Minerals for promoting enzyme activity such as lipase and cholesterol esterase, and methods for producing the same.

Abstract

Functional minerals produced from plant ash offer new applications, including the promotion of lipase enzyme activity, the promotion of cholesterol esterase or cholesterol oxidase enzyme activity, and the promotion of micronization of oil droplets of oily substances. [Solution] A plant ash selected from wood ash, grass ash, and seaweed ash obtained by burning trees, grasses, and seaweeds respectively is used as a raw material. A mixed ash obtained by mixing seaweed ash, or 50% by weight or more of seaweed ash, with at least one of wood ash and grass ash, is reheated, and a mineral-containing lipase enzyme activity enhancer, cholesterol esterase or cholesterol oxidase enzyme activity enhancer, and oil droplet micronizer are obtained by extracting the resulting reheated ash with water.

Description

Technical Field

[0001] The present invention relates to a functional mineral and a method for producing the same. More specifically, in addition to an enhanced effect of inhibiting the enzymatic activity of tyrosinase and an antioxidant effect as compared with conventional minerals, as a new effect, it has an effect of promoting the enzymatic activity of lipase, an effect of promoting the enzymatic activity of cholesterol esterase or cholesterol oxidase, and an effect of promoting the refinement of oil droplets. The present invention also relates to a method for producing such a mineral.

Background Art

[0002] The applicant has proposed a method for producing a mineral obtained from wood ash, grass ash, and seaweed ash, which can eliminate the generated melanin in humans and the like (Patent No. 6343367). The mineral is obtained by mixing each of wood ash, grass ash, and seaweed ash at a predetermined ratio, reheating the mixed ash, and extracting the reheated ash with water. And it has been found that this mineral has an effect of inhibiting the enzymatic activity of tyrosinase and an antioxidant effect in addition to the effect of eliminating the generated melanin pigment.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] For the sake of explanation, the conventional minerals produced from the three types of plant ash (wood ash, grass ash, and seaweed ash) mentioned above will be referred to as "conventional blended minerals." In the production of these minerals (conventional blended minerals), all three—wood ash, grass ash, and seaweed ash—were essential, as described above. However, in recent years, the supply of grass ash has become unstable, raising concerns about the stable production of these minerals. Therefore, there has been a need to establish a manufacturing method for these minerals that reduces the amount of grass ash used.

[0005] As a result of diligent research, the applicant discovered that, without using any grass ash, it is possible to produce minerals with performance equivalent to or better than conventionally formulated minerals by reheating a mixed ash obtained by mixing wood ash and seaweed ash in a predetermined ratio, and then extracting the reheated ash with water. It was found that the minerals obtained by this new formulation (hereinafter referred to as the new formulation minerals) not only possess the effects of the conventional formulation minerals described above, namely, the effect of eliminating generated melanin pigment, the effect of inhibiting the enzymatic activity of tyrosinase, and the antioxidant effect, but also exhibit higher effects in these areas than the conventional formulation minerals. Furthermore, the applicant newly discovered that both the conventional formulation minerals and the new formulation minerals have effects that have never been found before, such as the effect of promoting the enzymatic activity of lipase, the effect of promoting the enzymatic activity of cholesterol esterase and cholesterol oxidase, and the effect of promoting the micronization of oil droplets in solutions containing oily substances, leading to the present invention.

[0006] Furthermore, conventionally formulated minerals required a certain level of usage to be fully effective, which contributed to the higher price of products containing these minerals. Therefore, there was a demand for minerals that could be used in smaller quantities while achieving the same or even greater effects than conventionally formulated minerals.

[0007] Therefore, the object of the present invention is to provide a mineral that has enhanced effects in inhibiting tyrosinase enzyme activity and antioxidant effects compared to conventional minerals (conventional blended minerals and their products), and further, to provide a mineral that has new effects such as promoting lipase enzyme activity, promoting cholesterol esterase and cholesterol oxidase enzyme activity, and promoting the micronization of oil droplets, as well as a method for producing such a mineral inexpensively. In particular, the effect of the mineral of the present invention on promoting the activity of lipid-soluble enzymes such as lipase, cholesterol esterase, and cholesterol oxidase is the exact opposite of the previously known effect of inhibiting enzyme activity (effect on tyrosinase), and is expected to greatly expand the range of applications of this mineral. [Means for solving the problem]

[0008] To solve the above problems, the mineral of the present invention is characterized in that it is obtained by using one of the following plant ashes as a raw material: wood ash, grass ash, or seaweed ash, which are obtained by burning trees, grasses, and seaweed, respectively, and by reheating a mixed ash obtained by mixing seaweed ash, or 50% by weight or more of seaweed ash, with at least one of wood ash and grass ash, and extracting the mineral with water from the resulting reheated ash.

