JPWO2019156174A1 - Particles, particle-containing compositions and methods for producing particles - Google Patents

Abstract

By adding to the preparation, properties such as hardness and disintegration can be easily controlled, and in order to provide new particles having antioxidative power and immunostimulatory power, particles A having a maximum diameter of 1 nm or more and less than 150 nm or Particles B having a maximum diameter of 150 nm or more and 1000 nm or less were obtained from heat-treated yeast.

Description

The present invention is a particle obtained from heat-treated yeast having excellent thermal stability and other properties, as well as antioxidant power and immunostimulatory power, and having a maximum diameter of 1 nm or more and less than 150 nm or 150 nm or more and 1000 nm or less. The present invention relates to a composition containing the above particles and a method for producing the above particles.

So far, many fine particles have been used in various fields. As such fine particles, for example, nanoparticles such as fullerenes and carbon nanotubes and fine particles derived from natural products such as liposomes are known. The former has physical properties such as heat resistance and solvent resistance, and the latter is derived from natural products and is easily applied to the human body, and a wide variety of physical properties and properties are recognized.

However, in each technical field, it is currently desired to further improve the physical characteristics and properties of such fine particles. Further, it is difficult to manufacture fine particles as they become finer and nano-sized, and it is desired to improve their quality stability and productivity.

Among the technical fields using such fine particles, the present inventors have focused on the technical field of formulations. That is, in the technical field of formulations, excipients, stabilizers, preservatives, and molding aids are used in almost all formulations for the purpose of facilitating formulation, stabilizing quality, and enhancing usefulness. And other additives have been added (see Patent Document 1). However, when an additive for increasing the hardness of the preparation is used, the hardness of the preparation is increased, but the disintegration property tends to be lowered. On the contrary, if the disintegration property of the preparation is emphasized, there tends to be a problem that a desired hardness cannot be obtained.

By the way, among the preparations, over-the-counter drugs that do not require a prescription from a doctor or the like are displayed at pharmacies or the like, unlike medical drugs that are strictly managed, and can be easily purchased by the general public. However, since the temperature inside the store is not constant depending on the store structure of a pharmacy or the like, over-the-counter drugs tend to be required to have higher heat resistance and cold resistance than medical drugs. Similarly, foods, cosmetics, etc. tend to be required to have higher heat resistance and cold resistance.

JP 2015-193600

The present invention has been made in view of such circumstances, and by adding it to a preparation, properties such as hardness and disintegration can be easily controlled, and moreover, heat resistance and cold resistance are not impaired without impairing these properties. It provides new fine particles that can enhance the properties and exhibit sufficient properties even when blended in over-the-counter drugs.

In order to achieve the above object, the gist of the present invention is the following [1] to [8].
[1] Particles obtained from heat-treated yeast and having a maximum diameter of 1 nm or more and less than 150 nm.
[2] Particles obtained from heat-treated yeast and having a maximum diameter of 150 nm or more and 1000 nm or less.
[3] The particles of [1] or [2], wherein the particles are spheres.
[4] The particles according to any one of [1] to [3], wherein the particles contain sugar as a main component.
[5] A particle-containing composition containing any of the particles of [1] to [4].
[6] The particle-containing composition of [5], wherein the particles are contained in an amount of 10 to 99% by weight based on the entire composition.
[7] A method for producing particles according to any one of [1] to [4], which comprises a step of heating yeast and a step of separating particles from the heated product obtained by the above step. Method.
[8] The method for producing particles according to [7], wherein the heating in the step of heating the yeast is boiling.

That is, the present inventor has repeated various studies in order to obtain fine particles that can be applied to various uses. As a result, we found new fine particles that have not been clarified so far. As a result of subsequent research, it was found that these particles not only have excellent dispersibility, heat resistance, cold resistance, etc., but also have antioxidant power and immunostimulatory power.

As described above, since the particles of the present invention are obtained from heat-treated yeast, they can enjoy the health benefits derived from natural dietary fiber. Moreover, the maximum diameter is extremely small, 1 to 1000 nm, it has solubility resistance in both water and oil systems, and it is excellent in heat resistance, pressure resistance, cold resistance, drying resistance, etc., so that a wide range of formulations can be prepared. Can be blended with. Further, when the particles of the present invention are used in a preparation, properties such as hardness and disintegration can be easily controlled, and a preparation having desired properties can be obtained. Furthermore, since the particles of the present invention have antioxidant power and immune activating power, it can be expected that the particle-containing composition containing the particles suppresses active oxygen and regulates the immune system.

Among them, when the particles of the present invention are spheres, they are excellent in fluidity and dispersibility.

When the particles of the present invention contain sugar as a main component, the protein content that tends to become an allergen is relatively low or completely eliminated, so that the particles can be made safer when taken.

Further, among the particle-containing compositions of the present invention, those containing the above particles in an amount of 10 to 99% by weight based on the entire composition may further benefit from the antioxidant power and immunostimulatory power of the particles of the present invention. it can.

