble vitamins, nucleotides, and sugars.

The encapsulation process is not particularly limited as long as it is a process for encapsulating the component pt to be encapsulated in the first particle p1. For example, the encapsulation process may be a process for mixing and stirring the first particle p1 and the component pt to be encapsulated in a solvent. The solvent is, for example, distilled water. However, the solvent is not limited to distilled water. The solvent may be any liquid that can mix and disperse the first particle p1 and the component pt to be encapsulated. For example, the solvent may be a buffer solution, sugar solution, salt solution, seasoning solution, or soup stock. When the solvent contains salt, the amount of the component pt to be encapsulated in the first particle p1 can be increased compared to when the solvent does not contain salt.

The encapsulation process may include a process of drying a dispersion liquid obtained by mixing the first particles p1 and the component pt to be encapsulated in a solvent. By drying the dispersion liquid, the second particles p2 in a dry state are obtained. The process of drying the dispersion liquid is not particularly limited as long as the second particles p2 in a dry state can be obtained. For example, the dispersion liquid can be dried by spray drying, heat drying, freeze drying, or vacuum drying.

The encapsulation process is not limited to a process of mixing and stirring the first particles p1 and the component pt to be encapsulated in a solvent. For example, the encapsulation process may be a process of adjusting the environmental factors of the system in which the first particles p1 and the component pt to be encapsulated are dispersed. The environmental factors include heat, pressure, ion concentration, pH value, and the like. For example, by adjusting the ion concentration, the component pt to be encapsulated can be moved into the first particles p1 by the effect of coexisting ions. Specifically, the component pt to be encapsulated moves into the first particles p1 according to the second law of thermodynamics. When the first particles p1 are living yeast and the component pt to be encapsulated is zinc (a mineral), the encapsulation process may be, for example, a process of culturing the yeast in a medium or culture solution to which zinc has been added.

<Second observation step S6>
In the second observation step S6, the second particles p2 to be analyzed, which are the first particles p1 after the encapsulation process, are magnetophoresed and observed. Specifically, similarly to the first observation step S2, the magnetophoresed second particles p2 are observed using the analysis device 10 described with reference to FIG.

<Second migration speed measurement step S7>
In the second migration velocity measurement step S7, the magnetophoretic velocity v2 of the second particle p2 is measured from the results of the observation in the second observation step S6. Specifically, similar to the first migration velocity measurement step S3, the magnetophoretic velocity v2 of the second particle p2 is measured using the analysis device 10 described with reference to Fig. 2. In this embodiment, the particle diameter d2 of the second particle p2 is measured from the results of the observation in the second observation step S6 together with the magnetophoretic velocity v2 of the second particle p2.

<Second magnetic susceptibility measurement step S8>
In the second magnetic susceptibility measurement step S8, the volume magnetic susceptibility χs2 of the second particles p2 is measured based on the result of the measurement in the second migration velocity measurement step S7. Specifically, similar to the first magnetic susceptibility measurement step S4, the volume magnetic susceptibility χs2 of the second particles p2 is measured using the analysis device 10 described with reference to FIG.

<Magnetic susceptibility analysis step S9>
In the magnetic susceptibility analysis step S9, it is analyzed whether or not the component pt to be included is included in the second particle p2 based on the result of the measurement in the first magnetic susceptibility measurement step S4 and the result of the measurement in the second magnetic susceptibility measurement step S8. Specifically, the difference between the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2 is analyzed. When the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2 show different values, it is determined that the component pt to be included is included in the second particle p2.

More specifically, the volume magnetic susceptibility χs2 of the second particle p2 differs depending on the components of the second particle p2. Therefore, the volume magnetic susceptibility χs2 of the second particle p2 differs depending on whether the second particle p2 contains the component pt to be contained. In other words, the volume magnetic susceptibility χs2 of the second particle p2 containing the component pt to be contained differs from the volume magnetic susceptibility χs2 of the second particle p2 not containing the component pt to be contained. Specifically, by containing the component pt to be contained, the volume magnetic susceptibility χs2 of the second particle p2 becomes closer to the volume magnetic susceptibility χst of the component pt to be contained compared to the volume magnetic susceptibility χs2 of the second particle p2 not containing the component pt to be contained.

Therefore, by comparing the volume magnetic susceptibility χs1 of the first particle p1 with the volume magnetic susceptibility χs2 of the second particle p2, it is possible to determine whether or not the component pt to be encapsulated is contained in the second particle p2. More specifically, if the volume magnetic susceptibility χs2 of the second particle p2 is closer to the volume magnetic susceptibility χst of the component pt to be encapsulated than the volume magnetic susceptibility χs1 of the first particle p1, it is determined that the component pt to be encapsulated is contained in the second particle p2.

The second particles p2 may be dried after the measurement of the volume magnetic susceptibility χs2. For example, after the second observation step S6, the solution (dispersion liquid) in which the second particles p2, which are the solute (dispersoid), are dispersed is dried to obtain the second particles p2 in a dry state. The process for drying the dispersion liquid is not particularly limited as long as the second particles p2 in a dry state can be obtained. For example, the dispersion liquid can be dried by spray drying, heat drying, freeze drying, or vacuum drying.

<Analysis device 10>
Next, an analysis device 10 according to the present embodiment will be described with reference to Fig. 1 and Fig. 2. Fig. 2 is a schematic diagram of the analysis device 10 according to the present embodiment. The analysis device 10 analyzes whether or not a component pt to be included is included in the second particle p2.

As shown in FIG. 2, the analysis device 10 includes a magnetic field generating unit 20, an observation unit 30, and a calculation unit 40. A cell 21 is disposed near the magnetic field generating unit 20. The magnetic field generating unit 20 generates a magnetic field to magnetophoretically migrate particles p in the cell 21. The observation unit 30 observes the particles p in the cell 21. The calculation unit 40 measures the particle diameter d and magnetophoretic velocity v of the particles p from the results of observation by the observation unit 30. The calculation unit 40 also measures the volume magnetic susceptibility χs of the particles p based on the particle diameter d and magnetophoretic velocity v of the particles p.

In more detail, in the first observation step S2, the magnetic field generating unit 20 magnetophores the first particle p1 in the cell 21, and the observation unit 30 observes the first particle p1 in the cell 21. In the first migration velocity measurement step S3, the calculation unit 40 measures the magnetophoretic velocity v1 of the first particle p1 from the result of observation by the observation unit 30. In this embodiment, the calculation unit 40 measures the particle diameter d1 of the first particle p1 as well as the magnetophoretic velocity v1 of the first particle p1 from the result of observation by the observation unit 30. In addition, in the first magnetic susceptibility measurement step S4, the calculation unit 40 measures the volume magnetic susceptibility χs1 of the first particle p1 based on the magnetophoretic velocity v1 and particle diameter d1 of the first particle p1.

