Method for producing yeast-derived microparticles

Abstract

To provide a method for mass-producing novel yeast-derived microparticles at once without using special apparatus or reagents.SOLUTION: The method includes: at least one of a step (A1) of heating a yeast suspension in which yeast is suspended in an aqueous solvent and a step (A2) of suspending the heated yeast in an aqueous solvent to prepare a yeast suspension; a step (B) of fractionating the yeast suspension into a precipitate fraction and a floating fraction; and a step (C) of concentrating the floating fraction to prepare a floating concentrate.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing fine particles having excellent properties such as thermal stability, antioxidant activity and immune activation activity, which are obtained from heat-treated yeast and have a maximum diameter of 1 nm or more and 1,500 nm or less. [Background technology]

[0002] Conventionally, many fine particles have been used in various fields. For example, nanoparticles such as fullerenes and carbon nanotubes, and fine particles derived from natural products such as liposomes are known as such fine particles. The former have heat resistance and solvent resistance, and the latter are derived from natural products and can be easily applied to the human body. Fine particles are recognized to have a wide variety of physical properties and characteristics.

[0003] However, in each technical field, the physical properties and characteristics of such fine particles are currently desired to be further improved as technology advances. In addition, the finer and nano-sized particles are, the more difficult they are to manufacture, and there is a demand for improvements in the quality stability and productivity.

[0004] Among the technical fields using such fine particles, the present inventors focused on the technical field of formulations. That is, in the technical field of formulations, additives such as excipients, stabilizers, preservatives, and molding aids are added to almost all formulations for the purpose of facilitating formulation, stabilizing quality, and increasing usefulness (see Patent Document 1). However, when additives are used to increase the hardness of a formulation, although the hardness of the formulation is increased, there is a tendency for a problem to arise that the disintegration property is reduced. Conversely, when emphasis is placed on the disintegration property of a formulation, there is a tendency for a problem to arise that the desired hardness cannot be obtained.

[0005] Among preparations, over-the-counter drugs that do not require a doctor's prescription are displayed in pharmacies and can be easily purchased by the general public, unlike medical drugs that are strictly controlled. However, since the temperature inside a pharmacy or other store may not be constant depending on the store configuration, over-the-counter drugs tend to be required to be more heat-resistant and cold-resistant than medical drugs. Similarly, foods, cosmetics, etc. also tend to be required to be more heat-resistant and cold-resistant than medical drugs.

[0006] Therefore, the present inventors have conducted various studies to obtain fine particles that can be used for various purposes. As a result, they have found new fine particles that have not been disclosed so far (patent application filed in Japan as Patent Application No. 2018-22501). These new fine particles are not only excellent in dispersibility, heat resistance, cold resistance, etc., but also have antioxidant and immune activation properties. Therefore, there is a demand for the development of a manufacturing method for the above fine particles that can be mass-produced at low manufacturing costs. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2015-193600 A Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in consideration of the above circumstances, and provides a method for producing the above-mentioned novel yeast-derived fine particles in large quantities at once, without requiring any special equipment such as an ultracentrifuge, and without requiring any special reagents. [Means for solving the problem]

[0009] In order to achieve the above object, the present invention provides the following [1] to [6]. [1] A method for producing fine particles derived from yeast, comprising at least one of a step (A1) of heating a yeast suspension in an aqueous solvent and a step (A2) of suspending the heated yeast in an aqueous solvent to prepare a yeast suspension, a step (B) of fractionating the yeast suspension into a sediment fraction and a floating fraction, and a step (C) of concentrating the floating fraction to prepare a floating concentrate. [2] The method for producing yeast-derived fine particles according to [1], wherein the yeast suspension is heated in the step (A1) of heating the yeast suspension so that the temperature of the yeast suspension is 40 to 150°C. [3] The method for producing yeast-derived fine particles according to [1] or [2], wherein the pH of the yeast suspension in the step (A1) of heating the yeast suspension is 4 to 10. [4] The method for producing yeast-derived fine particles described in [1], wherein dry yeast is used as the yeast in the step (A2) of preparing the yeast suspension. [5] The method for producing yeast-derived fine particles according to [1] or [4], wherein the pH of the yeast suspension in the step (A2) of preparing the yeast suspension is 4 to 10. [6] A method for producing yeast-derived fine particles described in any one of [1] to [5], wherein fractionation of the yeast suspension in step (B) of fractionating the yeast suspension is carried out by sedimenting a sediment fraction in the yeast suspension.

[0010] That is, the present inventors discovered novel yeast-derived fine particles that had not been previously identified. However, because the production of these fine particles required special equipment such as an ultracentrifuge, they conducted research into a simpler method for mass production. Effect of the Invention

[0011] As described above, the method for producing yeast-derived fine particles of the present invention does not require special equipment such as an ultracentrifuge, so there is no need to purchase new expensive equipment, and yeast-derived fine particles can be produced at low cost. Furthermore, the method for producing yeast-derived fine particles of the present invention makes it possible to produce a large amount of these fine particles at once, which further reduces production costs. Furthermore, the method for producing yeast-derived fine particles of the present invention does not require special reagents in all steps, so the safety of the obtained fine particles to the human body can be guaranteed.

[0012] In particular, when the yeast suspension is heated in the step (A1) so that the temperature of the yeast suspension is 40 to 150° C., the production efficiency of yeast-derived fine particles can be further improved.

[0013] Furthermore, when the pH of the yeast suspension in the step (A1) of heating the yeast suspension is 4 to 10, denaturation of yeast-derived fine particles can be prevented, and the production efficiency of yeast-derived fine particles can be increased.