Further, the method for producing particles of the present invention, which comprises a step of heating yeast and a step of separating particles from the heated product obtained by the above step, can produce particles more safely. it can.

Above all, when the heating in the step of heating the yeast is boiling, the production efficiency of the particles of the present invention can be further improved.

In the present invention, the "main component" means a component that affects the characteristics of the material, and the content of the component is usually 50% by weight or more of the whole material.

It is a figure for demonstrating the manufacturing process of the particle A and the particle B which are one Embodiment of this invention. (A) is a photograph showing an aggregate of the particles A, (b) is a transmission electron micrograph of the particles A, and (c) is a transmission electron micrograph of the particles B. d) is a transmission electron micrograph of the particles contained in the separate fraction [liquid (III)] in the manufacturing process of the particles A and B, and (e) is another in the manufacturing process of the particles A and B. It is a transmission electron micrograph of the particles contained in the fraction [white suspended matter (IV)]. It is a figure which showed the distribution of the particle diameter of the said particle A. It is a figure which showed the distribution of the particle diameter of the said particle B. It is a figure which showed the distribution of the particle diameter of the particle contained in the said liquid (III). It is a figure which showed the distribution of the particle diameter of the particle contained in the said white suspended matter (IV). It is a figure which showed the Raman analysis spectrum of the said particle A. (A) is a photograph showing the state in which the particles A are dispersed in ultrapure water and dried, and (b) is a photograph showing the state in which the particles A are dispersed in ultrapure water again. Yes, (c) is a transmission electron micrograph of the particle A, and (d) is a diagram showing the distribution of the particle size of the particle A. (A) is a photograph showing a state in which the freeze-dried particles A are dispersed in ultrapure water again and autoclaved together with the ultrapure water, and (b) is a transmission electron micrograph of the particles A, (c). ) Is a diagram showing the distribution of the particle size of the particle A. (A) is a photograph showing the state after one week has passed by dispersing the freeze-dried particles A in ultrapure water again, and (b) is a transmission electron micrograph of the particles A, (c). ) Is a diagram showing the distribution of the particle size of the particle A.

Next, a mode for carrying out the present invention will be described. However, the present invention is not limited to this embodiment.

In the present invention, the "yeast" is a unicellular, almost spherical eukaryotic microorganism belonging to ascospores, basidiomycetes, etc., and a yeast that undergoes so-called fermentation in which most of the life cycle is unicellular. It refers to the whole, and means not only yeast itself but also various states such as frozen state and dry state. Yeasts belonging to the genus Saccharomyces and Schizosaccharomyces are particularly preferably used because it is easy to produce particles having high uniformity in large quantities.

In the present invention, the "particle" means a structure that looks like a two-layer structure, a double-layer structure, a multi-layer structure, or a multi-layer structure when observed with an electron microscope. That is, the particles of the present invention have at least different electron densities from the outermost layer and the inner part.

The particles according to one embodiment of the present invention have a maximum diameter of 1 to 1500 nm, preferably 40 to 1000 nm, more preferably 50 to 800 nm, and even more preferably 50 to 500 nm. In the present invention, the "maximum diameter of the particle" means the diameter of the particle when it is a sphere, and the maximum length of the particle when it has another shape. The diameter of the particles can be measured, for example, by dispersing the obtained particles in ultrapure water and measuring the dispersion using a concentrated particle size analyzer. When the particle size is measured using a concentrated particle size analyzer, the calculated average particle size should be the maximum size of the particles, and the calculated average particle size should be within the range specified by the maximum size. ..

The particles according to one embodiment of the present invention usually have a shape without sharp portions such as carbon nanotubes, and preferably have a spherical shape. In addition, the surface of the particles is smooth, and no wear marks formed by physical contact can be seen. The sphere includes not only a true sphere but also an oval shape, an ellipsoid, and the like. The shape of the particles can be determined, for example, by photographing the negatively stained particles with a transmission electron microscope and observing the appearance thereof. For example, particles collected in pellet form are dispersed in ultrapure water, this dispersion is adsorbed on a mesh, and a dyeing agent is placed on the mesh. Then, the appearance of the particles can be observed by sucking up the excess dyeing solution with a filter paper and drying it with a transmission electron microscope.

The particles according to one embodiment of the present invention have high dispersibility in an aqueous liquid. Moreover, its excellent dispersibility is maintained for a long period of time. The dispersibility of the particles can be determined, for example, by dispersing the obtained particles in ultrapure water, photographing the dispersion with a transmission electron microscope, and observing the degree of dispersion. Further, the degree of maintaining dispersibility can be determined by comparing the dispersion liquid with that after storage for a certain period of time.

The particles according to the embodiment of the present invention are excellent in pressure resistance and heat resistance, and the particle size does not change by pressurization up to at least 2 atm and heating up to 121 ° C. For the pressure resistance and heat resistance of the particles, for example, the particle size distribution of the particles dispersed in ultrapure water and the particles contained in the pressurized and heated dispersion is determined by using a concentrated particle size analyzer. When calculated and compared between the two, it can be determined from the fact that the distribution of the particle size does not fluctuate between the two.

