Nanoparticle production method

Abstract

[Problem] Provided is 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).

Description

Nanoparticle manufacturing method

[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 ultra-nanoparticles produced by said method.

[0002] Many plants contain fat-soluble components such as oils. Many animals obtain useful components from plants (vegetables, fruits, nuts, etc.). The digestive enzymes present in the body break down food chewed in the mouth, reducing it to a size that can be absorbed by intestinal cells. The intestinal bacteria that live in the intestines further break down components that cannot be broken down by digestive enzymes, producing nutrients necessary for life activities.

[0003] Meanwhile, throughout history, humans have processed and used a variety of plants in a variety of ways. From boiling and baking to crushing, steaming, and drying, we have devised delicious and efficient ways to absorb the nutrients they contain. Oils contained in soybeans and olives have long been extracted by applying mechanical pressure. In this way, the components obtained from plants are essential to our lives. In recent years, plant nutrients have been industrially synthesized and commercialized as nutritional supplements.

[0004] Such nutrients and supplements only become effective once they are absorbed by the body, but many properties are required for useful ingredients taken orally to be absorbed into the body. First, they must not be broken down by stomach acid, second, they must be small enough to be absorbed in the intestinal tract, and third, they must be highly efficient at being absorbed in the intestinal tract. Nutrients and supplements must have properties that meet these conditions. Nano-sized (10 -9 Nanoparticles (m) are small enough to be taken up by living cells. For this reason, many methods have been proposed for processing useful ingredients into nano-sized particles, but roughly speaking, two structures have been put into practical use. The first is a micellar structure (emulsion) that uses the principle of soap, and the second is a liposome structure formed from phospholipids that mimics living cells.

[0005] The present inventors proposed a method for producing nanoparticles using metasilicic acid in Patent Document 1. Specifically, soy milk is prepared using hot spring water containing metasilicic acid as preparation water, and this soy milk is used as a culture medium to culture lactic acid bacteria (AI-001). It was found that the medium becomes acidic due to the lactic acid produced by the lactic acid bacteria, and nanoparticles are obtained in which fat-soluble components contained in the soy milk (ceramide, equol, etc.) are enclosed by the metasilicic acid in the acidic culture medium.

[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 brain tissue in order to pass through the blood-brain barrier; Patent Document 5 describes an antibody preparation that prevents amyloid beta, the substance that causes Alzheimer's, from increasing in the brain; Patent Document 6 describes cosmetics that use liposomes; Patent Document 7 describes a treatment for pulmonary fungal infections that uses liposomes; and Patent Documents 8 and 9 describe methods of extracting cannabis extract (CBD) as a fat-soluble component.

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

[0009] According to Non-Patent Document 2, the intestines contain special immune tissues called Peyer's patches, and the M cells within these have the ability to take in many components. It has been reported that there are particle sizes that are easily taken up by M cells, but the smaller the particle size, the more it is taken up.

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

[0011] Japanese Patent No. 6898620 Japanese Patent No. 6190512 Japanese Patent No. 5197880 Japanese Patent No. 6889218 Japanese Patent Publication No. 2023-011002 Japanese Patent No. 2807840 Japanese Patent No. 3245955 Japanese Patent Publication No. 2023-078465 Japanese Patent Publication No. 2021-042233

[0012] Hiroyuki Kusuhara, "Molecular Structure of the Blood-Brain Barrier and Drug Transport," Drug Delivery System 27,5, 201; Hideo Hasegawa and Tatsuhiko Suga, "Long-Life Immunity and Lactic Acid Bacteria: The Role of Nano-Lactic Acid Bacteria in Inducing Th1 Cells," New Food Industry 2008 Vol. 50 No. 8; Yusuke Arai, Toshiya Matsubara, Hyun-ji Kim, Kazuhiko Yamada, "Development of Spherical Hollow Silica Nanoparticles," AGC Research Report 71 (2021); Norihiro Nakamura, "Preparation and Application of Organic Nanosilica Particles," Microscope Vol. 52, No. 3 (2017)