[0014] In the step (A2) of preparing the yeast suspension, the use of dry yeast as the yeast has the advantage that the yeast can be easily handled and the manufacturing process is simplified. In addition, since dry yeast is usually subjected to a heating process, there is no need to subject it to another heating process, and the time required for manufacturing can be shortened. Note that the yeast may be subjected to another heating process, in which case the manufacturing efficiency of the yeast-derived fine particles can be further improved.

[0015] Furthermore, when the pH of the yeast suspension in the step (A2) of preparing the yeast suspension is 4 to 10, the production efficiency of yeast-derived fine particles can be further improved.

[0016] Furthermore, if fractionation of the yeast suspension in the step (B) of fractionating the yeast suspension is carried out by sedimenting a sediment fraction in the yeast suspension, production costs can be further reduced. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a flow chart for explaining an outline of an embodiment of the present invention. [Diagram 2] 1(a) to 1(f) are diagrams showing the results of measuring the particle diameter of fine particles at each heating temperature in one step of one embodiment of the present invention. [Diagram 3] 1(a) to 1(d) are diagrams each showing the results of measuring the particle size of fine particles for each heating time in one step of one embodiment of the present invention. [Figure 4] FIG. 1(a) to (c) are diagrams showing the results of measuring the particle size of fine particles at each pH in one step of one embodiment of the present invention. [Diagram 5] 1A to 1C are diagrams showing the results of measuring the particle size of fine particles at each pH before washing with water in one step of one embodiment of the present invention, and 1D to 1F are diagrams showing the results of measuring the particle size of fine particles at each pH after washing with water. [Figure 6] FIG. 4 is a diagram showing the results of measuring the particle diameter of fine particles in one step of one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Next, an embodiment of the present invention will be described, but the present invention is not limited to this embodiment.

[0019] In the present invention, the term "yeast" refers to a single-celled, nearly spherical eukaryotic microorganism belonging to the Ascomycota and Basidiomycota, which undergoes most of its life cycle as a single cell and performs so-called fermentation, and includes not only yeast itself but also yeast in various states such as frozen and dried states. Yeasts belonging to the genera Saccharomyces and Schizosaccharomyces are particularly preferred because they can be easily mass-produced as highly uniform particles.

[0020] In the present invention, the term "fine particles" refers to particles that, when observed under an electron microscope, appear to have a two-layer structure, a double-membrane structure, a multi-layer structure, or a multi-membrane structure. In other words, the fine particles obtained by the production method of the present invention are considered to have different electron densities at least between the outermost layer and the inside, but may appear to have a two-layer structure, a double-membrane structure, a multi-layer structure, or a multi-membrane structure due to light refraction, etc.

[0021] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have a maximum diameter of 1 to 1,500 nm, preferably 40 to 1,000 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" refers to the diameter of the particle when the particle is spherical, and refers to the maximum length of the particle when the particle has another shape. The particle diameter 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 diameter is measured using a concentrated particle size analyzer, the calculated average particle diameter is taken as the maximum diameter of the particle, and the calculated average particle diameter may be within the range defined by the maximum diameter.

[0022] The fine particles obtained by the manufacturing method according to one embodiment of the present invention usually have a shape without sharp parts like carbon nanotubes, and preferably have a spherical shape. In addition, the surface of the fine particles is smooth, and no wear marks formed by physical contact are observed. The above-mentioned spheres include not only perfect spheres, but also egg shapes, ellipsoids, and other shapes. The shape of the fine particles can be determined, for example, by photographing negatively stained particles with a transmission electron microscope and observing their appearance. For example, fine particles aggregated in a pellet shape are dispersed in ultrapure water, the dispersion is adsorbed on a mesh, and a dye is placed on it. Then, the excess dye is absorbed with filter paper, and the dried product is photographed with a transmission electron microscope, whereby the appearance of the fine particles can be observed.

[0023] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have high dispersibility in aqueous liquids. Moreover, this excellent dispersibility is maintained for a long period of time. The dispersibility of the fine particles can be determined, for example, by dispersing the obtained fine particles in ultrapure water, photographing the dispersion with a transmission electron microscope, and observing the degree of dispersion. Moreover, the degree of dispersibility retention can be determined by comparing the dispersion with that after storage for a certain period of time.

[0024] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have excellent pressure resistance and heat resistance, and the particle size hardly changes even when pressurized up to at least 2 atmospheres and heated up to 121° C. The pressure resistance and heat resistance of the fine particles can be determined, for example, by calculating the particle size distribution of particles dispersed in ultrapure water and particles contained in a pressurized and heated dispersion using a concentrated particle size analyzer, and comparing the two, and finding that the particle size distribution does not change between the two.

[0025] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have excellent cold resistance and drying resistance, and the particle size hardly changes when cooled and dried from -50°C to -80°C. The cold resistance and drying resistance of the fine particles can be determined by, for example, calculating the particle size distribution of particles dispersed in ultrapure water and particles contained in the dispersion obtained by freeze-drying (-50°C) and dispersing the dried material again in ultrapure water using a concentrated particle size analyzer, and comparing the two to find that the particle size distribution does not change. In addition, when the freeze-dried material is stored at -80°C for 7 days and then dispersed in ultrapure water, the particle size distribution does not change when compared in the same manner.