The particles according to the embodiment of the present invention are excellent in cold resistance and drying resistance, and the particle size does not change almost by cooling and drying from -50 to -80 ° C. The cold resistance and drying resistance of the particles are included in, for example, the particles dispersed in ultrapure water and the dispersion liquid freeze-dried (-50 ° C.) and the dried product dispersed in ultrapure water again. The particle size distribution of each of these particles is calculated with a concentrated particle size analyzer, and it can be determined from the fact that the particle size distribution does not fluctuate when the two are compared. Further, even if the particles contained in the freeze-dried product after being stored at −80 ° C. for 7 days and then dispersed in ultrapure water are compared in the same manner, the distribution of the particle size does not change.

The particles according to one embodiment of the present invention have excellent antioxidant power. Among them, particles having a maximum diameter of 80 nm or more, preferably 150 nm or more and 1500 nm or less, preferably 1000 nm or less, more preferably 800 nm or less have excellent antioxidant power, and 80 nm or more and 1500 nm or less, preferably 150 nm or more and 1000 nm or less. Particles have more excellent antioxidant power, and particles of 150 nm or more and 800 nm or less have further excellent antioxidant power. The fact that the particles have antioxidant power is generated in the body by, for example, dispersing the obtained particles in ultrapure water and subjecting the dispersion to an ESR spin trapping method (hereinafter referred to as "ESR method"). The ability to scavenge superoxide radicals, hydroxyl radicals and singlet oxygen, which are active oxygen, can be measured and discriminated from the results.

The particles according to one embodiment of the present invention have excellent immunostimulatory activity. Among them, particles having a maximum diameter of 1 nm or more, preferably 50 nm or more and less than 250 nm, preferably less than 150 nm have excellent immunostimulatory activity, and particles having a maximum diameter of 1 nm or more and less than 250 nm, preferably 1 nm or more and less than 150 nm are more excellent. It has an immunostimulatory power, and particles having a diameter of 50 nm or more and less than 150 nm have an even more excellent immunostimulatory power. The fact that the particles have immunostimulatory activity means that, for example, when lipopolysaccharide (LPS) that activates immunocompetent cells such as macrophages is used as a positive control, interferon β (IFNβ) and interleukin- The production amounts of 6 (IL-6) and tumor necrosis factor α (TNFα) can be measured and discriminated from the results.

Even if pressurization, heating, cooling, and drying are performed, the structure of the particles according to the embodiment of the present invention does not change. This means that the particles are dispersed in ultrapure water, and the dispersion is pressurized and heated, or cooled and dried, and then dispersed again in ultrapure water, and the particles contained in them are measured with an electron microscope. It can be discriminated by observing the structure of. These are properties that could not be obtained with conventional nanoparticles such as liposomes.

Such particles can be produced, for example, by a method including a step of heating yeast and a step of separating the particles from the heated product obtained by the above step.

As a step of heating the yeast, for example, the yeast is placed in a heating / drying chamber set at around 95 ° C. together with the culture solution to perform heating and drying at the same time, or the yeast is immersed in a liquid and the yeast is immersed together with the liquid. It is more preferable to heat the yeast together with the liquid, and it is more preferable that the heating in the heating step is boiling. The heating of yeast together with the above liquid will be described in more detail. First, prepare yeast as a material. The yeast may be in any state, but it is preferably dried and pulverized from the viewpoint of increasing the production efficiency of particles. The prepared yeast is immersed in a separately prepared liquid and usually heated at 60 ° C. or higher for 3 minutes or longer to obtain a heated product in which the components constituting the yeast are dissolved in the liquid. The longer the heating time, the higher the yield of particles obtained from yeast. Examples of the liquid include liquids used as various solvents such as water and alcohol, which are used alone or in combination of two or more. However, considering taking particles and the like, water or a water-based liquid is preferably used from the viewpoint of health consideration.

In the present invention, "dissolving the components constituting yeast in a liquid" means that the structure of yeast is disrupted by separating the basic skeleton of the cell wall and the substrate, and the components of the yeast that have collapsed in the liquid are dispersed. It means to form a system of yeast, and it also means to disperse it in a liquid by decomposing a polysaccharide, which is one of the components of yeast, into a small one.

Next, examples of the step of separating the particles of the present invention from the heated product obtained by the above step include centrifugation, filter filtration, ultrafiltration, ultracentrifugation and the like. These are more suitable depending on the type of organism having a cell wall as a material and the like. Among them, centrifugation and filter filtration are preferable from the viewpoint of ease of operation, and it is preferable to use them in combination from the viewpoint of increasing the degree of purification.
When the yeast that has passed through the heating step is in a dry state, it is possible to immerse the yeast in a liquid and perform a step of separating the particles from the one in which the components of the yeast that has collapsed in the liquid are dispersed. preferable.