[0009] Furthermore, the present invention is characterized by being an enzyme activity enhancer for lipase containing the mineral, an enzyme activity enhancer for cholesterol esterase, an enzyme activity enhancer for cholesterol oxidase, or an oil droplet micronizer.

[0010] Furthermore, the minerals of the present invention may also be a mixed ash obtained by mixing only wood ash with 50% by weight or more of seaweed ash.

[0011] Furthermore, the present invention may also be an antioxidant containing minerals or a tyrosinase enzyme activity inhibitor produced using a mixed ash obtained by mixing 50% by weight or more of seaweed ash with only wood ash.

[0012] Furthermore, the present invention's method for producing minerals includes the step of selecting either wood ash or seaweed ash, obtained by burning trees and seaweed respectively, as a raw material, The process is characterized by comprising the steps of reheating seaweed ash, or a mixed ash obtained by mixing only wood ash with 50% or more by weight of seaweed ash, and extracting the ash after reheating with water. [Effects of the Invention]

[0013] The minerals of this invention exhibit even higher functionality compared to conventional minerals due to the discovery of a novel composition. This allows for the provision of minerals with enhanced tyrosinase enzyme activity inhibition and antioxidant effects compared to conventionally formulated minerals. Simultaneously, this novel composition eliminates the use of grass ash, which has an unstable supply, enabling stable production and supply of minerals. Furthermore, the enhanced effect allows for the use of a smaller amount to achieve the same or greater effect as conventional products, thus reducing the amount used in the production of mineral-containing products. This, in turn, lowers the production cost of the product. Moreover, the discovery of entirely new applications, such as the effect of promoting lipase enzyme activity and the effect of promoting cholesterol esterase or cholesterol oxidase enzyme activity, suggests that applications utilizing this function in various fields are expected in the future. In addition, according to this invention, minerals with such diverse effects can be produced inexpensively and easily. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 is a graph showing the difference in antioxidant capacity for each blending ratio of the minerals according to the present invention. (A) shows the conventional mineral blend consisting of wood ash, grass ash, and seaweed ash, and (B) shows the effect of the new mineral blend consisting of only wood ash and seaweed ash. [Figure 2]Figure 2 is a graph showing the inhibitory effect on tyrosinase enzyme activity of the minerals according to the present invention at different blending ratios. (A) shows the inhibitory effect on tyrosinase enzyme activity at a mineral concentration of 1%, (B) shows the inhibitory effect at a mineral concentration of 0.05%, and (C) shows a comparison of the inhibitory effect on tyrosinase enzyme activity of conventional and newly blended mineral compositions at different concentrations. [Figure 3] Figure 3 is a graph showing the effect of the mineral according to the present invention on the enzymatic activity of lipase. [Figure 4] Figure 4 is a graph showing the effects of the minerals according to the present invention on enzyme activity, specifically (A) cholesterol esterase, (B) cholesterol oxidase, and (C) horseradish peroxidase (HRP). [Figure 5] Figure 5 is an image showing the effect of the mineral according to the present invention on oil droplets. [Figure 6] Figure 6 is an image comparing the browning prevention effect of the minerals according to the present invention on foods, with (A) showing the browning prevention effect on avocados and (B) showing the browning prevention effect on apple juice. [Modes for carrying out the invention]

[0015] The present invention relates to a functional mineral obtained by using one of the following plant ashes as a raw material: wood ash, grass ash, or seaweed ash, which are obtained by burning trees, grasses, and seaweed, respectively. The ash is obtained by reheating a mixed ash, which is made by mixing seaweed ash or 50% by weight or more of seaweed ash with at least one of wood ash and grass ash, and then extracting the resulting reheated ash with water. Furthermore, the present invention relates to a functional mineral containing the functional mineral, which is an enzyme activity enhancer for lipase, an enzyme activity enhancer for cholesterol esterase, an enzyme activity enhancer for cholesterol oxidase, or an oil droplet micronizer. The present invention also relates to a functional mineral produced using a mixed ash obtained by mixing 50% by weight or more of seaweed ash with only wood ash as the mixed ash, and which may be an antioxidant or a tyrosinase enzyme activity inhibitor containing the mineral.