Furthermore, in the second observation step S6, the magnetic field generating unit 20 magnetophores the second particle p2 in the cell 21, and the observation unit 30 observes the second particle p2 in the cell 21. In the second migration velocity measurement step S7, the calculation unit 40 measures the magnetophoretic velocity v2 of the second particle p2 from the results of the observation by the observation unit 30. In this embodiment, the calculation unit 40 measures the particle diameter d2 of the second particle p2 as well as the magnetophoretic velocity v2 of the second particle p2 from the results of the observation by the observation unit 30. In addition, in the second magnetic susceptibility measurement step S8, the calculation unit 40 measures the volume magnetic susceptibility χs2 of the second particle p2 based on the magnetophoretic velocity v2 and particle diameter d2 of the second particle p2.

Furthermore, in the magnetic susceptibility analysis step S9, the calculation unit 40 analyzes whether or not the component pt to be contained is contained in the second particle p2 based on the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2.

The analysis device 10 will be described in more detail below. The magnetic field generating unit 20 generates a magnetic field gradient (magnetic flux density gradient) and applies a magnetic force to the particles p in the cell 21. As a result, the particles p undergo magnetic migration. In this embodiment, the magnetic field generating unit 20 includes two permanent magnets 20a, 20b (see FIG. 3) that generate a magnetic field gradient. The two permanent magnets 20a, 20b are arranged with a gap of a fixed distance, for example, 100 μm or more and 500 μm or less. The cell 21 is arranged in the gap between the two permanent magnets 20a, 20b.

In this embodiment, the cell 21 is a capillary tube. A capillary tube is an example of a tubular member. The material of the cell 21 is not particularly limited as long as it is a material that can transmit visible light or laser light. For example, the cell 21 can be made of glass or plastic.

Particles p are introduced into cell 21 together with medium m, for example, by a microsyringe, a micropump, or an autosampler. Alternatively, particles p may be introduced into cell 21 together with medium m based on the siphon principle. Alternatively, droplets containing particles p may be introduced into cell 21 (capillary tube) by capillary action. When droplets containing particles p are dripped onto one end of the capillary tube, the droplets flow through the capillary tube by capillary action.

Particle p is present in medium m. There may be one particle p in medium m, or multiple particles p in medium m. When multiple particles p are present in medium m, the multiple particles p may be dispersed in medium m, or may be unevenly distributed in medium m. Medium m is, for example, distilled water. The analysis device 10 measures the volume magnetic susceptibility χs of each of the multiple particles p present in medium m.

The observation unit 30 observes the particles p in the cell 21 and generates a signal indicating the observation result. The calculation unit 40 measures the magnetophoretic velocity v and particle diameter d of the particles p based on the signal generated by the observation unit 30. The calculation unit 40 includes a memory unit 41, a processing unit 42, and an output unit 43.

The memory unit 41 stores programs, setting information, and the like. The memory unit 41 may be configured, for example, with a storage device and a semiconductor memory. The storage device is, for example, a HDD (Hard Disk Drive). The memory unit 41 may have, as the semiconductor memory, for example, a RAM (Random Access Memory) and a ROM (Read Only Memory).

The processing unit 42 executes the programs stored in the memory unit 41 to perform various processes such as numerical calculations, information processing, and device control. The processing unit 42 is configured with a processor such as a CPU (Central Processing Unit) or a microcomputer. Alternatively, the processing unit 42 is configured with an integrated circuit such as an ASIC (Application Specific Integrated Circuit). The integrated circuit has a logic circuit. The processing unit 42 may be configured with a processor and an integrated circuit. Note that a general-purpose computer such as a personal computer may be used as the calculation unit 40.

The processing unit 42 measures the change over time of the position of the particle p in the cell 21 from the observation results of the observation unit 30 (the signal generated by the observation unit 30). For example, the processing unit 42 measures the position of the particle p in the cell 21 at each predetermined time interval. In other words, it measures the position of the particle p at different times. The processing unit 42 measures the magnetophoretic velocity v of the particle p from the change over time of the position of the particle p.

The processing unit 42 also measures the particle diameter d of the particle p from the signal generated by the observation unit 30. The processing unit 42 further measures the volume magnetic susceptibility χs of the particle p based on the magnetophoretic velocity v and particle diameter d of the particle p.

For example, the processing unit 42 calculates the volume magnetic susceptibility χs of the particle p based on the following formula (1).
v={2(χs-χm)r ² /9ημ _o }B(dB/dx)...(1)

In formula (1), v is the magnetophoretic velocity of particle p, χs is the volume magnetic susceptibility of particle p, χm is the volume magnetic susceptibility of medium m, r is the radius of particle p, η is the viscosity of medium m, _μo is the permeability of a vacuum, B is the magnetic flux density, and dB/dx is the magnetic field gradient (gradient of magnetic flux density). Formula (1) is derived from the fact that the difference in magnetic force acting on particle p and medium m in the axial direction (x direction) of cell 21 (capillary tube) is approximately equal to the viscous resistance force.

The magnetophoretic velocity v and the volume magnetic susceptibility χs are measured values measured by the processing unit 42. The radius r of the particle p is calculated, for example, by the processing unit 42 from the particle diameter d of the particle p measured. The volume magnetic susceptibility χm of the medium m, the viscosity η of the medium m, the magnetic permeability μ _o of the vacuum, the magnetic flux density B, and the magnetic field gradient dB/dx are stored in advance in the storage unit 41. For example, an analyst can operate an input device such as a keyboard, a mouse, or a touch display to store the volume magnetic susceptibility χm of the medium m, the viscosity η of the medium m, the magnetic permeability μ _o of the vacuum, the magnetic flux density B, and the magnetic field gradient dB/dx in the storage unit 41. The volume magnetic susceptibility χm of the medium m, the viscosity η of the medium m, and the magnetic permeability μ _o of the vacuum are, for example, literature values. The magnetic flux density B and the magnetic field gradient dB/dx are, for example, measured values.

The memory unit 41 stores the volume magnetic susceptibility χs1 of the first particle p1 measured in the first magnetic susceptibility measurement process S4. The memory unit 41 also stores the volume magnetic susceptibility χs2 of the second particle p2 measured in the second magnetic susceptibility measurement process S8. The processing unit 42 accesses the memory unit 41 and acquires the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2 from the memory unit 41. The processing unit 42 then compares the volume magnetic susceptibility χs1 of the first particle p1 with the volume magnetic susceptibility χs2 of the second particle p2 to analyze whether the component pt to be included is included in the second particle p2. More specifically, if the volume magnetic susceptibility χs2 of the second particle p2 is closer to the volume magnetic susceptibility χst of the component pt to be encapsulated than the volume magnetic susceptibility χs1 of the first particle p1, the processing unit 42 determines that the component pt to be encapsulated is encapsulated in the second particle p2.

The volume magnetic susceptibility χst of the inclusion target component pt is stored in advance in the storage unit 41. For example, the volume magnetic susceptibility χst of the inclusion target component pt can be a literature value. In this case, the analyst operates the input device to store the volume magnetic susceptibility χst (literature value) of the inclusion target component pt in the storage unit 41. Alternatively, the analyst may use the analysis device 10 to measure the volume magnetic susceptibility χst of the inclusion target component pt. In this case, the volume magnetic susceptibility χst of the inclusion target component pt measured by the processing unit 42 is stored in the storage unit 41.