The centrifugal separation includes, although it depends on the size of the particles, a method of centrifuging the heated product at 10,000 to 1 million G and collecting the supernatant (coarse separation step). Further, in order to further increase the degree of purification, for example, the supernatant may be filtered through a filter having a pore size of 0.22 to 0.45 μm to obtain a filtrate (precision separation step).

As described above, the particles according to the embodiment of the present invention can be prepared by using a method (for example, caustic soda, hydrochloric acid, etc.) for the purpose of substituting terminal molecules for components constituting yeast, for example, various sugars. Since it does not use the method used), it is also excellent in safety.

By blending the particles according to the embodiment of the present invention into, for example, preparations, foods, cosmetics, etc., it becomes possible to control each property, for example, hardness, disintegration, etc. as an additive. When considering an additive in a formulation or the like, it is important that the additive does not limit the blending amount of the main agent. However, since each characteristic of the particles of the present invention can be controlled by adding a relatively small amount to the composition constituting the preparation or the like, there is no limitation on the blending amount of the main agent, and the degree of freedom of blending is not limited. Can be enhanced.

When the particles according to the embodiment of the present invention are blended as an additive, they are preferably contained in an amount of 0.01 to 95% by weight, more preferably 0.01 to 90% by weight, based on the entire composition. %, More preferably 0.1 to 50% by weight, even more preferably 1 to 10% by weight.

In addition, the particles according to one embodiment of the present invention can be used as a composition having an antioxidant effect and a composition having an immunostimulatory effect by blending them in, for example, preparations, foods and the like.
When the above particles are blended in a composition having an antioxidant effect or a composition having an immunostimulatory effect, for example, the particles are preferably contained in an amount of 10% by weight or more, preferably 20% by weight or more, based on the entire composition. It is more preferably contained, more preferably 30% by weight or more, further preferably 40% by weight or more, and even more preferably 50% by weight or more. Further, the particle content is preferably 99% by weight or less, more preferably 90% by weight or less, further preferably 80% by weight or less, and 70% by weight or less with respect to the entire composition. Is even more preferable, and even more preferably 60% by weight or less. If the content is too low, it tends to require a large amount of intake to exert the desired effect, and conversely, if it is too high, it tends to be difficult to control to obtain the desired effect. is there.

Next, an embodiment will be described. First, the particles of the present invention themselves were examined (Example 1), and then a preparation using these particles was examined (Example 2). However, the present invention is not limited to this.

[Examination of the particles of the present invention]
Particles A and B, which are embodiments of the present invention, were produced by the procedure shown below. Then, the distribution of particle diameters of the obtained particles A and B was calculated as follows. Then, for particle A, observation of appearance, analysis of constituent sugars and lignin, raman analysis, evaluation of heat resistance, pressure resistance, cold resistance and drought resistance, evaluation of water dispersibility and storage stability are performed according to the following items. went.
Furthermore, the antioxidant power and the immunostimulatory power of the particles A and B were measured and evaluated according to the items described below.

[Example 1]
100 g of yeast (natural brewer's yeast manufactured by Nippon Garlic Co., Ltd.) was immersed in 1 L of water at 95 ° C. and heated continuously for 3 hours to obtain a heated product. This heated product was centrifuged at 13,250 G to obtain a supernatant from which impurities of a relatively large size were removed. As a result of centrifuging this supernatant at 140,000 G, it was fractionated into four as shown in FIG.
That is, in FIG. 1, a transparent pellet (I) adhering to the bottom of the centrifuge tube, a white muddy substance (II) floating on the bottom of the centrifuge in the vicinity of the pellet, and a brown liquid (III) in the middle of the centrifuge tube. And four fractions of white suspended matter (IV) in the upper layer of the centrifuge tube. The transparent pellet (I) is a collection of particles A in the form of pellets. FIG. 2A shows a sample obtained by taking out only the pellet (I). The muddy substance (II) is an aggregate of particles B. The liquid (III) is fractionated into the pellets (I) and the muddy substance (II) by performing ultracentrifugation again. Therefore, the liquid (III) includes the particles A and particles. It is considered that B is contained.
The obtained particles A and B were freeze-dried (manufactured by Tokyo Science Instruments Co., Ltd., EYEL4) according to a conventional method and subjected to the following studies.

(Calculation of particle size distribution)
The particles A and B are each dispersed in ultrapure water, and the dispersion is used to calculate the particle size distribution by the histogram method using a concentrated particle size analyzer (FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.). did. FIG. 3 shows a distribution map of the particle size of the particle A, and FIG. 4 shows a distribution map of the particle size of the particle B. All of these showed a normal distribution. In addition, in FIGS. 3 and 4, a very small amount of particles having a particle size deviating from the normal distribution appear, but it is considered that these particles that could not be fractionated are mixed. Therefore, when calculating the average particle diameter of the particles A or B, it is possible to exclude these mixed particles.
In addition, the particle size distribution was calculated in the same manner for the particles contained in the liquid (III) and the particles contained in the white suspended matter (IV). FIG. 5 shows a distribution map of the particle size of the particles contained in the liquid (III), and FIG. 6 shows a distribution map of the particle size of the particles contained in the white suspended matter (IV).