[0026] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have excellent antioxidant power. In particular, particles with a maximum diameter of 80 nm to 1,500 nm have excellent antioxidant power, particles with a maximum diameter of 150 nm to 1,000 nm have even better antioxidant power, and particles with a diameter of 150 to 800 nm have even better antioxidant power. The fact that the above particles have antioxidant power can be determined, for example, by dispersing the obtained particles in ultrapure water and measuring the ability of the dispersion to eliminate superoxide radicals, hydroxyl radicals, and singlet oxygen, which are active oxygen generated in the body, using the ESR spin trapping method (hereinafter referred to as the "ESR method").

[0027] The fine particles obtained by the manufacturing method according to one embodiment of the present invention have excellent immune activation power. In particular, particles with a maximum diameter of 1 nm or more and less than 250 nm have excellent immune activation power, particles with a maximum diameter of 1 nm or more and less than 150 nm have even better immune activation power, and particles with a maximum diameter of 50 nm or more and less than 150 nm have even better immune activation power. Whether the particles have immune activation power can be determined, for example, by measuring the production amounts of interferon β (IFNβ), interleukin-6 (IL-6), and tumor necrosis factor α (TNFα) when lipopolysaccharide (LPS), which activates immune cells such as macrophages, is used as a positive control.

[0028] The structure of the fine particles obtained by the manufacturing method according to one embodiment of the present invention does not change even if pressure, heating, cooling, or drying are performed. This can be determined by dispersing the particles in ultrapure water, and dispersing the dispersion liquid after pressure and heating, or after cooling and drying, in ultrapure water again, and comparing the particle structures contained therein with an electron microscope. These are characteristics that could not be obtained with conventional nanoparticles such as liposomes.

[0029] The method according to one embodiment of the present invention can produce a large amount of such novel fine particles having excellent properties at a low cost at once, and includes at least one of a step (A1) of heating a yeast suspension in which yeast is suspended in an aqueous solvent and a step (A2) of preparing a yeast suspension by suspending the heated yeast in an aqueous solvent, a step (B) of fractionating the yeast suspension into a precipitate fraction and a floating fraction, and a step (C) of concentrating the floating fraction to prepare a floating concentrate, as shown in the flow diagram of Figure 1. Note that (B') and (D) in parentheses in Figure 1 are optional steps. Each step of the present invention will be described in detail below.

[0030] <Step (A1) of heating a yeast suspension in which yeast is suspended in an aqueous solvent> In this step, for example, yeast that has been prepared in advance is suspended in an aqueous solvent to prepare a yeast suspension, and this yeast suspension is then heated.

[0031] The yeast may be in any state, such as dried yeast, live yeast, frozen yeast, etc., and commercially available yeast may also be used. These yeasts are preferably dried and pulverized in order to increase the production efficiency of fine particles.

[0032] The aqueous solvent may be, for example, water, alcohol, or other liquids used as aqueous solvents, either alone or in combination. However, in consideration of the ingestion of fine particles, water or a liquid containing 50% by weight or more of water is preferably used, more preferably water, from the viewpoint of health. As the water, distilled water, ion-exchanged water, tap water, etc. may be used, but distilled water and ion-exchanged water are preferably used, from the viewpoint of more efficient production of fine particles. In addition, the pH of the aqueous solvent is preferably 10 or less, more preferably near neutral (near pH 7).

[0033] The heating is performed by heating the yeast suspension to 40° C. or higher, preferably 50 to 150° C., more preferably 50 to 100° C., and even more preferably 50 to 75° C. When heating to 100° C. or higher, the heating is performed under pressure using, for example, an autoclave.

[0034] The heating time is preferably 30 minutes (min) or more, more preferably 1 to 3 hours (hr), and even more preferably 1 hr, in order to efficiently obtain fine particles from yeast.

[0035] <Step (A2) of suspending the heated yeast in an aqueous solvent to prepare a yeast suspension> In this process, for example, a dry yeast that has been previously subjected to a heating process is prepared, and the dry yeast is suspended in an aqueous solvent to produce a yeast suspension. That is, by heating the yeast in advance prior to suspension in the aqueous solvent, heating of the yeast suspension is not required.

[0036] The heated yeast may be, for example, a dried yeast that has been subjected to a heat drying step or a heat sterilization step, or a live yeast that has been subjected to heat drying, heat sterilization, etc. The aqueous solvent may be the same as that used in step (A1), and the preferred range is also the same. The temperature of the yeast suspension is not particularly limited, but is preferably 0 to 40°C, and more preferably 4 to 30°C.

[0037] Either one of the steps (A1) and (A2) may be performed, or both may be performed. That is, the dried yeast that has been subjected to the heating step may be suspended in an aqueous solvent to prepare a yeast suspension, and the yeast suspension may be heated, or the dried yeast that has been subjected to the heating step may be suspended in a heated aqueous solvent. Specifically, the dried yeast may be immersed in an aqueous solvent and the yeast may be heated together with the aqueous solvent. By performing both of the steps, the yeast is heated in a repeated manner, and therefore fine particles derived from the yeast can be obtained more efficiently. The heating temperature of the yeast suspension and the temperature of the heated aqueous solvent are not particularly limited, but are preferably 40 to 150°C, more preferably 40°C to 100°C, and even more preferably 40°C to 60°C.

[0038] <<Step (B) of fractionating the yeast suspension into a precipitate fraction and a floating fraction>> This step involves fractionating the yeast suspension obtained in at least one of steps (A1) and (A2) into a precipitate fraction and a floating fraction. This step may be carried out once or multiple times.