The output unit 43 outputs the analysis result (determination result) by the processing unit 42. Specifically, the output unit 43 outputs information indicating whether or not the component pt to be encapsulated is encapsulated in the second particle p2. The output unit 43 includes, for example, a display. In this case, the processing unit 42 displays information indicating the analysis result on the display. The output unit 43 may include a communication interface in addition to the display or instead of the display. The communication interface is, for example, a USB (Universal Serial Bus) connector. In this case, the processing unit 42 outputs a signal indicating the analysis result to an external device via the communication interface and a USB cable. The external device is, for example, a display or a printer. When the external device is a display, information indicating the analysis result is displayed on the external device. When the external device is a printer, an image indicating the analysis result is printed. Alternatively, the external device may be a USB memory. In this case, the processing unit 42 outputs a signal indicating the analysis result to the USB memory via the communication interface. As a result, information showing the analysis results is stored in the USB memory.

Next, the movement of particle p will be described with reference to Figures 3(a) and 3(b). Figures 3(a) and 3(b) are diagrams showing the movement of particle p. As shown in Figures 3(a) and 3(b), the magnetic field generating unit 20 includes a permanent magnet 20a with a north magnetic pole and a permanent magnet 20b with a south magnetic pole. The two permanent magnets 20a and 20b face each other with the cell 21 in between.

As shown in FIG. 3(a), when the volume magnetic susceptibility χs of particle p is smaller than the volume magnetic susceptibility χm of medium m, particle p moves in a direction away from the magnetic field (magnetic field generating unit 20). On the other hand, as shown in FIG. 3(b), when the volume magnetic susceptibility χs of particle p is larger than the volume magnetic susceptibility χm of medium m, particle p moves in a direction toward the magnetic field (magnetic field generating unit 20).

As shown in Figures 3(a) and 3(b), the movement of particle p is determined according to the relationship between the volume magnetic susceptibility χs of particle p and the volume magnetic susceptibility χm of medium m. Note that particle p is subjected to force in the vicinity of the ends of permanent magnets 20a and 20b. For example, particle p is subjected to force within a range of about ±200 μm from the vicinity of the ends of permanent magnets 20a and 20b.

Next, the analysis device 10 will be further described with reference to FIG. 4. FIG. 4 is a diagram showing the configuration of the analysis device 10. As shown in FIG. 4, the analysis device 10 further includes a light source 50. In addition, the observation unit 30 includes a magnification unit 32 and an imaging unit 34.

The light source 50 emits light of a relatively high intensity that includes a visible light component. The light source 50 irradiates the cell 21 with light. As a result, the particle p is irradiated with light. The wavelength spectrum of the light emitted from the light source 50 may be relatively broad. For example, a halogen lamp is preferably used as the light source 50.

The particle p introduced into the cell 21 is magnified at an appropriate magnification by the magnifying unit 32 and imaged by the imaging unit 34. The position of the particle p can be identified from the imaging result of the imaging unit 34 (the image captured by the imaging unit 34). For example, the magnifying unit 32 includes an objective lens, and the imaging unit 34 includes a charge coupled device (CCD). Alternatively, each pixel of the imaging unit 34 may be composed of a photodiode or a photomultiplier tube. The imaging unit 34 images the particle p at, for example, a predetermined time interval. The imaging unit 34 may image the light emitted from the light source 50 and transmitted through the cell 21, or may image the light emitted from the light source 50 and scattered by the particle p.

The calculation unit 40 (processing unit 42) measures the change in the position of the particle p over time from the imaging results of the imaging unit 34, and measures the magnetic migration velocity v of the particle p from the change in the position of the particle p over time.

The calculation unit 40 (processing unit 42) also measures the particle diameter d of the particle p from the imaging result of the particle p. For example, the calculation unit 40 (processing unit 42) executes the following process. That is, first, the image captured by the imaging unit 34 is converted to monochrome and its brightness is quantified. Next, the differential value of the brightness value is compared with a threshold value to set the boundary of the particle p. Next, the area of the particle p is detected from the set boundary, and the particle diameter d is calculated from the radius of the circle corresponding to that area. Alternatively, the center of the particle p is specified, multiple straight lines are drawn that pass through the center of the particle p, and the average distance between two points where each straight line intersects with the boundary of the particle p is calculated.

The first embodiment of the present invention has been described above with reference to Figs. 1 to 4. According to this embodiment, it is possible to analyze whether or not the component pt to be included is included in the particle to be analyzed (second particle p2) for each analysis target.

The particles p for which the volume magnetic susceptibility χs1 is measured may be all or a part of the first particles p1 prepared in the first particle preparation process S1. Similarly, the particles p for which the volume magnetic susceptibility χs2 is measured may be all or a part of the second particles p2 obtained in the encapsulation process S5.

[Embodiment 2]
Next, a second embodiment of the present invention will be described with reference to Fig. 5. However, differences from the first embodiment will be described, and a description of the same matters as in the first embodiment will be omitted. The second embodiment differs from the first embodiment in that the second particles p2 are spectroscopically analyzed.

Figure 5 is a flowchart showing the particle manufacturing method according to this embodiment. The particle manufacturing method according to this embodiment further includes a spectroscopic analysis step S10.

<Spectroscopic analysis step S10>
In the spectroscopic analysis step S10, the second particle p2 is observed spectroscopically to analyze whether or not the component pt to be included is included in the second particle p2. Specifically, the second particle p2 is irradiated with laser light, the scattered light scattered from the second particle p2 is detected, and the spectrum of the scattered light is analyzed. By analyzing the spectrum of the scattered light scattered from the sample, the molecular structure of the sample can be identified, and therefore, it is possible to analyze whether or not the component pt to be included is included in the second particle p2 from the scattered light scattered from the second particle p2. Specifically, it is analyzed whether or not the spectrum of the scattered light includes a spectrum indicating the component pt to be included.

For example, by analyzing the Raman spectrum using a Raman spectrometer, it is possible to analyze whether or not the component pt to be included is included in the second particle p2. The Raman spectrometer may include a microscope. In other words, the Raman spectrometer may be a confocal Raman microscope.

Specifically, the Raman spectroscopy device includes a laser light source, a detector, and a calculation unit. The laser light source irradiates the second particle p2 with laser light to generate scattered light from the second particle p2. The detector detects the scattered light. The calculation unit of the Raman spectroscopy device analyzes the spectrum of the scattered light based on the output of the detector. In detail, the calculation unit identifies the molecular structure of the second particle p2 by analyzing the spectrum of the scattered light. The calculation unit includes, for example, a microcomputer or an ASIC.

The calculation unit 40 described with reference to FIG. 2 may be used to analyze the spectrum of the scattered light. In particular, the output from the detector of the Raman spectrometer is input to the calculation unit 40. The processing unit 42 of the calculation unit 40 analyzes the spectrum of the scattered light based on the output of the detector, and analyzes from the analysis results whether the spectrum of the scattered light contains a spectrum indicating the component pt to be included.