(Observation of appearance)
The particles A and B, the particles contained in the liquid (III), and the particles contained in the white suspended matter (IV) were each negatively stained and photographed with a transmission electron microscope. A photograph of particle A is shown in FIG. 2 (b), and a photograph of particle B is shown in FIG. 2 (C). Further, a photograph of the particles contained in the liquid (III) is shown in FIG. 2 (d), and a photograph of the particles contained in the white suspended matter (IV) is shown in FIG. 2 (e). In these, each of the obtained particles is dispersed in ultrapure water, the dispersion is adsorbed on a collodion-attached mesh (manufactured by Nissin EM), and uranyl acetate (dyeing agent) is placed on the dispersion. Then, the excess uranyl acetate was sucked up with a filter paper and dried, which was photographed with a transmission electron microscope (JEM-1400TC, manufactured by JEOL Ltd.). A typical particle A was a sphere having a maximum diameter of about 70 nm, as shown in FIG. 2 (b). Further, as shown in FIG. 2C, a typical particle B was a sphere having a maximum diameter of more than about 500 nm.

(Analysis of constituent sugars)
The constituent sugars of the particles A were analyzed. The analysis of the constituent sugars was performed as follows. That is, first, particles A that have been assembled into pellets and have an increased degree of purification are dried in a vacuum dryer at 60 ° C. for about 1 day to prepare a test sample (anhydrous base), and an appropriate amount (about) of this test sample is used. 0.3 g) was weighed into a beaker with a balance, 3 mL of 72% sulfuric acid was added, and the mixture was left to stand for 1 hour with stirring at 30 ° C. This reaction solution was completely transferred to a pressure-resistant bottle while being mixed with 84 mL of purified water, and then decomposed by heating in an autoclave at 120 ° C. for 1 hour. After decomposition by heating, the decomposition solution and the residue were separated by filtration, the filtrate and the washing solution of the residue were added, and the volume was adjusted to 100 mL as a test solution. In addition, in order to correct the overdecomposition of sugar during decomposition, a recovery rate test using monosaccharide was conducted in parallel. The monosaccharides (rhamnose, ribose, xylose, arabinose, fructose, mannose, glucose, galactose) in the test solution were quantified by a high-speed liquid chromatograph method (fluorescence detector). The device used for the analysis is a GL-7400 HPLC system manufactured by GL Sciences. The amount of constituent sugar in the sample was calculated from the monosaccharide concentration of the obtained decomposition liquid and the amount of sample decomposition. The results obtained are shown in Table 1 below. The results in Table 1 are obtained by correcting the amount of constituent sugars using the sugar overdecomposition correction coefficient (Sf) at the time of decomposition obtained from the recovery rate test of monosaccharides. Further, since fructose is easily over-decomposed, Sf becomes a large value, and the included error is large. Therefore, the amount of fructose after over-decomposition correction is treated as a reference value (for example, it is described as "* 2" in Table 1).

As can be seen from the results shown in Table 1 above, almost all of the constituent sugars of the particles A are glucose. This result seems to correlate with the fact that the yeast cell wall is composed mainly of β-glucan and contains almost no hemicellulose and lignin. However, since the constituent sugars of the particle A also contain galactose, rhamnose, mannose and the like, the particle A is not a mere isolate of the component.

(Analysis of lignin)
The analysis of the lignin of the particle A was performed as follows. Originally, in the measurement of lignin, it is a standard method to remove soluble components (oil, tannin, polyphenol, etc.) in the sample in advance with an organic solvent or the like, but the above analysis of lignin does not perform extraction. That is, in the above-mentioned lignin analysis, as a quantification of acid-insoluble lignin, the residue obtained by filtration in the above-mentioned constituent sugar analysis was dried at 105 ° C., weighed, the decomposition residue ratio was calculated, and the ash content in the residue was measured. The acid-insoluble lignin concentration was calculated by the correction. Further, as a quantification of acid-soluble lignin, the filtrate obtained by filtration through the above constituent sugar analysis was measured at a wavelength of 210 nm using a double-beam spectrophotometer (Hitachi High-Tech Science Co., Ltd., U-2001 type), and the following The concentration was calculated using the extinction coefficient of acid-soluble lignin of hippo (plant name) according to the formula (1) of. The results obtained are shown in Table 2 below. It is known that the extinction coefficient of hippopotamus lignin is around 110 L · g ^-1 cm ^-1 .

As shown in Table 2 above, it was found that the particles A contained a small amount of lignin.

(Raman analysis)
The Raman analysis of the particles A was carried out under the following conditions using an inVia Reflex Raman microscope on a small amount (about 0.5 mm square) of the particles A placed on a slide glass. That is, using the LD pumped grains laser (wavelength 532 nm), using an objective lens 50 times, the irradiation diameter of the laser beam 1.5 [mu] m, less illumination laser power 1 mW, photometric Raman shift range 4000～150Cm ^-1, wavenumber resolution 6 cm ^-1, accumulating The number of times was set to 10 times, and the analysis was performed by searching the library by comparing and collating the spectrum wavelength with the database. The obtained spectrum is shown in FIG.

