Nanoparticle production method

Abstract

To provide a lactobacillus bioreactor for producing ultrafine nanoparticles containing a fat-soluble component.SOLUTION: Emulsion particles containing a fat-soluble component are formed, as shown in Fig. 2, by sequentially adding a vegetable surfactant (saponin) and a fat-soluble component to a culture medium in which lactic acid bacteria can grow. Sodium hydrogen carbonate (Na2HCO3) and sodium metasilicate nonahydrate (SiO2) are further added. As the lactic acid bacteria added thereto grow, a hydrophilic part and a lipophilic part of the surfactant are gradually decomposed. As a result, the emulsion breaks down, and a fat-soluble component leaks out from the emulsion particles. The leaked fat-soluble component is surrounded by metasilicic acid in an acidic state due to the growth of lactic acid bacteria and constitutes a lactobacillus bioreactor having ultrafine nanoparticles (micelle structure).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing ultra-nanoparticles encapsulating fine fat-soluble components that can be taken up by intestinal cells (M cells) and the like, and to the ultra-nanoparticles produced by the method.

Background Art

[0002] Many plants contain fat-soluble components such as oils. Many animals obtain useful components from plants (such as vegetables, fruits, and nuts). Digestive enzymes in the living body break down the food chewed in the mouth with digestive enzymes and change it to a size that can be absorbed from intestinal cells. Intestinal bacteria living in the intestine create nutrients necessary for life activities by further decomposing components that cannot be decomposed by digestive enzymes.

[0003] On the other hand, throughout history, humans have processed and utilized various plants in various ways. They have devised methods to deliciously and efficiently take in the nutrients of plants by boiling, roasting, crushing, steaming, and drying. Oils contained in soybeans and olives, for example, have long been extracted by applying mechanical pressure. Components obtained from plants in this way are essential for our lives. In recent years, nutrients of plants have been industrially synthesized and commercialized as nutritional agents and supplements.

[0004] Such nutrients and supplements become effective components only after being absorbed by the living body. However, many characteristics are required for useful components ingested orally to be taken up by the living body. First, they must not be decomposed by gastric acid. Second, their size must be small enough to be absorbed in the intestine. Third, they must have excellent absorption efficiency in the intestine. Nutrients and supplements must have characteristics that satisfy such conditions. Nano size (10 -9The microparticles of (m) are small enough to be taken up by living cells. For this reason, many methods have been proposed for processing useful components into nanoparticles, and broadly speaking, two structures have been put into practical use. First, there is the micelle structure (emulsion) using the principle of soap, and second, there is the liposome structure formed from phospholipids that mimics living cells.

[0005] The present inventor proposed a method for producing nanoparticles using metasilicic acid as Patent Document 1. Specifically, soy milk is made using hot spring water containing metasilicic acid as preparation water, and lactic acid bacteria (AI-001) are cultured using this soy milk as a culture medium. Then, it was found that the medium becomes acidic due to the lactic acid produced by the lactic acid bacteria, and nanoparticles surrounding fat-soluble components (such as ceramide and equol) contained in the soy milk are obtained by the metasilicic acid in the acidic culture solution.

[0006] Patent Document 2 describes a method for producing lactic acid bacteria (AI-001) that control M cells, and Patent Document 3 describes a method for producing filamentous lactic acid bacteria that adsorb IgE antibodies, which are allergic antibodies.

[0007] Patent Document 4 describes a method of binding a drug to a receptor expressed on the blood-brain barrier and transferring it to the brain tissue in order to pass through the blood-brain barrier. Patent Document 5 describes an antibody preparation in which amyloid-β, which is the causative substance of Alzheimer's disease, does not increase in the brain. Patent Document 6 describes cosmetics using liposomes. Patent Document 7 describes a therapeutic agent for pulmonary fungal infections using liposomes. Patent Documents 8 and 9 describe an extraction method for cannabis extract (CBD) as a fat-soluble component.