[0039] The above fractionation can be carried out by sedimenting the precipitate fraction. For example, the yeast suspension can be left to stand and the residue other than the target fine particles can be allowed to naturally settle. Alternatively, the yeast suspension can be centrifuged at a low speed to allow the residue other than the target fine particles to naturally settle. From the viewpoint of cost, it is preferable to carry out the fractionation by allowing the residue other than the target fine particles (precipitate fraction) to naturally settle, but from the viewpoint of time saving and easy processing such as residue removal, it is preferable to use a centrifuge.

[0040] When fractionation is performed by natural sedimentation, the time for which the yeast suspension is allowed to stand is not particularly limited, but may be 24 hours or more from the viewpoint of more clearly separating the floating fraction and the precipitate fraction. On the other hand, from the viewpoint of preventing the proliferation of unnecessary bacteria, the time is preferably 1 to 24 hours, more preferably 2 to 7 hours, and even more preferably 2 to 4 hours. Furthermore, when the yeast suspension is allowed to stand, from the viewpoint of preventing the proliferation of unnecessary bacteria, the time is preferably performed in a room at a temperature of -10 to 50°C, more preferably 0 to 40°C, and even more preferably 5 to 35°C. The floating fraction can be removed, for example, by leaving the yeast suspension to stand for a certain period of time until the suspension is eliminated and the supernatant and sediment are separated. Then, the supernatant (floating fraction) located above the yeast suspension can be aspirated or discharged from above or the side by a pump, a siphon, or by directly discharging the supernatant from a tank.

[0041] When centrifuging, it is preferable to carry out the centrifugation at a low speed, and the centrifugal force is usually about 10,000 G, preferably 1,000 to 20,000 G. After centrifugation, the precipitate fraction adheres to the bottom in the form of a pellet, and the floating fraction appears as the supernatant, so the floating fraction can be taken out by discharging the supernatant from above with a pump or the like. Alternatively, the precipitate fraction and the floating fraction can be separated by the centrifugal force using a disk-type centrifuge that naturally discharges the supernatant, and the floating fraction can be taken out as the supernatant.

[0042] <<Step (B-1) of obtaining a floating fraction containing the target fine particles from the above-mentioned precipitate fraction>> This step is carried out to recover fine particles remaining in the precipitate fraction, and the precipitate fraction is further subjected to solid-liquid separation to obtain a filtrate containing the fine particles. This step can be carried out not only once, but also repeatedly. Specifically, a filter aid (diatomaceous earth or perlite, etc.) and, if necessary, an aqueous solvent are added to the precipitate fraction obtained in the fractionation step (B), and the mixture is stirred, and then subjected to filter cloth filtration to separate the residue and filtrate. The filtrate contains the fine particles and can be treated in the same manner as the floating fraction. The filter cloth filtration may be used in combination with fractionation using a centrifuge or a filter press filter, or fractionation using these may be performed instead of filter cloth filtration. Note that this step (B-1) is an optional step and does not necessarily have to be performed.

[0043] Furthermore, the residue obtained in the fractionation step (B-1) can be suspended in an aqueous solvent, and the suspension can be allowed to stand to allow residue other than the target fine particles to settle naturally, or the suspension can be centrifuged again to obtain a supernatant (floating fraction) further containing the target fine particles.

[0044] <Step (B') of purifying the floating fraction> This step is a step of purifying the floating fraction (filtrate) obtained in steps (B) and (B-1) to remove impurities such as yeast residue. This step can be performed not only once but also repeatedly. Specifically, this step is a step of purifying the floating fraction (filtrate) obtained in steps (B) and (B-1) by passing it through a filtration filter such as diatomaceous earth, activated carbon, perlite, filter paper, hemp bag, hollow fiber filter, etc., and it is particularly preferable to use a filtration filter. The pore size of the filtration filter is preferably 0.01 to 10 μm, more preferably 0.1 to 1 μm. By setting the pore size as above, impurities in the floating fraction (filtrate) can be efficiently removed, and the purity of the target fine particles can be increased. Furthermore, the impurities from the above purification step (B') can be rinsed with an aqueous solvent, and the rinse liquid can be purified again through the above-mentioned diatomaceous earth or the like to obtain a floating fraction (filtrate) containing the target fine particles. The purification step (B') may be carried out on the floating concentrate after passing through the floating concentrate preparation step (C) described below.

[0045] <Step (B'-1) of purifying the floating fraction> This step is a step for further purifying the floating fraction (filtrate) obtained in steps (B), (B-1), and (B') to remove substances that cause coloring, etc., and is necessary in order to obtain fine particles that can be used in products where color is an important selling point, such as beverages. However, this step does not have to be included if it is not necessary to remove coloring, etc., from the fine particles. This step can be carried out not only once, but also repeatedly many times. Specifically, it is a step of purifying the floating fraction (filtrate) obtained in step (B) or the like through a hollow fiber filter, a ceramic membrane, diatomaceous earth, perlite, activated carbon, an ion exchange resin, or the like, and in particular, a hollow fiber filter or activated carbon can be preferably used, and a hollow fiber filter is more preferable. The pore size of the hollow fiber filter is preferably 1 to 1,000 nm, and more preferably 5 to 100 nm. This makes it possible to increase the purity of the target fine particles without impairing their properties. The purification step (B'-1) may be carried out on the floating concentrate after passing through the floating concentrate preparation step (C) described below.

[0046] <<Step (C) of concentrating the floating fraction to prepare a floating concentrate>> This step is carried out in order to remove the aqueous solvent from the floating fraction (filtrate) obtained in step (B) [including (B-1)] or step (B') [including (B'-1)] and increase the content ratio of fine particles, and can be carried out, for example, by vacuum concentration, evaporation concentration, membrane concentration, filtration through a hollow fiber filter without adding water, etc. The solid content of the thus obtained suspended concentrated liquid contains about 30 to 60% by weight of fine particles derived from yeast.