Above, the second embodiment of the present invention has been described with reference to FIG. 5. According to this embodiment, in addition to the analysis based on the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2, it is possible to spectroscopically analyze whether or not the component pt to be included is included in the second particle p2. Therefore, it is possible to more reliably analyze for each analysis target whether or not the component pt to be included is included in the particle to be analyzed (second particle p2).

The particles p to be analyzed spectroscopically may be all or a portion of the second particles p2 obtained in the encapsulation process S5.

In the flowchart shown in FIG. 5, the spectroscopic analysis step S10 is performed after the magnetic susceptibility analysis step S9, but the order in which the spectroscopic analysis step S10 is performed is not limited to after the magnetic susceptibility analysis step S9. The spectroscopic analysis step S10 may be performed, for example, before the second observation step S6.

In addition, in this embodiment, a Raman spectrometer is used, but the spectrometer is not limited to a Raman spectrometer. For example, an ultraviolet-visible spectrometer or an infrared spectrometer may be used. When an ultraviolet-visible spectrometer is used, the ultraviolet-visible absorption spectrum is analyzed to analyze whether the component pt to be included is included in the second particle p2. When an infrared spectrometer is used, the infrared absorption spectrum is analyzed to analyze whether the component pt to be included is included in the second particle p2. Alternatively, a near-infrared spectrum may be obtained using an infrared spectrometer, and the near-infrared spectrum may be analyzed by statistical processing to analyze whether the component pt to be included is included in the second particle p2. In other words, the near-infrared spectrum may be analyzed chemometrically to analyze whether the component pt to be included is included in the second particle p2.

[Embodiment 3]
Next, a third embodiment of the present invention will be described with reference to Fig. 6. However, differences from the first and second embodiments will be described, and a description of the same aspects as the first and second embodiments will be omitted. The third embodiment differs from the first and second embodiments in that the zeta potentials of the first particle p1 and the second particle p2 are analyzed.

Figure 6 is a flowchart showing the particle manufacturing method according to this embodiment. The particle manufacturing method according to this embodiment further includes a first zeta potential measurement step S11, a second zeta potential measurement step S12, and a zeta potential analysis step S13.

<First zeta potential measurement step S11>
In the first zeta potential measurement step S11, the zeta potential of the first particles p1 is measured. More specifically, the first particles p1 are electrophoresed in a medium m, and the electrophoretic velocity of the first particles p1 is measured. Then, the zeta potential of the first particles p1 is measured based on the electrophoretic velocity.

Specifically, the zeta potential is measured using a zeta potential measuring device. The zeta potential measuring device has a positive electrode and a negative electrode. A cell is placed between the positive electrode and the negative electrode. A solution in which the first particles p1, which are solutes (dispersoids), are dispersed is enclosed in the cell. The zeta potential measuring device applies a voltage to the positive electrode to generate an electric field between the positive electrode and the negative electrode. As a result, the first particles p1 electrophoretically migrate. Zeta potential measuring devices include devices that measure the average value of the zeta potential of all the particles p dispersed in the cell, and devices that measure the individual zeta potentials of the particles p dispersed in the cell.

The device for measuring the average value of the zeta potential further includes a laser light source, a detector, and a calculation unit. The laser light source irradiates the entire electrophoretic first particle p1 with laser light to generate scattered light from the entire first particle p1. The detector detects the scattered light. More specifically, the frequency of the scattered light generated from the entire electrophoretic first particle p1 shifts (changes) due to the Doppler effect. The detector detects the frequency of the scattered light that shifts (changes) due to the Doppler effect.

The calculation unit of the device for measuring the average value of the zeta potential measures the average value of the electrophoretic velocity based on the output of the detector. In particular, the amount of shift (change) in the frequency of the scattered light is proportional to the average value of the electrophoretic velocity. The calculation unit measures the average value of the electrophoretic velocity of the entire first particle p1 by measuring the amount of shift in the frequency of the scattered light. Then, the calculation unit measures the average value of the zeta potential based on the average value of the electrophoretic velocity. The calculation unit includes, for example, a microcomputer or an ASIC. Note that the calculation unit 40 described with reference to FIG. 2 may be used as the calculation unit.

The device for measuring the individual zeta potentials of particles p further includes a laser light source, a detector, and a calculation unit, similar to the device for measuring the average value of the zeta potential. The laser light source irradiates the entire electrophoretic first particle p1 with laser light to generate scattered light from the entire first particle p1. The detector includes an objective lens and an image sensor. The image sensor may be a semiconductor sensor such as a CCD image sensor or a CMOS image sensor. The detector captures the light magnified by the objective lens using the image sensor. As a result, the movement of each of the first particles p1 is captured.

The calculation unit of the device that measures the individual zeta potentials of the particles p measures the individual electrophoretic velocities of the first particles p1 based on the image signals output from the detector. The calculation unit then measures the individual zeta potentials of the first particles p1 based on the electrophoretic velocities. The calculation unit includes, for example, a microcomputer or an ASIC. Note that the calculation unit 40 described with reference to FIG. 2 may be used as the calculation unit.

<Second zeta potential measurement step S12>
In the second zeta potential measurement step S12, the zeta potential of the second particles p2 is measured. The second zeta potential measurement step S12 is similar to the first zeta potential measurement step S11, except that the measurement target is the second particles p2.

<Zeta potential analysis step S13>
In the zeta potential analysis step S13, it is analyzed whether or not the component pt to be included is included in the second particle p2 based on the result of the measurement in the first zeta potential measurement step S11 and the result of the measurement in the second zeta potential measurement step S12. Specifically, the zeta potential of the first particle p1 is compared with the zeta potential of the second particle p2 to analyze the difference between the zeta potential of the first particle p1 and the zeta potential of the second particle p2. When the zeta potential of the first particle p1 and the zeta potential of the second particle p2 show the same value, it is determined that the component pt to be included is included in the second particle p2.

In more detail, if the component pt to be encapsulated does not enter the second particle p2 but adheres to the surface of the second particle p2, the zeta potential of the second particle p2 will have a different value from the zeta potential of the first particle p1. In other words, if the component pt to be encapsulated does not adhere to the surface of the second particle p2 but enters the second particle p2, the zeta potential of the second particle p2 will have the same value as the zeta potential of the first particle p1. Therefore, by comparing the zeta potential of the first particle p1 with the zeta potential of the second particle p2, it is possible to analyze whether the component pt to be encapsulated is encapsulated in the second particle p2.