[0008] Non-Patent Document 1 describes that the brain has central barrier structures called the blood-brain barrier, blood-spinal cord barrier, blood-brain-spinal cord barrier, and blood-arachnoid barrier, and that even nanoparticles often have difficulty passing through these barriers and reaching the brain tissue.

[0009] In Non-Patent Document 2, it is stated that there is a special immune tissue called Peyer's patches in the intestine, and M cells within them have the ability to take in many components. Although there is a particle size that is easily taken in by M cells, it has been reported that the smaller the particle size, the more likely it is to be taken in.

[0010] Non-Patent Documents 3 and 4 describe that many conditions are required for nutrients that reach the intestine to be taken into the living body.

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

[0013] Nanoparticles using metasilicic acid remain as fine particles in an acidic environment, but when placed in an alkaline environment, the fine particles break down, releasing the components contained within. Because the blood of the living body is kept weakly alkaline, nanoparticles that enter the blood break down and the components contained within are released into the blood. The metasilicic acid that formed the nanoparticles dissolves in the blood and is used to bind collagen in hair and skin. These nanoparticles have the characteristic that only fat-soluble components are taken up as the nucleus of the nanoparticles. For this reason, these nanoparticles have the ideal properties for allowing functional fat-soluble components to be absorbed by the human body.

[0014] However, the size of the nanoparticles disclosed in Patent Document 1 is limited to 50 nm to 150 nm, and at this size, the functional ingredient may not reach the target organ or tissue. Furthermore, other patent documents and prior art documents do not suggest the production of particles smaller than the above.

[0015] Furthermore, in Patent Document 1, the fat-soluble components that form the core of the nanoparticles are limited to those contained in soy milk, and no verification is made as to whether fine particles can be formed from fat-soluble components that are not contained in soy milk. [Means for solving the problem]

[0016] To solve the above problems, a lactic acid bacteria bioreactor for producing ultra-nanoparticles encapsulating a fat-soluble component according to the present invention cultures lactic acid bacteria in a culture solution containing emulsion particles in which the fat-soluble component is held by a surfactant and metasilicic acid, and gradually makes the culture solution acidic by lactic acid produced by the lactic acid bacteria, thereby disrupting the emulsion structure, leaking the fat-soluble component from the emulsion particles, and wrapping the leaked fat-soluble component with metasilicic acid to form ultra-nanoparticles.

[0017] Examples of the fat-soluble component include ceramide, equol, polyphenol, aroma oil, cannabidiol (CBD), etc. contained in plants such as soybeans, peanuts, grapes, oranges, herbs, and hemp. Examples of the surfactant include saponin.

[0018] The ultra-nanoparticles encapsulating a fat-soluble component according to the present invention are produced by the above method and are ultra-nanoparticles with a size (particle diameter) of 1.0 nm to 10.0 nm.

Advantages of the Invention

[0019] According to the present invention, it is possible to produce ultra-nanoparticles encapsulating a fat-soluble component that can be easily absorbed by, for example, intestinal cells. Therefore, it is possible to obtain pharmaceuticals and functional foods effective for alleviating allergic symptoms, improving skin conditions, having a soothing effect, and reducing pain.

Brief Description of the Drawings

[0020]

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Best Mode for Carrying Out the Invention

[0021] Examples of the present invention will be described below. As shown in FIG. 1, the outline of the present invention is to culture lactic acid bacteria in a culture solution in which metasilicic acid is dissolved with an emulsion containing a fat-soluble component formed of saponin, whereby ultranano fine particles encapsulating a lipidic component are expressed in the culture solution.

[0022] Metasilicic acid (H 2 SiO 3 ) is obtained by adding water to silicon dioxide, and in this example, commercially available sodium metasilicate nonahydrate (described as SiO2 in this specification) was used. Sodium hydrogen carbonate was added to dissolve more sodium metasilicate nonahydrate in water.