[0013] Nanoparticles made from metasilicic acid remain as fine particles in an acidic environment, but disintegrate in an alkaline environment, releasing the components contained within. Because the blood of the living body is kept slightly alkaline, nanoparticles that enter the bloodstream disintegrate, releasing the components contained within into the bloodstream. The metasilicic acid that formed the nanoparticles dissolves in the bloodstream and is used to bind collagen in hair and skin. These nanoparticles have the unique property that only fat-soluble components are taken up as the core of the nanoparticles. This makes them ideal nanoparticles 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 ingredients may not reach the target organs or tissues. Furthermore, other patent documents and prior art documents do not propose 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 there is no verification as to whether fine particles can be formed from fat-soluble components that are not contained in soy milk.

[0016] In order to solve the above problems, the lactic acid bacteria bioreactor of the present invention for producing ultra-nano particles encapsulating fat-soluble components is characterized in that lactic acid bacteria are cultured in a culture solution containing emulsion particles in which the fat-soluble components are encapsulated with a surfactant and metasilicic acid, and the culture solution is gradually made acidic by the lactic acid produced by the lactic acid bacteria, thereby breaking down the emulsion structure and causing the fat-soluble components to leak from the emulsion particles, and the leaked fat-soluble components are then encapsulated in metasilicic acid to form ultra-nano particles.

[0017] Examples of the fat-soluble components include ceramides, equol, polyphenols, aroma oils, cannabidiol (CBD), and the like, which are contained in plants such as soybeans, peanuts, grapes, oranges, herbs, and hemp, and examples of surfactants include saponins.

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

[0019] According to the present invention, it is possible to produce ultra-nanoparticles encapsulating fat-soluble components of a size that can be easily absorbed by intestinal cells, for example, and thus to obtain medicines and functional foods that are effective in alleviating allergic symptoms, improving skin conditions, providing healing effects, and alleviating pain.

[0020] Graph showing the relationship between the solubility and temperature of sodium metasilicate nonahydrate solution; Graph showing the relationship between the mixing ratio of sodium metasilicate nonahydrate and sodium bicarbonate and the solubility; Graph showing the separation function of sodium metasilicate nonahydrate for fat-soluble components (oil); Graph showing the components of a culture obtained by culturing lactic acid bacteria AI-001; Graph showing particle distribution under different conditions; Graph showing particle distribution under different conditions; Graph showing particle distribution under different conditions; Graph showing particle distribution under different conditions; Graph showing particle distribution under different conditions; Graph showing the pain relief effect of different cannabidiol (CBD) formulations;

[0021] Examples of the present invention are described below. As shown in Figure 1, the outline of the present invention is that lactic acid bacteria are cultured in a culture medium in which metasilicic acid is dissolved and an emulsion containing fat-soluble components formed by saponin is added, thereby producing ultra-nano particles encapsulating lipid components in the culture medium.

[0022] Metasilicic acid (H ２ SiO ３ ) is silicon dioxide plus water, and in this example, commercially available sodium metasilicate nonahydrate (referred to as SiO2 in this specification) was used. Sodium bicarbonate was added to dissolve more of the sodium metasilicate nonahydrate in water.

[0023] Figure 4 compares solubility as a function of temperature, showing that at room temperature the solution is cloudy and has low solubility, but when boiled sodium metasilicate nonahydrate (SiO2) and sodium bicarbonate (Na2HCO3) are used, no cloudiness is observed and the solubility is high. Therefore, when dissolving sodium metasilicate nonahydrate and sodium bicarbonate, it is preferable to boil the solution.

[0024] FIG. 5 shows the results of a test conducted on the mixing ratio of sodium metasilicate nonahydrate to sodium hydrogen carbonate. A ratio of 2:1 of sodium metasilicate nonahydrate to sodium hydrogen carbonate results in the least amount of precipitation, and this ratio is preferred.