The calculation unit 40 described with reference to FIG. 2 may be used to analyze the zeta potentials of the first particle p1 and the second particle p2. In more detail, the analyst operates the input device to store data indicating the zeta potential of the first particle p1 and data indicating the zeta potential of the second particle p2 in the memory unit 41. Alternatively, the zeta potential measuring device and the calculation unit 40 are connected via a transmission cable such as a USB cable, and the data indicating the zeta potential of the first particle p1 and data indicating the zeta potential of the second particle p2 are transmitted from the zeta potential measuring device to the calculation unit 40. The processing unit 42 of the calculation unit 40 compares the zeta potential of the first particle p1 with the zeta potential of the second particle p2 to analyze whether the component pt to be included is included in the second particle p2.

Above, the third embodiment of the present invention has been described with reference to FIG. 6. According to this embodiment, in addition to the analysis based on the volume magnetic susceptibility χs1 of the first particle p1 and the volume magnetic susceptibility χs2 of the second particle p2 and the spectroscopic analysis, it is possible to analyze whether or not the component pt to be included is included in the second particle p2 from the zeta potentials of the first particle p1 and the second particle p2. Therefore, it is possible to more reliably analyze for each analysis target whether or not the component pt to be included is included in the particle to be analyzed (second particle p2).

The particles p whose zeta potential is to be measured in the first zeta potential measurement step S11 may be all or a part of the first particles p1 prepared in the first particle preparation step S1. Similarly, the particles p whose zeta potential is to be measured in the second zeta potential measurement step S12 may be all or a part of the second particles p2 obtained in the encapsulation process S5.

In the flowchart shown in FIG. 6, the first zeta potential measurement step S11 is performed after the first magnetic susceptibility measurement step S4, but the order in which the first zeta potential measurement step S11 is performed is not limited to after the first magnetic susceptibility measurement step S4. The first zeta potential measurement step S11 may be performed, for example, before the first observation step S2.

Similarly, in the flowchart shown in FIG. 6, the second zeta potential measurement step S12 is performed after the second magnetic susceptibility measurement step S8, but the order in which the second zeta potential measurement step S12 is performed is not limited to after the second magnetic susceptibility measurement step S8. The second zeta potential measurement step S12 may be performed, for example, before the second observation step S6.

In the flowchart shown in FIG. 6, the zeta potential analysis step S13 is performed after the spectroscopic analysis step S10, but the order in which the zeta potential analysis step S13 is performed is not limited to after the spectroscopic analysis step S10. The zeta potential analysis step S13 may be performed after the second zeta potential measurement step S12. For example, the zeta potential analysis step S13 may be performed before the magnetic susceptibility analysis step S9.

In addition, in this embodiment, the spectroscopic analysis process S10 is performed, but the spectroscopic analysis process S10 may be omitted.

The above describes an embodiment of the present invention with reference to the drawings. Note that the present invention is not limited to the above embodiment, and can be implemented in various aspects without departing from the spirit of the present invention.

For example, in the embodiment of the present invention, the particle diameter d of the particle p is measured by image analysis, but the particle diameter d of the particle p may also be measured by analyzing the Brownian motion of the particle p.

Specifically, the diffusion coefficient is calculated from the variance of the change (displacement) in the position of particle p in the direction (y direction) perpendicular to the axial direction (x direction) of cell 21 (capillary tube), and the particle diameter d of particle p can be measured from this diffusion coefficient. In more detail, particle p is affected by the magnetic field gradient in the axial direction (x direction) of cell 21 (capillary tube), but is hardly affected by the magnetic field gradient in the direction (y direction) perpendicular to the axial direction of cell 21. Therefore, the diffusion coefficient D can be calculated from the variance of the displacement of the position of particle p in the y direction. Specifically, the diffusion coefficient D can be calculated by dividing the square of the movement distance in the y direction of particle p performing Brownian motion by twice the time.

The processing unit 42 measures the particle diameter d of the particle p from the diffusion coefficient D based on the following formula (2): In formula (2), d is the particle diameter of the particle p, k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the medium m.
d=kT/(3πηD)...(2)

In the embodiment of the present invention, the analysis device 10 includes the light source 50, but the analysis device 10 may include a laser light source instead of the light source 50, or may further include a laser light source in addition to the light source 50. When the analysis device 10 includes the light source 50 and the laser light source, when the light source 50 emits light, the laser light emission from the laser light source is stopped, and when the laser light source emits laser light, the light emission from the light source 50 is stopped. When the laser light source is used, the laser light is irradiated onto the particle p introduced into the cell 21. The observation unit 30 observes the particle p by the laser light (scattered light) scattered by the particle p in the cell 21. For example, the imaging unit 34 described with reference to FIG. 3 images the laser light scattered by the particle p via the magnification unit 32.

When a laser light source is used, the particle diameter d of the particle p may be measured, for example, based on dynamic light scattering or static light scattering. When the particle p is irradiated with laser light, the capillary tube is preferably a square capillary with a square cross-sectional shape perpendicular to the axial direction. By using a square capillary, it becomes easier to give a mirror finish to the side surface of the cell 21 that is irradiated with the laser light.

In addition, in the embodiment of the present invention, the calculation unit 40 (processing unit 42) measures the particle diameter d of the particle p, but the image captured by the imaging unit 34 may be displayed on a display, and the analyst may measure the particle diameter d of the particle p from the image displayed on the display. Alternatively, the image captured by the imaging unit 34 may be printed, and the analyst may measure the particle diameter d of the particle p from the printed image. In this case, the analyst operates the input device to store data indicating the particle diameter d in the memory unit 41.

In the embodiment of the present invention, the particle diameter d is measured, but a literature value may be used as the particle diameter d. In this case, the analyst operates the input device to store data indicating the particle diameter in the memory unit 41.

In addition, in the embodiment of the present invention, the imaging unit 34 measures the magnetophoretic velocity v of the particle p by capturing an image of the particle p at a predetermined time interval, but the magnetophoretic velocity v of the particle p may also be measured using a laser light source, for example, based on the laser Doppler method.

The present invention will be described in more detail below using examples. Note that the present invention is not limited to the scope of the examples.

[Example 1]
<Production of yeast-derived capsule base material>
First, a capsule substrate D11 derived from yeast was produced. Hereinafter, the capsule substrate derived from yeast may be referred to as a "yeast capsule substrate". In Example 1, soluble components were removed from baker's yeast by hot water treatment to obtain a paste-like solid component, and the solid component was sterilized and dried to obtain the yeast capsule substrate D11. The yeast capsule substrate D11 was in a dry state. Then, the yeast capsule substrate D11 was made into a paste. Specifically, the dried yeast capsule substrate D11 was added to distilled water to prepare a solution L1, and the solution L1 was heated and sterilized. Then, the solution L1 was cooled to room temperature to make the yeast capsule substrate D11 into a paste.

<Cleaning treatment>
The washing process was performed twice. Specifically, the yeast capsule substrate D11 in the form of a paste was added to distilled water and stirred to prepare a solution L2. Then, the distilled water was centrifuged from the solution L2 using a centrifuge to obtain the yeast capsule substrate D11 after the first washing process. Then, the yeast capsule substrate D11 after the first washing process was added to distilled water again and stirred to prepare a solution L3. Then, the distilled water was centrifuged from the solution L3 using a centrifuge to obtain the yeast capsule substrate D11 after the second washing process. Hereinafter, the "yeast capsule substrate D11 after the second washing process" and the like may be described as "yeast capsule substrate D11 after washing" and the like. The yeast capsule substrate D11 after washing was in the form of a paste.