[0023] Figure 4 is a diagram comparing solubility by temperature. At room temperature, it becomes cloudy and the solubility is low. However, when using boiled sodium metasilicate nonahydrate (SiO2) and sodium hydrogen carbonate (Na2HCO3), no cloudiness is observed and it can be seen that the solubility is high. Therefore, when dissolving sodium metasilicate nonahydrate and sodium hydrogen carbonate, it is preferable to boil the solution.

[0024] Figure 5 shows the test results regarding the mixing ratio of sodium metasilicate nonahydrate and sodium hydrogen carbonate. For sodium metasilicate nonahydrate:sodium hydrogen carbonate, 2:1 has the least precipitation and this ratio is preferable.

[0025] Figure 6 is a diagram showing the separation effect of fat-soluble components (oil). To 1 liter of boiled water, 2 g of sodium metasilicate nonahydrate and 1 g of sodium hydrogen carbonate were added. 30 g of orange peel (Natt Herb man, ETSY, USA) powder was added to this boiling solution and it was further boiled for 30 minutes. The left photo in Figure 6 shows the state after 0 hours of boiling, and the right photo shows the state after 24 hours maintained at 40°C. Suspended matter can be confirmed in the right photo, and it can be inferred that metasilicic acid decomposes the orange peel and has a high separation effect on fat-soluble components (oil).

[0026] Instead of the above water, soy milk containing nutrients that can support the growth of lactic acid bacteria was used. Sodium metasilicate nonahydrate, sodium hydrogen carbonate, and orange peel were added to this soy milk, boiled, and then maintained at 40°C. Next, lactic acid bacteria (AI-001) were added to the solution (culture solution) at 40°C and cultured for 6 days.

[0027] The above-mentioned treated culture was dried and a series of cannabidiol (CBD) analyses were performed by high performance liquid chromatography (HPLC) (left table in Figure 7). On the other hand, only orange peel was analyzed in the same way (right table in Figure 7). As a result, it was found that a small amount of CBD was detected in the treated culture in which lactic acid bacteria AI-001 was cultured.

[0028] Incidentally, CBD is a functional fat-soluble ingredient that has been approved in many countries for use in suppressing excessive neurotransmission, relieving pain, and improving sleep, and is currently being discussed in the Diet in Japan so that it can be used clinically as an anti-epileptic drug. In addition, undetectable THC is treated as a narcotic ingredient.

[0029] Next, the plant-derived surfactant saponin, cannabidiol (CBD), sodium bicarbonate (Na2HCO3), and sodium metasilicate nonahydrate (SiO2) were added sequentially to a medium (soy milk) in which lactic acid bacteria could grow, and the particle size and number of particles formed at each step were measured using a zeta potential, particle size, and molecular weight measurement system ELSZ-2000 series (Otsuka Electronics, Kyoto).

[0030] FIG. 8 shows the change in the particle distribution described above, with the horizontal axis representing particle size, the left vertical axis representing number distribution %, and the right vertical axis representing cumulative frequency distribution %. When fat-soluble CBD was added to a solution containing saponin (1) (2), it dissolved due to the surfactant properties of saponin. Furthermore, Na2HCO3 and SiO2 were mixed into this solution in the same ratio as above, and the particles were measured (3)(4). As a result, nanoparticles with a particle size of about 160 nm were formed in (1) and (2), and about 80 nm in (3)(4).

[0031] In the above test, solutions were prepared with and without the addition of fat-soluble CBD, and the particle size and number distribution % were measured in the same manner as above. As a result, as shown in Figure 9, when fat-soluble CBD was added, the number of fine particles of about 200 nm seen in [(5)-CBD] decreased, and fine particles mainly with a size of about 80 nm were formed (5). It was speculated that the presence of fat-soluble CBD formed an emulsion surrounded by the plant surfactant saponin.