[0047] The above-mentioned floating concentrated liquid can be made into a dried product containing fine particles derived from yeast by further carrying out a drying step (D) such as spray drying or freeze drying. In this way, by providing the drying step (D) and drying the above-mentioned floating concentrated liquid into a dried product with a moisture content of 8% or less, it is possible to suppress the growth of unnecessary bacteria and to obtain a food raw material with excellent preservability, but the moisture content is not limited to this. The above-mentioned dried product usually contains about 30 to 60% by weight of fine particles. Note that this drying step (D) is an optional step and does not necessarily have to be carried out.

[0048] According to the production method of one embodiment of the present invention, since a method for replacing terminal molecules of fine particles obtained from yeast (e.g., a method using caustic soda, hydrochloric acid, etc.) is not adopted, the obtained fine particles are excellent in safety. Furthermore, a special device such as an ultracentrifuge is not required for the production of fine particles, and a large amount of fine particles can be produced at one time. EXAMPLES

[0049] Next, examples of the present invention will be described, but prior to that, conditions suitable for each step were examined, although the present invention is not limited to these.

[0050] Experiments were conducted as follows for the process (A1) of heating a yeast suspension in an aqueous solvent and the process (A2) of suspending the heated yeast in an aqueous solvent to prepare a yeast suspension (hereinafter, these processes are collectively referred to as the "fine particle extraction process (A)").

[0051] <Fine particle extraction process (A)> [Heating temperature] In the fine particle extraction process (A), the following experiment was conducted to examine the relationship between the heating temperature and the yield of fine particles. That is, 40 g of brewer's yeast manufactured by Japan Garlic Co., Ltd. was suspended in 400 mL of ion-exchanged water, heated for 1 hour while stirring under the temperature conditions shown in Table 1, and then cooled by standing in a room at 4°C for 2 hours. After cooling, the suspension was centrifuged at 13,250G for 30 minutes to separate the suspension into a floating fraction and a sediment fraction. The optical density (OD600) and particle size distribution of the floating fraction were measured. The optical density (OD600) is shown in Table 1. The particle size distribution of sample 1 (heated at 4°C) is shown in Figure 2(a), the particle size distribution of sample 2 (heated at 22°C) is shown in Figure 2(b), and the particle size distribution of sample 3 (heated at 50°C) is shown in Figure 2(c). Moreover, the particle size distribution of sample 4 (heated at 75°C) is shown in Figure 2(d), the particle size distribution of sample 5 (heated at 100°C) is shown in Figure 2(e), and the particle size distribution of sample 6 (heated at 125°C) is shown in Figure 2(f).

[0052] [Table 1]

[0053] From the results in Table 1, the optical density (OD600), which is an index of the concentration of fine particles, showed the maximum value in sample 3 (heated at 50°C). In addition, from the results shown in Figures 2(a) to (f), it was found that the diameter of the fine particles tended to become slightly larger in sample 5 (heated at 100°C) and sample 6 (heated at 125°C), but the diameter of the fine particles was almost the same in all samples 1 to 6. From these results, it can be said that in order to efficiently obtain fine particles from yeast, heating at 50°C or higher is preferable, and heating at 50 to 75°C is more preferable.

[0054] [Heating time] In the extraction process (A) of fine particles, the following experiment was carried out to examine the relationship between the heating time and the yield of fine particles. That is, 40 g of brewer's yeast manufactured by Japan Garlic Co., Ltd. was suspended in 400 mL of ion-exchanged water, heated at 50°C while stirring for the time shown in Table 2, and then cooled by standing in a room at 4°C for 2 hours. After cooling, the suspension was centrifuged at 13,250G for 30 minutes to separate the suspension into a floating fraction and a sediment fraction, and the optical density (OD600) and particle size distribution of the floating fraction were measured. The optical density (OD600) is shown in Table 2. The particle size distribution of sample 7 (heating time 0 hr) is shown in Figure 3(a), the particle size distribution of sample 3 (heating time 1 hr) is shown in Figure 3(b), the particle size distribution of sample 8 (heating time 3 hr) is shown in Figure 3(c), and the particle size distribution of sample 9 (heating time 5 hr) is shown in Figure 3(d).

[0055] [Table 2]

[0056] From the results in Table 2, the optical density (OD600), which is an index of the concentration of fine particles, showed the maximum value in sample 3 (heating time 1 hr). Furthermore, from the results in Figures 3(a) to (d), it was found that the diameter of the fine particles in sample 7 (heating time 0 hr) tended to be slightly smaller, but the diameter of the fine particles in samples 3, 8, and 9 was almost the same. From these results, it can be said that in order to efficiently obtain fine particles from yeast, heating for 1 hr or more is preferable, and heating for 1 hr is more preferable.

[0057] [Aqueous solvent pH] In the extraction step (A) of fine particles, the following experiment was carried out to examine the relationship between the pH of the aqueous solvent and the yield of fine particles. That is, 40 g of brewer's yeast manufactured by Nippon Garlic Co., Ltd. was suspended in 400 mL of the solvent shown in Table 3 below, heated at 100°C while stirring for 1 hour, and then left to stand in a room at 4°C for 2 hours to cool. After cooling, the suspension was centrifuged at 13,250G for 30 minutes to separate the suspension into a floating fraction and a sediment fraction, and the optical density (OD600) and particle size distribution of the floating fraction were measured as shown below. The optical density (OD600) is shown in Table 3. The particle size distribution of sample 10 (pH 4) is shown in Figure 4(a), the particle size distribution of sample 11 (pH 6.6) is shown in Figure 4(b), and the particle size distribution of sample 12 (pH 10) is shown in Figure 4(c).