As a result of the above analysis, it was found that the particle A surprisingly has a spectrum close to that of starch. Also in FIG. 7, mainly broad peak around the 3500～3300Cm ^-1 attributable to the hydroxyl group was observed, the peak in the vicinity of 2900 cm ^-1 attributable to the C-H bond was observed. When the particles A were subjected to an iodine-starch reaction, no reaction was observed. Therefore, it has been confirmed that the particles A are not starch.

<Heat resistance, pressure resistance, cold resistance and drying resistance>
First, the particles A dispersed in ultrapure water were freeze-dried [FIG. 8 (a)]. Then, the freeze-dried product dispersed in ultrapure water again [FIG. 8 (b)] was observed in the same manner as the observation of the appearance [FIG. 8 (c)], and the concentrated particle size analyzer was used. The distribution of particle size was calculated [Fig. 8 (d)].
Next, the freeze-dried product dispersed in ultrapure water [FIG. 9 (a)] is further heated and pressurized by an autoclave (2 atm, 121 ° C., 20 minutes) in the same manner as in the observation of the appearance. After observing [FIG. 9 (b)], the particle size distribution was calculated by the above-mentioned concentrated particle size analyzer [FIG. 9 (c)].
As a result of comparing and considering these results, no significant change was observed in the appearance and particle size of the particles A regardless of whether freeze-drying or heating and pressurization was performed, and the particles A had heat resistance and cold resistance. And it was found to be excellent in drought resistance. The same experiment as that for particle A was carried out for particle B, but it had excellent heat resistance, cold resistance and drying resistance as well as particle A.

<Water dispersibility and storage stability>
FIG. 10 (a) shows a lyophilized product dispersed in ultrapure water [FIG. 8 (b)] stored at 4 ° C. for 1 week. Further, FIG. 10B shows particles A contained in the dispersion liquid after storage observed in the same manner as in the above-mentioned observation of appearance. Then, the distribution of the particle size of the particles A contained in the dispersion liquid after storage calculated by the above-mentioned concentrated particle size analyzer is shown in FIG. 10 (c). All of these results indicate that the appearance and diameter of the particles A do not change significantly with time, and the particles A can be kept water-dispersible even after long-term storage and have excellent storage stability. all right. Further, the same experiment as that for particle A was carried out for particle B, but it had excellent water dispersibility and storage stability as well as particle A.

<Antioxidant power>
For the particles A and B prepared in Example 1, (1) superoxide radical scavenging activity value (units SOD / g) was measured.
Further, with respect to the particles B prepared in Example 1, (2) hydroxyl radical scavenging activity value (μmoL DMSO / g) and (3) singlet oxygen scavenging activity value (μmoL histidine / g) were measured.
The measurements (1) to (3) above are all performed by the ESR spin trapping (ESR) method, and the measurement conditions thereof are as shown below.

(1) Superoxide radical scavenging activity value (units SOD / g)
The superoxide radical scavenging activity value (units SOD / g) was measured for each of the particles A and B prepared in Example 1. The results are shown in Table 3 below. The measurement method and measurement conditions are as shown below.

[Measuring method]
To produce superoxide radicals by reacting hypoxanthine with xanthine oxidase, and to measure how much free radicals resonating with the frequency of the superoxide radicals are present in the test solution containing particles A or B. Was done by. That is, dimethyl sulfoxide (DMSO) was used as a superoxide radical scavenger, and the superoxide radical scavenging activity of each of the above test solutions was measured using the following protocols.
-Test solution (40% by weight): 50 μL
8.55M 5,5-dimethyl-1-pyrroline-N-oxide (DMPO): 30 μL · 1.25 mM hypoxanthine: 50 μL
4.37 mM DMSO: 20 μL
50 μL of 0.1 U / mL xanthine oxidase was added, and the mixture was reacted for 60 seconds after stirring for measurement.
[Measurement condition]
The prepared test solution was collected in an ESR flat cell, and ESR measurement was performed under the following measurement conditions.
・ Field: 335 ± 5mT
・ Power: 3mW
-Modulation Width: 0.079mT
-Time Constant: 0.1 sec
・ Sweep Time: 1min
-Amplify: 250

From the results in Table 3, it was found that both Particle A and Particle B had excellent superoxide radical scavenging activity. Among them, the superoxide radical scavenging activity of the particles B having a large particle size was more excellent.

(2) Hydroxyl radical scavenging activity value (μmoL DMSO / g)
The hydroxyl radical scavenging activity value (μmoL DMSO / g) of particle B was measured and compared with the value of lemon registered in the database. The results are shown in Table 4 below. The measurement method and measurement conditions are as shown below.