[0032] Thus, the particle distribution can be changed depending on the presence or absence of the fat-soluble component CBD. Without adding CBD, the particles circled by the ○ in [(5)-CBD] of Fig. 9 match the particle size in Fig. 8(1) when only saponin is added. When CBD is added, the particles in this range decrease, and instead, particles of a size that seems to be an emulsion containing the fat-soluble component CBD increase.

[0033] Lactic acid bacterium AI-001 was added to the solution in Fig. 9(5), and aerobic culture was performed at 40 °C for 6 days. As a result, as shown in Fig. 10(6), even tinier nano microparticles (average 1.9 nm) were observed.

[0034] On the other hand, in Fig. 10 [(6)-CBD] without added CBD, only microparticles with an average of 51 nm were observed. It was speculated that the emulsion of saponin with CBD as the core formed even tinier nano microparticles (ultra-nano microparticles) by lactic acid bacterium AI-001.

[0035] When cultured together with lactic acid bacterium (AI-001), it was observed that the culture solution gradually became acidic and the particles of the emulsion (4) changed to particles of a smaller size (6). Also, without adding CBD, no further smaller microparticles were formed. This is presumably because even if saponin is broken down, no ultra-nano microparticles made of metasilicic acid are produced since no fat-soluble component is contained.

[0036] Whether ultra-nano microparticles are formed depending on the presence or absence of SiO2 was observed. The results are shown in Fig. 11. Lactic acid bacterium AI-001 was added to a solution without adding SiO2, and culture was performed at 40 °C for 6 days. As a result, no ultra-nano microparticles were formed (7). The result was almost the same when lactic acid bacterium AI-001 was added to a solution of only soy milk without adding anything and cultured at 40 °C for 6 days (negative control) (8). From this, it can be seen that ultra-nano microparticles are not formed without adding SiO2. Therefore, it can be concluded that ultra-nano microparticles are made of SiO2 (made of dissolved metasilicic acid).

[0037] The culture solution in which ultrananoparticles were formed had a pH of 4.1. This solution was adjusted to a solution with a pH of 9.0 using a sodium hydroxide solution. The results of measuring this solution with a particle counter are shown in Fig. 12(9). As a result of making it alkaline, the ultrananoparticles disappeared, and instead, particles with a bimodal distribution having peaks at 70 nm and 700 nm were detected. Next, a hydrochloric acid solution was added to this solution to adjust the pH to 3.0 (10). As a result, ultrananoparticles with an average of 1.5 nm were detected.

[0038] That is, ultrananoparticles are stable in an acidic solution, but in an alkaline solution, the ultrananoparticles may dissolve and aggregate to form large aggregates.

[0039] CBD was added to the solution containing saponin so that the CBD concentration became 10 g / L. In order to form an emulsion of the dissolved saponin, this solution was treated with a homogenizer (Marusan Machine, Shizuoka), and Na2HCO3 and SiO2 were sequentially added, and lactic acid bacterium AI-001 was cultured. As a result, ultrananoparticles with an average of 1.0 nm were detected in Fig. 13(11).

[0040] Fig. 14 is a graph verifying the effect of using ultrananoparticles. A liquid containing saponin, CBD, Na2HCO3, and SiO2 was prepared with soy milk, and soy milk yogurt was prepared by inoculating and culturing lactic acid bacterium AI-001 in this. 100 ml of this was continuously ingested by subject A every day. Subject A has persistent low back pain due to severe sciatica. Subject A was asked to evaluate the reduction of pain by soy milk yogurt ingestion on a 5-point scale (4: not painful, 3.5: sometimes painful, 3: painful when pressed, 2.5: conscious of pain, 2: always painful). Yogurts of various combinations of saponin, CBD, Na2HCO3, and SiO2 were ingested and evaluated. One pattern of ingestion was continued for 7 days, the highest and lowest scores were deleted, and the average of the remaining 5-day scores was used as the evaluation value. As a result, the one in which saponin, CBD, Na2HCO3, and SiO2 were sequentially dissolved was excellent in relieving low back pain.