[0058] [Table 3]

[0059] From the results in Table 3, the optical density (OD600), which is an index of the concentration of fine particles, showed the maximum value in sample 12 (pH 10). However, from the results in Figs. 4(a) to (c), the particle size of the fine particles in sample 12 was considerably larger, and the appearance (color) of sample 12 was a dark brown color different from the appearance (white) of samples 10 and 11, so there is a possibility that something has adhered to the fine particles in sample 12, causing the fine particles to deteriorate. Therefore, in order to efficiently obtain fine particles from yeast, it is preferable that the aqueous solvent in which the yeast is suspended has a pH of less than 10, and more preferably is close to or below neutral (pH 7).

[0060] Next, regarding the step (B) of fractionating the yeast suspension into a sediment fraction and a floating fraction, an experiment was carried out as follows. [Relationship with heating time in extraction process (A)] In the fractionation of yeast suspension, the following experiment was carried out to examine the relationship between the heating time in the extraction step (A) and the ease of fractionation. That is, 40 g of brewer's yeast manufactured by Japan Garlic Co., Ltd. was suspended in 400 mL of ion-exchanged water, and heated at a temperature of about 90°C for 0 hr, 1 hr, 2 hr, 3 hr, 4 hr, and 5 hr, respectively, while stirring. Then, the mixture was left to stand in a room at 4°C, and its appearance was visually observed over time (immediately after standing, after 1 hr, after 2 hr, after 3 hr, and after 3 days). As a result, it was found that the shorter the heating time in the extraction step (A), the faster the natural sedimentation of fine particles, and the easier it was to fractionate into a precipitate fraction and a floating fraction. This is presumably because the cell walls of the yeast were broken down finely by the long heating time in the extraction step (A), and the fine particles became finer and less likely to settle. Therefore, in terms of the balance between the amount of extraction and the ease of fractionation, the heating time in the extraction step (A) is preferably 2 hours or less, and more preferably about 1 hour.

[0061] [Relationship with heating temperature in extraction process (A)] In the fractionation of the yeast suspension, the following experiment was carried out to examine the relationship between the heating temperature in the extraction step (A) and the ease of fractionation. That is, 40 g of brewer's yeast manufactured by Japan Garlic Co., Ltd. was suspended in 400 mL of ion-exchanged water, and heated with stirring at each temperature of 25°C, 40°C, 50°C, and 60°C for 1 hr, and then allowed to stand in a room at 4°C, and the appearance was visually observed over time (immediately after standing, after 1.5 hr, after 2 hr, after 3 hr, after 4 hr). As a result, it was found that the lower the heating temperature, the faster the natural sedimentation of fine particles, and the easier it was to separate into a precipitate fraction and a floating fraction. This is presumably because the yeast cell wall is finely decomposed by heating at a high temperature, and the fine particles become finer and less likely to settle. Therefore, from the viewpoint of the balance between the amount of extraction and the ease of fractionation, the heating temperature in the extraction step (A) is preferably 60°C or less, more preferably 50 to 60°C.

[0062] Then, for the step (B') of purifying the floating fraction of the yeast suspension, an experiment was carried out as follows. [pH of aqueous solvent in extraction step (A)] The following experiment was carried out to investigate the effect of the pH of the aqueous solvent in the extraction step (A) on purification of the floating fraction. That is, 150 g of brewer's yeast manufactured by Nippon Garlic Co., Ltd. was suspended in 1,500 mL of the aqueous solvent shown in Table 4 below, heated at 100°C with stirring for 2 hours, and then allowed to stand in a room at 4°C for 1.5 hours to cool. After cooling, the yeast suspension was centrifuged at 13,250G for 30 minutes to fractionate it into a precipitate fraction and a floating fraction. The floating fraction was then washed with water using a hollow fiber filter (pore size of hollow fiber filter: 50 nm), and the appearance (color) of the washed floating fraction was visually observed. The results are also shown in Table 4 below.

[0063] [Table 4]

[0064] From the results in Table 4, sample 15, which was extracted at pH 10, remained colored light brown even after washing with a hollow fiber filter, suggesting that something was attached to the fine particles and could not be removed.

[0065] Next, to examine whether fine particles are affected by washing with water using a hollow fiber filter, the optical density (OD600) and particle size of the floating fraction of samples 13 to 15 were measured before and after washing with water. The optical density of each sample is shown in Table 5 below, and the particle size distribution of sample 13 (before washing with water) is shown in Figure 5(a), the particle size distribution of sample 14 (before washing with water) is shown in Figure 5(b), and the particle size distribution of sample 15 (before washing with water) is shown in Figure 5(c). In addition, the particle size distribution of sample 13 (after washing with water) is shown in Figure 5(d), the particle size distribution of sample 14 (after washing with water) is shown in Figure 5(e), and the particle size distribution of sample 15 (after washing with water) is shown in Figure 5(f).

[0066] [Table 5]

[0067] The results in Table 5 and Figures 5(a) to (f) show that there is no change in the trends in optical density and particle size before and after washing with water, which suggests that washing with water has almost no effect on fine particles.