<Volume magnetic susceptibility measurement of yeast-derived capsule base material>
The individual volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing) was measured. Specifically, the first sample SP1 (yeast capsule substrate D11 after washing) was added to distilled water, and the distilled water was irradiated with ultrasonic waves (ultrasonic treatment) to prepare a solution L4. After ultrasonic treatment, the solution L4 was sealed in a cell, and the individual volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing) was measured. In addition, after measuring the volume magnetic susceptibility, the solution L4 was sealed in a hemocytometer, and the number of the first sample SP1 (yeast capsule substrate D11 after washing) was counted using a microscope.

<Encapsulation treatment>
The encapsulation process was carried out using the yeast capsule substrate D11 in the form of a paste, d-limonene, and distilled water. The mixing ratio of the yeast capsule substrate D11 in the form of a paste, d-limonene, and distilled water was 2:1:7 by weight. Here, the mixing ratio of d-limonene indicates the weight of d-limonene when the weight of the yeast capsule substrate D11 is set to "2". Similarly, the mixing ratio of distilled water indicates the weight of distilled water when the weight of the yeast capsule substrate D11 is set to "2".

Specifically, d-limonene was added to distilled water, and the temperature was then raised. Then, paste-like yeast capsule substrate D11 was added to the distilled water and stirred to prepare solution L5. After stirring, solution L5 was heated and sterilized. Then, solution L5 was cooled to room temperature to prepare paste-like microcapsules D12. Microcapsules D12 are yeast capsule substrate D11 after encapsulation treatment. Note that d-limonene is a hydrophobic substance, and yeast capsule substrate D11 has a hydrophobic region inside.

<Cleaning treatment>
The washing process was performed twice. Specifically, the paste-like microcapsules D12 were added to distilled water and stirred to prepare a solution L6. Then, the distilled water was centrifuged from the solution L6 using a centrifuge to obtain the microcapsules D12 after the first washing process. Then, the microcapsules D12 after the first washing process were added to distilled water again and stirred to prepare a solution L7. Then, the distilled water was centrifuged from the solution L7 using a centrifuge to prepare the microcapsules D12 after the second washing process. Hereinafter, the "microcapsules D12 after the second washing process" and the like may be described as the "microcapsules D12 after washing" and the like. The microcapsules D12 after washing were in a paste form.

<Volume magnetic susceptibility measurement of microcapsules>
The volume magnetic susceptibility of each of the second sample SP2 (microcapsules D12 after washing) was measured. Specifically, the second sample SP2 was added to distilled water, and the distilled water was irradiated with ultrasonic waves (ultrasonic treatment) to prepare solution L8. After ultrasonic treatment, solution L8 was sealed in a cell, and the volume magnetic susceptibility of each of the second sample SP2 (microcapsules D12 after washing) was measured. In addition, after measuring the volume magnetic susceptibility, solution L8 was sealed in a hemocytometer, and the number of the second sample SP2 (microcapsules D12 after washing) was counted using a microscope.

Table 1 shows the measurement results of Example 1.

Figure 7 is a diagram showing the measurement results of Example 1. In detail, Figure 7 shows the measurement results of the volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing) and the second sample SP2 (microcapsule D12 after washing). The first sample SP1 and the second sample SP2 correspond to the first particle p1 and the second particle p2, respectively, in the above embodiment. In Figure 7, the horizontal axis shows the volume magnetic susceptibility, and the vertical axis shows the percentage (%) of the number of particles. Also, in Figure 7, the solid line graph shows the measurement results of the volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing), and the dashed line graph shows the measurement results of the volume magnetic susceptibility of the second sample SP2 (microcapsule D12 after washing).

Specifically, the solid line graph was created by measuring the individual volume magnetic susceptibilities of the first sample SP1 and calculating the proportion of the number of first samples SP1 for each measured volume magnetic susceptibility. Similarly, the dashed line graph was created by measuring the individual volume magnetic susceptibilities of the second sample SP2 and calculating the proportion of the number of second samples SP2 for each measured volume magnetic susceptibility.

As shown in Table 1 and FIG. 7, the volume magnetic susceptibility is different between the first sample SP1 and the second sample SP2. The difference in volume magnetic susceptibility is due to the encapsulation of d-limonene in the microcapsules D12. Specifically, the volume magnetic susceptibility of d-limonene is "-8.48 x 10 ^-6 ". As shown in Table 1, the average value of the volume magnetic susceptibility of the second sample SP2 (microcapsules D12 after washing) is closer to the volume magnetic susceptibility of d-limonene than the average value of the volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing). Also, as shown in FIG. 7, the peak of the volume magnetic susceptibility of the second sample SP2 (microcapsules D12 after washing) is closer to the volume magnetic susceptibility of d-limonene than the peak of the volume magnetic susceptibility of the first sample SP1 (yeast capsule substrate D11 after washing). Therefore, it was possible to quantitatively analyze that d-limonene is encapsulated in the microcapsules D12.

[Example 2]
In Example 2, similarly to Example 1, the volume magnetic susceptibility of the first sample SP11 (yeast capsule substrate D21 after washing) and the volume magnetic susceptibility of the second sample SP12 (microcapsule D22 after washing) were measured. Example 2 is similar to Example 1 except that the type of yeast capsule substrate is different. In detail, in Example 2, after obtaining a paste-like solid component from baker's yeast by hot water treatment, an emulsifier was added to the paste-like solid component. Then, protease was further added to the paste-like solid component. Glycerol fatty acid ester was used as the emulsifier. An endo-type protease was used as the protease. The yeast capsule substrate D21 has a hydrophobic region inside.

Table 2 shows the measurement results of Example 2.

Figure 8 is a diagram showing the measurement results for Example 2. In detail, Figure 8 shows the measurement results of the volume magnetic susceptibility of the first sample SP11 and the second sample SP12. In Figure 8, the horizontal axis shows the volume magnetic susceptibility, and the vertical axis shows the percentage (%) of the particle number. In addition, in Figure 8, the solid line graph shows the measurement results of the volume magnetic susceptibility of the first sample SP11 (yeast capsule substrate D21 after washing), and the dashed line graph shows the measurement results of the volume magnetic susceptibility of the second sample SP12 (microcapsules D22 after washing).

As shown in Table 2 and FIG. 8, the first sample SP11 and the second sample SP12 have different volume magnetic susceptibilities. Specifically, as shown in Table 2, the average value of the volume magnetic susceptibility of the second sample SP12 (microcapsules D22 after washing) is closer to the volume magnetic susceptibility of d-limonene than the average value of the volume magnetic susceptibility of the first sample SP11 (yeast capsule substrate D21 after washing). Also, as shown in FIG. 8, the peak of the volume magnetic susceptibility of the second sample SP12 (microcapsules D22 after washing) is closer to the volume magnetic susceptibility of d-limonene than the peak of the volume magnetic susceptibility of the first sample SP11 (yeast capsule substrate D21 after washing). Therefore, it was possible to quantitatively analyze that d-limonene is encapsulated in the microcapsules D22.