[0041] A clinical evaluation was conducted to confirm the tissue migration of the fat-soluble component contained in the ultra-nano particles, and the tissue migration of the fat-soluble component to the nerves was confirmed. Although various causes are considered for sciatica, it is thought that the function of CBD, which suppresses excessive neurotransmission at nerve synapses, reduces pain. Ultra-nano particles containing a fat-soluble component can contribute to the efficiency of intestinal absorption and the effect of tissue migration.

[0042] Summarizing the above, by sequentially adding the plant surfactant saponin and CBD to a medium (soy milk) in which lactic acid bacteria can grow, emulsion particles incorporating CBD are formed as shown in Figure 2. The same was carried out using aroma oil in addition to CBD as the fat-soluble component, and ultra-nano particles could be detected. It is necessary to add saponin, the fat-soluble component, Na2HCO3, and SiO2, and then culture lactic acid bacteria in the above order to produce ultra-nano particles.

[0043] Furthermore, since the above solution is used as a culture solution containing nutrients for growing lactic acid bacteria such as soy milk, the culture solution becomes acidic due to the lactic acid produced by the lactic acid bacteria. As the lactic acid bacteria grow, as shown in Figure 3, it is decomposed into the hydrophilic and lipophilic parts of the surfactant, and due to the disintegration of the emulsion particles, the fat-soluble component leaks out from the emulsion particles. The leaked fat-soluble component becomes surrounded by metasilicic acid and forms ultra-nano particles (micelle structures) of 1.0 to 10.0 nm because the culture solution is an acidic metasilicic acid solution due to the lactic acid of the lactic acid bacteria.

[0044] Here, since the soy milk is merely a solution that supplies nutrients for the growth of lactic acid bacteria, any solution other than soy milk that allows the growth of lactic acid bacteria is acceptable. Also, lactic acid bacteria gradually change the environment to acidic with lactic acid in a tiny reaction space and function as a bioreactor capable of performing a series of reactions such as the disintegration of the emulsion by saponin and the formation of ultra-fine particles by the leaked fat-soluble components and metasilicic acid at an optimal pH in the same container. Therefore, the type of lactic acid bacteria is not limited to AI-001. However, since the reaction space is formed as the cell size of lactic acid bacteria increases with growth, lactic acid bacteria larger than other lactic acid bacteria like AI-001 are preferred.

[0045] Furthermore, sodium hydrogen carbonate facilitates the dissolution of sodium metasilicate nonahydrate. It also has the effect of reducing the particle size of the emulsion containing fat-soluble components [Fig. 8(3)]. It is not necessarily sodium hydrogen carbonate, but sodium hydrogen carbonate is preferred.

Claims

1. A method for producing ultrananoparticles, comprising culturing lactic acid bacteria in a culture solution containing emulsion particles in which a fat-soluble component is held by a surfactant and metasilicic acid, allowing the lactic acid bacteria to grow to cause leakage of the fat-soluble component from the emulsion particles, and encapsulating the leaked fat-soluble component with metasilicic acid in the acidified culture solution to form ultrananoparticles.

2. The method for producing ultrananoparticles encapsulating a fat-soluble component according to claim 1, wherein the fat-soluble component is a fat-soluble component contained in a plant or a fat-soluble component added to a solution, and the surfactant is saponin.

3. Ultrananoparticles produced by the method for producing ultrananoparticles encapsulating a fat-soluble component according to claim 1, wherein the particle size is 1.0 nm to 10 nm.

4. A lactic acid bacteria bioreactor that uses lactic acid bacteria capable of disrupting an emulsion within a pH range from an alkaline solution formed by sodium metasilicate and sodium hydrogen carbonate to an acidic solution produced by lactic acid bacteria over a period of 6 days or more at 40°C for forming the ultrananoparticles described in claim 3.

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