[0068] Furthermore, when a portion of Samples 13 to 15 after washing with water was freeze-dried and the properties of the dried matter were examined, it was found that Sample 15 (pH 10) was more susceptible to static electricity than Samples 13 and 14. This suggests that the properties of the fine particles in Sample 15 may have changed due to something adhering to them. The weights of the freeze-dried products of Samples 13 to 15 after the above water washing were measured, and the results are shown in Table 6 below. From the results in Table 6, Sample 15 has a heavier dry weight than the others, and at first glance it appears that a large amount of fine particles was obtained. However, as mentioned above, it is highly likely that something is attached to the particles of Sample 15, and therefore it is believed that the increase in weight is due to this attachment, and it is presumed that a large amount of fine particles themselves was not obtained.

[0069] [Table 6]

[0070] [Examples 1 to 3] The fine particles that are the subject of the present invention were produced through the steps described below. <Example 1 and Example 2> [Fine particle extraction process (A)] 50 kg of dried brewer's yeast (Asahi Group Foods) was added to 1,000 L of tap water (based on the Japanese Pharmacopoeia) to prepare a yeast suspension (pH 7). The yeast suspension was heated to 50°C and stirred for 1 hour while maintaining that temperature.

[0071] [Step (B) of fractionating the yeast suspension into a sediment fraction and a floating fraction] The yeast suspension was then allowed to stand for 12 hours while being cooled in a room at 12.5° C. After confirming that the suspension had been separated into an upper layer (floating fraction) and a lower layer (sediment fraction), 800 L of the upper layer (floating fraction) was collected using a stainless steel rotary pump and centrifuged at 18,000 rpm in a Sharpless centrifuge to recover 723 L of supernatant. On the other hand, 37 kg of diatomaceous earth was added to 200 L of the lower layer (precipitate fraction) and stirred, and the mixture was filtered through a filter cloth in a top-discharge centrifuge, and 143 L of filtrate was recovered. The supernatant and filtrate were combined to give 866 L of floating fraction.

[0072] [Step (C) of concentrating the floating fraction to prepare a floating concentrate] The floating fraction obtained above was concentrated under reduced pressure at a temperature of 60° C. or less and a vacuum of about 90 kPa using a coil type concentrator to obtain 66 L of a floating concentrate.

[0073] [Step (B') of purifying the floating fraction] The Brix value of the above floating concentrated liquid was measured using a sugar refractometer, and 11.5 L of water was added until the Brix reached 20°Bx. 3.75 kg of activated carbon was added to this, stirred for 1 hour, and filtered through a 150 mesh sieve to obtain a filtrate. The activated carbon remaining on the mesh sieve was rinsed with water, and the rinse was again filtered through a 150 mesh sieve to obtain a filtrate. These filtrates were combined and centrifuged at 18,000 rpm in a Sharpless centrifuge to remove the activated carbon from the filtrate, and 73.8 kg of an upper layer (floating fraction) was obtained. An appropriate amount of diatomaceous earth was mixed with the above obtained upper layer (floating fraction), stirred and mixed, and then filtered through a filter paper to obtain 68.6 kg of filtrate.

[0074] [Step of purifying the floating fraction (B'-1)] The filtrate was filtered through cartridge filters (Millipore) of 1 μm and then 0.45 μm to obtain 65.9 kg of filtrate.

[0075] [Drying process (D)] A portion (50 g) of the filtrate obtained through each of the above steps was freeze-dried in a conventional manner without adding dextrin or the like, to obtain 5.94 g of a freeze-dried product (Example 1) containing the desired fine particles. In addition, 6 kg of dextrin was added to a portion of the filtrate (60 kg), and the mixture was sent to a spray dryer (Okawahara Kakoki Co., Ltd., L-8i) and evaporated to dryness at a rotation speed of 18,000 rpm, an inlet temperature of 140°C, and an outlet temperature of 90°C, to obtain approximately 10 kg of a spray-dried product containing the desired fine particles (Example 2).

[0076] <Example 3> [Fine particle extraction process (A)] 100 kg of dried brewer's yeast (Asahi Group Foods Co., Ltd.) was added to 5,000 L of normal water to prepare a yeast suspension (pH 7). The yeast suspension was heated to 50° C. and stirred for 1 hour while maintaining the temperature.

[0077] [Step (B) of fractionating the yeast suspension into a sediment fraction and a floating fraction] The yeast suspension was then cooled to 30°C and centrifuged at 12,000G using a disk centrifuge to recover 4982 kg of supernatant. This supernatant was filtered using a filter press to obtain a filtrate (floating fraction). The particle size distribution of this filtrate (floating fraction) is shown in Figure 6. The average particle size of the fine particles in this filtrate (floating fraction) was 147.5 nm.

[0078] [Step (C) of concentrating the floating fraction to prepare a floating concentrate] The filtrate (floating fraction) was concentrated under reduced pressure using a plate-type vacuum concentrator to obtain a floating concentrate, the final Brix value of which was 11.3.

[0079] [Step (B') of purifying the floating fraction] The above-mentioned suspended concentrated liquid was continuously filtered through a cartridge filter having a filtration accuracy of 1 μm to obtain 255 kg of filtrate.

[0080] A portion of the filtrate was purified using a hollow fiber filter (Spectrumlabs, N04-E100-05N) and then concentrated 1.5-fold by standard methods.

[0081] A portion (20 g) of the concentrated liquid obtained through the above process was freeze-dried in a conventional manner without adding dextrin or the like, to obtain 0.54 g of a freeze-dried product (Example 3) containing the desired fine particles.

[0082] The immune stimulating power of the fine particles contained in Examples 1 to 3 obtained above was evaluated as follows.