[Measuring method]
Hydroxyl radicals were generated by irradiating hydrogen peroxide with ultraviolet rays, and the amount of free radicals resonating with the frequency of the hydroxyl radicals was measured in the test solution containing the particles B. That is, DMSO was used as a hydroxyl radical scavenger, and the hydroxyl radical scavenging activity of the above test solution was measured using the following protocol.
-Test solution (25% by weight): 50 μL
5.7M DMPO: 20μL
-2.5 mM hydrogen peroxide: 90 μL
-Measurement was performed after irradiation with ultraviolet rays for 30 seconds.
[Measurement condition]
The prepared test solution was collected in an ESR flat cell, and ESR measurement was performed under the following measurement conditions.
・ Field: 335 ± 5mT
・ Power: 3mW
・ Modulation With: 0.1mT
-Time Constant: 0.1 sec
・ Sweep Time: 1min
・ Amplify: 50

From the results in Table 4, it was found that particle B has much better hydroxyl radical scavenging activity than lemon and red paprika, which are said to have high antioxidant power.

(3) Singlet oxygen scavenging activity value (μmoL histidine / g)
The singlet oxygen scavenging activity value (μmoL histidine / g) of particle B was measured and compared with the values of lemon and red paprika registered in the database. The results are shown in Table 5 below. The measurement method and measurement conditions are as shown below.

[Measuring method]
Riboflavin was irradiated with ultraviolet rays to produce singlet oxygen, and the amount of free radicals resonating with the frequency of the singlet oxygen was measured in the test solution containing the particles B. That is, 2,2,4-trimethyl-1,3-pentanediol (TMPD) was used as a singlet oxygen scavenger, and the singlet oxygen scavenging activity of the above test solution was measured using the following protocol.
-Test solution (11.1% by weight): 25 μL
44.4 mM TMPD: 50 μL
2.5 mM DMSO: 100 μL
55.5 mM riboflavin: 50 μL
-Measurement was performed after irradiation with ultraviolet rays for 20 seconds.
[Measurement condition]
The prepared test solution was collected in an ESR flat cell, and ESR measurement was performed under the following measurement conditions.
-Field: 336.4 ± 5 mT
・ Power: 3mW
・ Modulation With: 0.1mT
-Time Constant: 0.1 sec
・ Sweep Time: 1min
-Amplify: 250

From the results in Table 5, it was found that particle B has a singlet oxygen scavenging activity far superior to that of lemon and red paprika, which are said to have high antioxidant power.

<Immune activating power>
In order to examine whether the particles A and B prepared in Example 1 have an immunostimulatory ability against RAW264.7 cells (hereinafter referred to as "RAW264.7 cells") which are mouse macrophage cells, the following measurement method and According to the conditions, the production amounts of (1) interferon β (IFNβ), (2) interleukin-6 (IL-6) and (3) tumor necrosis factor α (TNFα) were measured.

[Measurement method and conditions]
First, ultrapure water (control), particles A and B (all having a final concentration of 10 mg / mL), and lipopolysaccharide (LPS: final concentration of 1 ng / mL) were prepared as test substances.
Next, a cell solution prepared by dispersing RAW264.7 cells in a culture solution so as to have a concentration of 5.0 × 10 ⁷ cells / mL was prepared, and 200 μL of the test substance was added to 1.8 mL of this cell solution, respectively, and 37 Incubated for 3 hours, 6 hours, 9 hours and 24 hours under the conditions of ° C. and 5% CO ₂ . These cells were then harvested using an RLT lysis buffer. RNA was extracted from each of the collected cells, and cDNA for each of the cells was prepared from the obtained RNA.
Then, the prepared cDNA was subjected to Realtime PCR, the production amounts of IFNβ, IL-6 and TNFα were measured, and evaluated according to each of the following items.

(1) Interferon β (IFNβ)
Particle A was allowed to act on RAW264.7 cells and LPS was allowed to act on RAW264.7 cells. The results are shown in Table 6 below. Table 6 shows the amount of IFNβ produced in the supernatant of each of the particles A and LPS, where 1 is the amount of IFNβ produced in the supernatant of ultrapure water (control).

From the results in Table 6, it can be seen that particle A has higher activity in IFNβ production than LPS, which is a positive control. That is, particle A has the ability to promote IFNβ production and enhance antitumor properties.

(2) Interleukin-6 (IL-6)
Particle A was allowed to act on RAW264.7 cells and LPS was allowed to act on RAW264.7 cells. The comparison results are shown in Table 7 below. Table 7 shows the amount of IL-6 produced in each of the supernatants of LPS and particle A, where 1 is the amount of IL-6 produced in the supernatant of ultrapure water (control). Shown.

From the results in Table 7, it can be seen that particle A has much higher activity in IL-6 production than LPS, which is a positive control. That is, particle A has an extremely high ability to promote IL-6 production and enhance antiviral properties.

(3) Tumor necrosis factor α (TNFα)
The one in which particle A or particle B was allowed to act on RAW264.7 cells was compared with the one in which LPS was allowed to act on RAW264.7 cells. The results are shown in Table 8 below. Table 10 shows the amount of TNFα produced in each of the supernatants of LPS, particle A, and particle B, where 1 is the amount of TNFα produced in the supernatant of ultrapure water (control). ing.