[0016] The wood ash, grass ash, and seaweed ash used in this invention are produced by burning natural plant materials until they are reduced to ash. Wood ash is produced by burning trees and reducing them to ash, and there are no particular restrictions on the type of tree. For example, it may be made from branches and leaves cut from trees in a forest, and may also include parts of the tree trunk. Grass ash may be produced by burning grass, commonly known as weeds, and there are no particular restrictions on the type of grass. For example, grasses that grow naturally on the slopes of riverbanks (embankments) can be used as raw materials. Seaweed ash may be produced by burning plant materials, generally known as seaweed, and reducing them to ash. There are no particular restrictions on the type of seaweed used for seaweed ash either.

[0017] The mixed ash used in this invention is a mixture of seaweed ash and one or more materials selected from wood ash and grass ash, in a ratio containing 50% by weight or more of seaweed ash. Here, this mixed ash can also be replaced with seaweed ash only (100% seaweed ash). Furthermore, when mixing two or more materials, as long as seaweed ash is included at 50% by weight or more, the other wood ash and grass ash can be in any proportion. When using all of wood ash, grass ash, and seaweed ash, the preferred mixing ratio is wood ash (25-37.5%), grass ash (12.5-25%), and seaweed ash (50-75%), and these can be appropriately selected within this range so that the total is 100%. Here, it is also possible to use a blending ratio that does not use any grass ash, which has been in an unstable supply in recent years. In that case, the blending ratio is 50% by weight or more of seaweed ash and 50% by weight or less of wood ash, preferably wood ash (4-33%) and seaweed ash (67-96%), and particularly preferably wood ash (8-25%) and seaweed ash (75-92%), and can be appropriately selected within this range so that the total is 100%.

[0018] When the amount of calcined mixed ash used in the present invention is about several tens of Kg per firing, the reheating of the mixed ash is preferably carried out by setting the reheating conditions as a heating temperature of 600 - 700 °C (particularly preferably 650 °C) and a heating time of 2 - 6 hours (particularly 5 hours), and setting oxygen-blocking heating in an electric furnace or the like. Particularly preferably, it is heating at 650 °C for 5 hours. However, when the amount of calcined mixed ash per firing is an extremely small amount (the amount of ash is from several grams to about 100 grams), the heating time can be about 2 hours at the same temperature. Such adjustment of heating conditions can be easily adjusted by those skilled in the art by confirming the effects of the produced minerals.

[0019] By reheating after mixing a plurality of plant ashes in this way, it becomes possible to extract the minerals of the present invention. The applicant has conducted various tests and studies to elucidate this mechanism, but has not yet reached an elucidation. The reasons considered are that when the plant materials are only wood ash, grass ash, and seaweed ash in the first firing, there may be parts where the firing is uneven and complete firing has not been achieved, or there may be some changes (chemical reactions) occurring due to heating in a state where a plurality of plant ashes are mixed.

[0020] The minerals of the present invention are obtained by extracting with water from the reheated ash obtained by reheating. For the extraction, it is preferable to set the water temperature to 5 - 60 °C (room temperature may also be used), dissolve the reheated ash in the water, heat it to boiling, and then filter it using filter paper or a filter cloth to generate an aqueous solution containing minerals. Also, the water can be evaporated from the aqueous solution to generate powdered minerals.

[0021] [[ID=ll]] Hereinafter, embodiments of the present invention will be specifically described based on several examples, but the present invention is not limited thereto.

Example

[0022] <Antioxidant test> Conventional mineral formulations are known to have antioxidant effects. To confirm the antioxidant effect of the new mineral formulation of this invention, tests were conducted based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method. [Test Method] The minerals used as samples were obtained by burning trees, grasses, and seaweed to produce wood ash, grass ash, and seaweed ash, respectively. These were mixed according to the mixing ratios shown in Table 1, and the mixed ashes were reheated in an electric furnace at 650°C for 2 hours using oxygen-free heating. The reheated ashes were mixed with room temperature water in a weight ratio of approximately 1:3, heated to a boil, and then filtered to extract the minerals. The water was evaporated from the resulting aqueous solution to obtain a powder, which was then used.