[Example 3]
In Example 3, similarly to Example 1, the volume magnetic susceptibility of the first sample SP21 (yeast capsule substrate D31 after washing) and the volume magnetic susceptibility of the second sample SP22 (microcapsule D32 after washing) were measured. Example 3 is similar to Example 1, except for the type of yeast and the process for releasing intracellular components from the yeast. More specifically, in Example 3, torula yeast was used. In addition, intracellular components were released from torula yeast by an enzymatic decomposition method. The yeast capsule substrate D31 has a hydrophobic region inside.

Table 3 shows the measurement results of Example 3.

Figure 9 shows the measurement results for Example 3. In detail, Figure 9 shows the measurement results of the volume magnetic susceptibility of the first sample SP21 and the second sample SP22. In Figure 9, the horizontal axis shows the volume magnetic susceptibility, and the vertical axis shows the percentage (%) of the particle number. In addition, in Figure 9, the solid line graph shows the measurement results of the volume magnetic susceptibility of the first sample SP21 (yeast capsule substrate D31 after washing), and the dashed line graph shows the measurement results of the volume magnetic susceptibility of the second sample SP22 (microcapsules D32 after washing).

As shown in Table 3 and FIG. 9, the first sample SP21 and the second sample SP22 have different volume magnetic susceptibilities. Specifically, as shown in Table 3, the average value of the volume magnetic susceptibility of the second sample SP22 (microcapsules D32 after washing) is closer to the volume magnetic susceptibility of d-limonene than the average value of the volume magnetic susceptibility of the first sample SP21 (yeast capsule substrate D31 after washing). Also, as shown in FIG. 9, the peak of the volume magnetic susceptibility of the second sample SP22 (microcapsules D32 after washing) is closer to the volume magnetic susceptibility of d-limonene than the peak of the volume magnetic susceptibility of the first sample SP21 (yeast capsule substrate D31 after washing). Therefore, it was possible to quantitatively analyze that d-limonene is encapsulated in the microcapsules D32.

[Example 4]
In Example 4, the mixing ratio of the yeast capsule substrate D21 in the form of a paste, d-limonene, and distilled water was varied to carry out the encapsulation process, and the volume magnetic susceptibility of the microcapsule D22 (sample SP3) after washing was measured for each mixing ratio. Specifically, the mixing ratio of the yeast capsule substrate D21 in the form of a paste, d-limonene, and distilled water was varied in the range of "2:0:8" to "2:2.8:5.2" by weight ratio. Specifically, the mixing ratio of the yeast capsule substrate D21, d-limonene, and distilled water was set to "2:0:8", "2:0.125:7.875", "2:0.25:7.75", "2:0.5:7.5", "2:1:7", "2:2:6", "2:2.5:5.5", and "2:2.8:5.2" by weight ratio.

Figure 10 shows the first measurement results for Example 4. In detail, Figure 10 shows the measurement results of the volume magnetic susceptibility of sample SP3 (microcapsules D22 after washing). In Figure 10, the horizontal axis shows the blending ratio (weight ratio) of d-limonene, and the vertical axis shows the volume magnetic susceptibility (average value).

As shown in Figure 10, as the blending ratio of d-limonene increased, the volume magnetic susceptibility of sample SP3 (microcapsule D22 after washing) approached that of d-limonene. This result suggests that as the blending ratio of the component to be encapsulated increased, the amount of the component to be encapsulated in the microcapsules increased, causing a change in the volume magnetic susceptibility of the microcapsules.

In Example 4, the amount of d-limonene contained in sample SP3 (microcapsules D22 after washing) was further measured using high performance liquid chromatography (HPLC). Specifically, sample SP3 was added to ethanol and stirred for 30 minutes using a vortex mixer to prepare solution L9. After stirring, solution L9 was centrifuged. The supernatant was then measured using high performance liquid chromatography (HPLC). The HPLC conditions are shown below.

<HPLC conditions>
High-performance liquid chromatograph: Hitachi High-Technologies Corporation Column: Poroshell 120 EC-C18 (particle size: 2.7 μm, dimensions: 4.6 mm × 100 mm) manufactured by Agilent Technologies, Inc.
Column temperature: 40°C
Eluent: A mixture of acetonitrile and water (the mixing ratio of acetonitrile and water is 50% by weight:50% by weight)
Flow rate: 1 mL/min Detector: UV light with a wavelength of 210 nm Injection volume: 10 μL

Figure 11 shows the second measurement results for Example 4. Specifically, Figure 11 shows the measurement results for the amount of d-limonene contained in sample SP3. In Figure 11, the horizontal axis shows the blending ratio (weight ratio) of d-limonene, and the vertical axis shows the amount of d-limonene contained in sample SP3. Specifically, the vertical axis shows the ratio of the amount of d-limonene per 1 gram of microcapsules as a percentage.

As shown in Figure 11, the amount of d-limonene contained in sample SP3 (microcapsules D22 after washing) increased as the blending ratio of d-limonene increased. Therefore, by increasing the blending ratio of d-limonene, it was possible to increase the amount of d-limonene encapsulated in the microcapsules by the encapsulation process.

Figure 12 shows the third measurement result for Example 4. Specifically, Figure 12 shows the relationship between the amount of d-limonene contained in sample SP3 and the volume magnetic susceptibility of sample SP3. In Figure 12, the horizontal axis shows the amount of d-limonene contained in sample SP3. Specifically, the horizontal axis shows the ratio of the amount of d-limonene per 1 g (unit weight) of microcapsules as a percentage. The vertical axis shows the volume magnetic susceptibility (average value).

As shown in FIG. 12, in the range where the proportion of d-limonene per unit weight was 9% or less, the volume magnetic susceptibility of sample SP3 approached that of d-limonene as the amount of d-limonene per unit weight increased. On the other hand, in the range where the proportion of d-limonene per unit weight was more than 9%, the volume magnetic susceptibility of sample SP3 decreased as the amount of d-limonene per unit weight increased. In other words, the volume magnetic susceptibility was saturated. From this result, it was confirmed that the amount of d-limonene encapsulated in the microcapsules can be quantitatively analyzed using the volume magnetic susceptibility until the volume magnetic susceptibility is saturated. Hereinafter, the amount of d-limonene encapsulated in the microcapsules will be referred to as the "amount of limonene encapsulated."

Figure 13 shows the fourth measurement result for Example 4. In detail, Figure 13 shows the relationship between the blending ratio of d-limonene and the ratio of the amount of limonene encapsulated to the total amount of d-limonene used in the encapsulation process. Hereinafter, the ratio of the amount of limonene encapsulated to the total amount of d-limonene used in the encapsulation process will be referred to as the "ratio of the amount of limonene encapsulated." In Figure 13, the horizontal axis shows the blending ratio (weight ratio) of d-limonene, and the vertical axis shows the ratio (%) of the amount of limonene encapsulated.