From the results in Table 8, it can be seen that both particle A and particle B have higher activity in TNFα production than LPS, which is a positive control. That is, the particles of the present invention have the ability to promote TNFα production and enhance antitumor properties regardless of the size of the particles.

[Study on Formulation Using Particles of the Present Invention]
Next, a preparation using the particles A prepared in Example 1 (Example 2) and a preparation using no particles of the present invention (Comparative Example 1) were prepared, and the elution rate (%) of each was calculated. .. In addition, hardness and disintegration property were also measured for these.

[Example 2, Comparative Example 1]
The particles A prepared in Example 1 and the following materials were prepared, and the mixture obtained by stirring and mixing these materials was compressed at 8 kN using a tableting machine (HANDTAB-100, manufactured by Ichihashi Seiki Co., Ltd.). Example 2 was obtained as a 200 mg formulation (diameter 8 mm, radius of curvature 12 mm). Further, as Comparative Example 1, a 200 mg preparation (a preparation not using the particles of the present invention) was prepared in the same manner as in Example 2 except that lactose was used instead of the particles A. Table 9 below shows the formulations of Example 2 and Comparative Example 1.

(Calculation of elution rate (%))
The dissolution rate (%) was calculated according to the 16th revised Japanese Pharmacopoeia dissolution test method using an dissolution tester (NTR-3000 manufactured by Toyama Sangyo Co., Ltd.). The above test was carried out by the paddle method using 900 mL of purified water as a test solution. Then, the test solution was periodically sampled until 70 minutes after the start of the test, and the sample passed through a 0.45 μm membrane filter was used as the measurement sample. The absorbance of each of the obtained measurement samples at 275 nm was measured using an ultraviolet-visible spectrophotometer (UV-1800, manufactured by Shimadzu Corporation). The obtained absorbance was applied to the following formula (2) to calculate the elution rate (%) t minutes after the start of the test. The results are shown in Table 10 below.
Elution rate (%) = Absorbance t minutes after the start of the test / Absorbance 70 minutes after the start of the test x 100 ... (2)

From the results shown in Table 10 above, the addition of only 10% by weight of particles A to the entire mixture increased the elution rate by about 2 times from the time point 5 minutes after the start of the test to the time point 40 minutes after the start of the test. Was confirmed. From this, it was found that the particles A have a function as an additive for the fast-dissolving / disintegrating tablet.

Further, for Example 2 and Comparative Example 1, the hardness and the decay time were measured according to the following items. The results are shown in Table 11 below.

(Measurement of hardness)
Using a load cell type tablet hardness tester (portable checker PC-30 manufactured by Okada Seiko Co., Ltd.), a load was gradually applied in the diameter direction of the preparation, and the load when the preparation was crushed was measured as the hardness of the preparation. The measurement was performed at n = 5, and the average was adopted as the hardness of the preparation.

(Measurement of collapse time)
Using a disintegration tester (manufactured by Toyama Sangyo Co., Ltd., NT-200), the disintegration time (collapse property) was measured according to the 16th revised Japanese Pharmacopoeia disintegration test method. In the above test, 1000 mL of purified water was used as the test solution, and the measurement temperature was 37 ± 2 ° C. The time required for the preparation to disintegrate and disperse in the test solution was defined as the disintegration time.

From the results shown in Table 11 above, it was found that Example 2 had a higher hardness than Comparative Example 1. However, although the hardness of Example 2 is high, the time required for the preparation to disintegrate is shorter than that of Comparative Example 1. That is, it has been found that when the particles of the present invention are used in a formulation, they can be an extremely excellent additive that controls properties such as hardness, disintegration property, and drug dissolution property. Similar to the particle A, the particle B also showed a good elution rate and the like, and it was found that the particle B could be a very excellent additive.

Although the specific embodiments of the present invention have been shown in the above examples, the above examples are merely examples and are not to be interpreted in a limited manner. Various variations apparent to those skilled in the art are intended to be within the scope of the present invention.

The particles of the present invention are typically suitable as additives for formulations, foods, cosmetics and the like. It can also be used as an antioxidant and an immunostimulatory agent.

A particle B particle

Claims (8)

Particles obtained from heat-treated yeast and having a maximum diameter of 1 nm or more and less than 150 nm. Particles obtained from heat-treated yeast and having a maximum diameter of 150 nm or more and 1000 nm or less. The particle according to claim 1 or 2, wherein the particle is a sphere. The particle according to any one of claims 1 to 3, wherein the particle contains sugar as a main component. A particle-containing composition comprising the particles according to any one of claims 1 to 4. The particle-containing composition according to claim 5, wherein the particles are contained in an amount of 10 to 99% by weight based on the entire composition. The method for producing particles according to any one of claims 1 to 4, wherein the method comprises a step of heating yeast and a step of separating particles from the heated product obtained by the above steps. How to make particles. The method for producing particles according to claim 7, wherein the heating in the step of heating the yeast is boiling.