[0025] Figure 6 shows the separation effect of fat-soluble components (oil). 2 g of sodium metasilicate nonahydrate and 1 g of sodium bicarbonate were added to 1 liter of boiling water, and 30 g of orange peel powder (Natt Herbst, ETSY, USA) was added to the boiled solution and boiled for an additional 30 minutes. The left photograph in Figure 6 shows the state 0 hours after boiling, and the right photograph shows the state after 24 hours of maintaining at 40°C. Floating matter can be seen in the right photograph, suggesting that metasilicic acid decomposes orange peel and has a high separation effect on fat-soluble components (oil).

[0026] Instead of the water, soy milk containing nutrients that enable the growth of lactic acid bacteria was used, and sodium metasilicate nonahydrate, sodium bicarbonate, and orange peel were added to this soy milk, brought to a boil, and then maintained at 40° C. Lactic acid bacteria (AI-001) were then added to the 40° C. solution (culture solution) and cultured for 6 days.

[0027] The treated culture was dried and subjected to a series of cannabidiol (CBD) analyses using high-performance liquid chromatography (HPLC) (see the left table in Figure 7). Similarly, orange peel alone was analyzed (see the right table in Figure 7). The results show that trace amounts of CBD were detected in the treated culture in which Lactobacillus AI-001 was cultivated.

[0028] CBD is a functional fat-soluble compound that has been approved in many countries for use as a pain reliever and sleep aid, suppressing excessive neurotransmission, and is currently under discussion in the Diet in Japan to allow its clinical use as an anti-epileptic drug. Furthermore, undetectable THC is treated as a narcotic component.

[0029] Next, the plant 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 the ELSZ-2000 series zeta potential, particle size, and molecular weight measurement system (Otsuka Electronics, Kyoto).

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

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

[0032] Thus, the particle distribution can change depending on whether or not the fat-soluble component CBD is present. When CBD is not added, the particles circled in [(5)-CBD] in Figure 9 match the particle size shown in Figure 8(1) when only saponin is added. When CBD is added, the number of particles in this range decreases, and instead, there is an increase in particles of a size that is thought to be an emulsion containing the fat-soluble component CBD.

[0033] Lactic acid bacteria AI-001 was added to the solution shown in Figure 9 (5) and aerobically cultured for 6 days at 40°C. As a result, even smaller nanoparticles (average 1.9 nm) were observed, as shown in Figure 10 (6).

[0034] On the other hand, in the case of the CBD-free sample (Fig. 10 [(6)-CBD]), only particles with an average size of 51 nm were observed. It is speculated that the emulsion of saponin with CBD as the core caused the lactic acid bacteria AI-001 to form even smaller nanoparticles (ultra-nanoparticles).

[0035] When lactic acid bacteria (AI-001) are cultured together, the culture medium gradually becomes acidic, and it is observed that the particles of the emulsion (4) change into even smaller particles (6). Furthermore, without the addition of CBD, no further small particles are formed. This is presumably because even though saponin is broken down, it does not contain any fat-soluble components, so the ultra-nano particles made from metasilicic acid are not produced.

[0036] We observed whether ultra-nano particles were formed with or without SiO2. The results are shown in Figure 11. Lactic acid bacteria AI-001 was added to a solution without SiO2 and cultured at 40°C for 6 days. As a result, no ultra-nano particles were formed (7). The results were almost the same as when lactic acid bacteria AI-001 was added to a solution containing only soy milk and nothing else was added, and cultured at 40°C for 6 days (negative control) (8). This shows that no ultra-nano particles were formed without the addition of SiO2. Therefore, ultra-nano particles are made of SiO2 (they are made of dissolved metasilicic acid).

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

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

[0039] CBD was added to the saponin-containing solution to a concentration of 10 g / L. To form an emulsion of the dissolved saponin, the solution was homogenized (Marusan Machinery, Shizuoka), and then NaHCO3 and SiO2 were added sequentially. Lactic acid bacteria AI-001 was then cultivated. As a result, ultra-nano particles with an average size of 1.0 nm were detected (Figure 13 (11)).