[0023] The minerals used in the test were produced using mixed ash obtained by mixing wood ash, grass ash, and seaweed ash in the mixing ratios shown in Table 1. Sample No. 1, used for comparison, is BIE Mineral, a plant mineral product manufactured and sold by Plant Power Research Institute Co., Ltd. BIE Mineral is a mineral produced using mixed ash obtained by mixing wood ash, grass ash, and seaweed ash in a ratio of 1:1:2. Samples No. 2 to 6 were used to confirm the difference in functionality when the amount of grass ash used was reduced compared to the conventional formulation which uses all three: wood ash, grass ash, and seaweed ash. For comparison, Sample No. 5 did not use grass ash, and Sample No. 6 used only seaweed ash. Samples No. 7 to 13 were used to confirm the functionality of a new formulation which uses only wood ash and seaweed ash without using any grass ash. For comparison, Sample No. 7 uses only seaweed ash. This test was conducted on a laboratory scale, so the total ash amount for Samples No. 2 to 6 was 40g, and the total ash amount for Samples No. 7 to 13 was 24g. Specifically, for example, Sample No. 2 was prepared by mixing 10g of wood ash, 10g of grass ash, and 20g of seaweed ash in the specified mixing ratio (2:2:4) to obtain a total ash amount of 40g, while Sample No. 10 was prepared by mixing 4g of wood ash and 20g of seaweed ash in the specified mixing ratio (4:20) to obtain a total ash amount of 24g. In this test, minerals were produced by reheating small amounts of mixed ash in this manner on a laboratory scale, so the reheating was performed at a heating temperature of 650°C for 2 hours to produce each mineral.

[0024] [Table 1]

[0025] Trolox, an antioxidant water-soluble vitamin E derivative, was used as the standard for the antioxidant activity test. Trolox was prepared in 49.8% ethanol at concentrations ranging from 0 μM to 500 μM, and various mineral aqueous solutions were prepared at a concentration of 1 mg / mL. DPPH was dissolved in 99.5% ethanol to a concentration of 1 mM, and 200 mM MES (2-morpholinoethanesulfonic acid) (pH 6.0) was used as the buffer. Equal volumes of the Trolox dilution series or mineral aqueous solutions, MES, and DPPH were dispensed into a 96-well microplate (clear), mixed, and allowed to stand at room temperature for 20 minutes. Subsequently, the absorbance at 540 nm (OD540nm) was measured using a multimode microplate reader (Tecan, Infinite 200 PRO M Plex). A calibration curve was created from the results of the Trolox dilution series, and the antioxidant activity of each mineral was calculated in terms of Trolox equivalent. The test results are shown in the graph in Figure 1.

[0026] The graph in Figure 1A shows the results of comparing a conventional mixture of three types of plant ash—wood ash, grass ash, and seaweed ash—by varying the mixing ratio of each ash. Generally, minerals produced using a mixture with a reduced grass ash ratio and a higher seaweed ash ratio tended to exhibit higher antioxidant power (Samples No. 2-5). In particular, the high oxidizing power of Sample No. 5, which had a grass ash ratio of 0, is noteworthy. Furthermore, while a higher seaweed ash ratio tended to increase oxidizing power, Sample No. 6, which contained only seaweed ash, showed slightly lower oxidizing power compared to Samples No. 2-5, which also contained other plant ashes (wood ash and grass ash), indicating that mixing multiple plant ashes enhances functionality. Note that Samples No. 1 and No. 2 had the same simple mixing ratio of 1:1:2, but their oxidizing power results in this test were different. This is thought to be because, since the minerals of the present invention are manufactured from natural raw materials, slight differences in effectiveness inevitably occur depending on the manufacturing lot, and because sample No. 1, the BIE mineral, is a product manufactured on a factory scale, resulting in slight differences in the effect of reheating compared to samples No. 2-6, which were prepared on a laboratory scale. In any case, it was confirmed that it is possible to produce minerals that exhibit antioxidant power equivalent to or greater than that of the conventionally formulated BIE mineral, even when the proportion of grass ash used is reduced.