[0083] In order to examine whether the fine particles contained in Examples 1 to 3 have the ability to activate the immune system of RAW264.7 cells (hereinafter referred to as "RAW264.7 cells"), which are mouse macrophage cells, the gene expression levels of (1) interferon β (Ifnβ), (2) tumor necrosis factor α (Tnfα), (3) interleukin 12 (IL-12), and (4) interleukin 10 (IL-10) were measured and evaluated according to the measurement method and conditions described below.

[0084] [Measurement method and conditions] First, the fine particles of Examples 1 to 3 (final concentration 1 mg / mL or 10 mg / mL) and lipopolysaccharide (LPS: final concentration 1 ng / mL) were prepared as test substances. Next, RAW264.7 cells were cultured at 9.0 × 10 5 A cell suspension was prepared by dispersing cells in culture medium at 1.0 cells / mL. 1.0 mL of the test substance was added to 1.0 mL of this cell suspension, and the cells were incubated at 37°C, 5% CO 2 The cells were incubated for 9 hours under the conditions of . After that, the cells were harvested using RLT lysis buffer (QIAGEN). Total RNA was extracted from each of the harvested cells using RNeasy Kit (QIAGEN), and cDNA was prepared from the total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo).

[0085] The prepared cDNA was subjected to real-time PCR using a QuantStudio 7 Flex Real-Time PCR System (Thermo) to measure the gene expression levels of Ifnβ, Tnfα, IL-12, and IL-10, and evaluated according to the following items. The expression level of each gene was corrected by the expression level of β-actin (Actb), and the relative value was calculated based on the expression level in the negative control (no particles added).

[0086] (1) Interferon-β (Ifnβ) The fine particles of each Example were applied to RAW264.7 cells, and the RAW264.7 cells were applied to LPS for comparison. The results of Example 1 and Example 3 (final concentration 10 mg / mL) are shown in Table 7 below. Table 7 shows the expression levels of the Ifnβ gene in the cells to which LPS, Example 1, and Example 3 were added, when the expression level of the Ifnβ gene in the negative control cells was set to 1.

[0087] [Table 7]

[0088] (2) Tumor necrosis factor α (Tnfα) The fine particles of each Example were applied to RAW264.7 cells, and the RAW264.7 cells were applied to LPS for comparison. The results of Example 1 and Example 3 (final concentration 10 mg / mL) are shown in Table 8 below. Table 8 shows the Tnfα gene expression levels of the cells to which LPS, Examples 1, 2, and Example 3 were added, when the Tnfα gene expression level of the negative control cells was set to 1.

[0089] [Table 8]

[0090] (3) Interleukin 12 (IL-12) The fine particles of each Example were applied to RAW264.7 cells, and the RAW264.7 cells were applied to LPS for comparison. The results of Examples 1, 2, and 3 (final concentration 1 mg / mL) are shown in Table 9 below. Note that Table 9 shows the IL-12 gene expression levels of the cells to which Examples 1, 2, and 3 were added, respectively, when the IL-12 gene expression level of the negative control cells, which did not express the IL-12 gene, expressed by LPS, was set to 1.

[0091] [Table 9]

[0092] (4) Interleukin 10 (IL-10) The fine particles of Example 3 (final concentration 1 mg / mL) were applied to RAW264.7 cells, and the RAW264.7 cells were applied to LPS for comparison. The results are shown in Table 10 below. Table 10 shows the IL-10 gene expression levels of the cells to which LPS and Example 3 were added, when the IL-10 gene expression level of the negative control cells was set to 1.

[0093] [Table 10]

[0094] As can be seen from these results, the fine particles of Examples 1 and 3 were both shown to have higher activity than the positive control LPS, and were shown to have excellent immune activation. The fine particles of Example 2 also had excellent immune activation comparable to Examples 1 and 3. It is therefore evident that the process of the present invention makes it possible to mass-produce fine particles having the above-mentioned various excellent properties at low cost.

[0095] In the above embodiment, specific embodiments of the present invention are shown, but the above embodiment is merely illustrative and should not be interpreted as being limiting. Various modifications that are obvious to those skilled in the art are intended to be within the scope of the present invention. [Industrial Applicability]

[0096] The process of the present invention can safely produce fine particles derived from yeast and having various excellent properties at low cost.

Claims

1. A method for producing fine particles derived from yeast, comprising at least one of a step (A1) of heating a yeast suspension in an aqueous solvent and a step (A2) of suspending the heated yeast in an aqueous solvent to prepare a yeast suspension, a step (B) of fractionating the yeast suspension into a precipitate fraction and a floating fraction, and a step (C) of concentrating the floating fraction to prepare a floating concentrate.

2. 2. The method for producing yeast-derived fine particles according to claim 1, wherein the yeast suspension is heated in the step (A1) so that the temperature of the yeast suspension is 40 to 150°C.

3. 3. The method for producing yeast-derived fine particles according to claim 1 or 2, wherein the yeast suspension has a pH of 4 to 10 in the step (A1) of heating the yeast suspension.

4. 2. The method for producing yeast-derived fine particles according to claim 1, wherein in the step (A2) of preparing the yeast suspension, dry yeast is used as the yeast.

5. 5. The method for producing yeast-derived fine particles according to claim 1 or 4, wherein the pH of the yeast suspension in the step (A2) of preparing the yeast suspension is 4 to 10.

6. The method for producing yeast-derived fine particles according to any one of claims 1 to 5, wherein the fractionation of the yeast suspension in the step (B) of fractionating the yeast suspension is carried out by sedimenting a sediment fraction in the yeast suspension.

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