[0040] Figure 14 is a graph verifying the effects of ultra-nanoparticles. Soy milk yogurt was made by inoculating and culturing a solution containing saponin, CBD, Na2HCO3, and SiO2 in soy milk. Lactic acid bacteria AI-001 was then inoculated and cultured to create soy milk yogurt. Subject A consumed 100 ml of this solution daily. Subject A suffers from persistent lower back pain due to severe sciatic nerve damage. Subject A was asked to evaluate the pain relief caused by consuming soy milk yogurt by scoring it on a five-point scale (4: no pain, 3.5: occasional pain, 3: pain when pressed, 2.5: pain is conscious, 2: constant pain). Soy milk yogurt containing various combinations of saponin, CBD, Na2HCO3, and SiO2 was consumed and evaluated. Subjects continued consuming each pattern for seven days. The highest and lowest scores were discarded, and the average score for the remaining five days was used as the evaluation value. As a result, the solution containing Saponin, CBD, Na2HCO3, and SiO2 in succession was found to have an excellent effect in relieving lower back pain.

[0041] Clinical evaluation was conducted to confirm the tissue delivery of the lipid-soluble components encapsulated in ultra-nano particles, and the delivery of lipid-soluble components to nerve tissues was confirmed. Sciatica has various possible causes, but it is thought that CBD's ability to suppress excessive neurotransmission at nerve synapses may contribute to pain relief. Ultra-nano particles containing lipid-soluble components may contribute to the efficiency of intestinal absorption and tissue delivery.

[0042] In summary, by sequentially adding the plant-derived 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. Similar experiments were performed using aroma oils as fat-soluble components in addition to CBD, and ultra-nano particles were detected. The addition of saponin, fat-soluble components, Na2HCO3, and SiO2, followed by the cultivation of lactic acid bacteria, in the above order, is necessary to produce ultra-nano particles.

[0043] Furthermore, since the above solution is a culture medium containing nutrients for the growth of lactic acid bacteria, such as soy milk, the culture medium becomes acidic due to the lactic acid produced by the lactic acid bacteria. As the lactic acid bacteria grow, the surfactant is decomposed into a hydrophilic portion and a lipophilic portion, as shown in Figure 3, and the emulsion particles collapse, causing the fat-soluble components to leak out from the emulsion particles. Since the culture medium has become an acidic metasilicic acid solution due to the lactic acid from the lactic acid bacteria, the leaked fat-soluble components are surrounded by metasilicic acid and form ultra-nano particles (micelle structures) of 1.0 to 10.0 nm.

[0044] Here, soy milk is merely a solution that provides nutrients for the growth of lactic acid bacteria, so any solution other than soy milk will suffice as long as the lactic acid bacteria can grow in it. Furthermore, lactic acid bacteria function as a bioreactor capable of carrying out a series of reactions in the same container: gradually acidifying the environment with lactic acid in a tiny reaction space, disintegrating the emulsion with saponin at an optimal pH, and forming ultrafine particles from the leaked fat-soluble components and metasilicic acid. Therefore, the type of lactic acid bacteria is not limited to AI-001, but since the reaction space is formed by the growth of the lactic acid bacteria's bacterial cell size, a lactic acid bacterium such as AI-001 that is larger than other lactic acid bacteria is preferred.

[0045] Furthermore, sodium bicarbonate makes sodium metasilicate nonahydrate more soluble, and also has the effect of reducing the particle size of emulsions containing fat-soluble components [Figure 8 (3)]. Although sodium bicarbonate is not necessarily required, sodium bicarbonate is preferred.

Claims

1. A method for producing nanoparticles, comprising culturing lactic acid bacteria in a culture solution containing emulsion particles in which fat-soluble components are encapsulated with a surfactant and metasilicic acid, causing the fat-soluble components to leak out of the emulsion particles as the lactic acid bacteria grow, and the leaked fat-soluble components are encapsulated in metasilicic acid in the acidified culture solution to form nanoparticles with a particle size of 1.0 nm to 10 nm.

2. A method for producing nanoparticles encapsulating a fat-soluble component as described in claim 1, characterized in that 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.

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