As shown in Figure 13, the amount of d-limonene contained in sample SP3 (microcapsules D22 after washing) increased as the proportion of d-limonene increased. On the other hand, as the proportion of d-limonene increased, the proportion of limonene encapsulated decreased. This result suggests that as the proportion of the component to be encapsulated increases, the amount of the component to be encapsulated that is not encapsulated in the microcapsules increases. Furthermore, even when the proportion of d-limonene was such that, in theory, all of it would be encapsulated in microcapsules D22, the proportion of limonene encapsulated did not reach 100%. This result suggests that the process of encapsulating the component to be encapsulated in microcapsules follows the theory of chemical equilibrium.

[Example 5]
In Example 5, salt was added to distilled water to carry out the encapsulation process, and the volume magnetic susceptibility of the microcapsules D22 (sample SP4) after washing was measured. In other words, the encapsulation process was carried out using saline as a solvent. The mixing ratio of the paste-like yeast capsule base material D21, d-limonene, distilled water, and salt was 2:1:7:1 by weight.

Figure 14 is a diagram showing the first measurement results according to Example 5. In detail, Figure 14 shows the measurement results of the volume magnetic susceptibility of sample SP4. In Figure 14, the horizontal axis shows the volume magnetic susceptibility, and the vertical axis shows the percentage (%) of the particle number. In Figure 14, the solid line graph shows the measurement results of the volume magnetic susceptibility of sample SP4. Note that in Figure 14, as a reference example, the dashed line graph shows the measurement results of the volume magnetic susceptibility of the second sample SP12 (microcapsules D22 after cleaning) described in Example 2.

As shown in Figure 14, by adding salt, the volume magnetic susceptibility of microcapsule D22 after washing became closer to the volume magnetic susceptibility of d-limonene.

In Example 5, the amount of d-limonene contained in sample SP4 (amount of d-limonene per 1 g of microcapsules) was measured using high performance liquid chromatography in the same manner as in Example 4.

Figure 15 shows the second measurement results for Example 5. In detail, Figure 15 shows the ratio of limonene encapsulation in sample SP4. Note that Figure 15 also shows, as a reference example, the ratio of limonene encapsulation in the second sample SP12 (microcapsules D22 after washing) described in Example 2.

As shown in Figure 15, the amount of d-limonene encapsulated in microcapsules D22 after washing increased by adding salt. Therefore, by adding salt to the solvent to reduce the solubility of d-limonene in the solvent, the amount of the target component encapsulated in microcapsules D22 after washing increased. This result suggests that the amount of the target component encapsulated in the microcapsules is affected by the solubility of the target component in the solvent.

[Example 6]
In Example 6, ethyl hexanoate was encapsulated in the yeast capsule substrate D21 as a component to be encapsulated, to prepare sample SP5 (microcapsules D22 after washing), and the volume magnetic susceptibility of sample SP5 was measured. In Example 6, the mixing ratio of the yeast capsule substrate D21 in the form of a paste, ethyl hexanoate, and distilled water was varied in the range of "2:0:8" to "2:2:6" by weight ratio to perform the encapsulation process. Specifically, the mixing ratio of the yeast capsule substrate D21, ethyl hexanoate, and distilled water was set to "2:0:8", "2:0.125:7.875", "2:0.25:7.75", "2:0.5:7.5", "2:1:7", and "2:2:6" by weight ratio. Here, the mixing ratios of ethyl hexanoate and distilled water respectively indicate the weights of ethyl hexanoate and distilled water when the weight of the yeast capsule substrate D11 is "2". Ethyl hexanoate is a hydrophobic substance.

Figure 16 shows the measurement results for Example 6. In detail, Figure 16 shows the measurement results for the volume magnetic susceptibility of sample SP5. In Figure 16, the horizontal axis shows the blend ratio (weight ratio) of ethyl hexanoate, and the vertical axis shows the volume magnetic susceptibility (average value).

16, as the blending ratio of ethyl hexanoate increased, the volume magnetic susceptibility of sample SP5 (microcapsule D22 after washing) approached the volume magnetic susceptibility of ethyl hexanoate, which is −6.94×10 ⁻⁶ . This result suggests that as the blending ratio of the component to be encapsulated increased, the amount of the component to be encapsulated in the microcapsules increased, causing a change in the volume magnetic susceptibility of the microcapsules.

The present invention is useful, for example, for evaluating encapsulating materials used in DDS.

10 Analysis device 20 Magnetic field generating unit 21 Cell 30 Observation unit 40 Calculation unit 41 Storage unit 42 Processing unit

Claims (6)

a first observation step of magnetophoretically observing the first particles;
a first migration velocity measurement step of measuring a magnetophoretic velocity of the first particles from a result of the observation in the first observation step;
a first magnetic susceptibility measurement step of measuring a volume magnetic susceptibility of the first particles based on a result of the measurement in the first migration velocity measurement step;
an inclusion process for performing an inclusion process to cause the first particles to include a component to be included that is smaller than the first particles;
a second observation step of magnetically phoresing the second particles to be analyzed, which are the first particles after the encapsulation treatment, and observing the second particles;
a second migration velocity measuring step of measuring a magnetophoretic velocity of the second particles from a result of the observation in the second observation step;
a second magnetic susceptibility measurement step of measuring the volume magnetic susceptibility of the second particles based on a result of the measurement in the second migration velocity measurement step;
and analyzing whether or not the component to be included is included in the second particle based on a result of the measurement in the first magnetic susceptibility measurement step and a result of the measurement in the second magnetic susceptibility measurement step,
a first zeta potential measurement step of measuring the zeta potential of the first particles;
a second zeta potential measurement step of measuring the zeta potential of the second particles;
and analyzing whether or not the component to be included is included in the second particle based on a result of the measurement in the first zeta potential measurement step and a result of the measurement in the second zeta potential measurement step.
determining that the component to be encapsulated is encapsulated in the second particle when the volume magnetic susceptibility of the second particle is closer to the volume magnetic susceptibility of the component to be encapsulated compared to the volume magnetic susceptibility of the first particle;
A particle analysis method , wherein the volume magnetic susceptibility of the component to be encapsulated is stored in advance in a memory unit provided in the analysis device . The particle analysis method according to claim 1, further comprising a step of spectroscopically observing the second particles to analyze whether the component to be included is included in the second particles. A particle manufacturing method including the particle analysis method according to claim 1 or 2. The particle manufacturing method according to claim 3, wherein the first particles contain solid components of the yeast that remain after intracellular components are released from the yeast. The method further includes a first particle production step of producing the first particles,
The particle production method according to claim 4 , wherein the first particle production step includes a step of releasing the intracellular components from the yeast. The particle manufacturing method according to any one of claims 3 to 5, wherein the encapsulation process includes a process of mixing the first particles and the component to be encapsulated in a solvent.

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