[0027] The graph in Figure 1B shows the results of tests using minerals prepared from a new two-component mixed ash composition, which uses only wood ash and seaweed ash without grass ash. Samples No. 7 to 13 all showed higher antioxidant power than the BIE mineral product (Sample No. 1) manufactured from the conventional three-component mixed ash composition. However, when using this two-component mixed ash, the antioxidant power did not increase with a higher proportion of seaweed ash; high antioxidant power was observed at a ratio of 1:23 to 8:16 (seaweed ash 95.8% to 66.7%), and particularly high antioxidant power was observed at a ratio of 2:22 to 6:18 (seaweed ash 91.7% to 75.0%). From the above, it was confirmed that the minerals of the present invention act as antioxidants. [Examples]

[0028] <Confirmation of the inhibitory effect on tyrosinase enzyme activity> It is known that tyrosinase (melanin-producing enzyme) is involved in melanin production. In contrast, the applicant has disclosed that conventionally formulated minerals have the effect of inhibiting the activity of tyrosinase (Patent No. 6343367). The following tests were conducted to determine whether the new formulation of minerals also has the effect of inhibiting the enzymatic activity of tyrosinase.

[0029] This study investigated whether the mineral according to the present invention can be administered during the melanin production process to suppress melanin production. Equal volumes of a diluted solution of the mineral according to the present invention, a 0.03% 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA) solution, and a 135 U / mL tyrosinase solution were dispensed into a 96-well microplate (clear), stirred, and then allowed to stand at room temperature in the dark for 30 minutes. Subsequently, the absorbance at 450 nm (OD450) was measured using a multimode microplate reader (Tecan, Infinite 200 PRO M Plex) and compared with the enzyme activity of samples containing each concentration of the mineral. For the negative control, a dilution buffer (50 mM phosphate buffer, pH 6.5) was used instead of the tyrosinase solution, and the enzyme activity rate (%) of tyrosinase was calculated using the following formula. Tyrosinase enzyme activity rate (%) = (Measured value of sample - Measured value of negative control) / (Measured value of 0% sample - Measured value of negative control) × 100 In this test, minerals produced in factory-scale quantities using the same mixing ratio as Sample No. 11 shown in Table 1 were used. Specifically, minerals extracted from an aqueous solution (wood ash 6: seaweed ash 18) obtained by reheating 24 kg of mixed ash (6 kg wood ash and 18 kg seaweed ash) at 650°C for 5 hours, and adding 60 L of water, were used as the minerals of the present invention (newly formulated minerals).

[0030] Figure 2 shows a graph illustrating the inhibition of tyrosinase enzyme activity by the minerals according to the present invention. Figure 2(A) shows the results of tests with a mineral concentration of 1%, and (B) shows the results of tests with a mineral concentration of 0.05%. Figure 2(C) compares the results when the concentrations of conventional BIE minerals, the newly formulated minerals (wood ash 6: seaweed ash 18), and kojic acid are varied. Here, kojic acid is known as a substance that inhibits melanin production. From the test results, it was found that the minerals according to the present invention can suppress tyrosinase enzyme activity in a concentration-dependent manner. In particular, it can be seen that the newly formulated minerals of the present invention show an excellent inhibitory effect on tyrosinase enzyme activity even at low mineral concentrations. This effect is particularly pronounced in minerals produced from mixed ash of wood ash and seaweed ash in a ratio of 6:18. With conventional BIE minerals, a significant inhibitory effect on tyrosinase activity is seen at a concentration of 1% (A), but almost no inhibitory effect on enzyme activity is seen at a concentration of 0.05% (B). In contrast, the newly formulated mineral mixture (particularly wood ash:seaweed ash = 6:18) showed the same enzyme inhibitory effect at a concentration of 0.05% as at a concentration of 1%, indicating enhanced functionality. Therefore, it has been confirmed that the mineral mixture of this invention acts as an enzyme activity inhibitor of tyrosinase. [Examples]

[0031] <Confirmation of the effect on lipase enzyme activity> Lipase is known as an enzyme that breaks down neutral fats (triglycerides), and it works by acting on the ester bonds in triglycerides to hydrolyze them into glycerol and fatty acids. To investigate the effect of the mineral of the present invention on lipase, we measured the activity of lipase using 4-methylumbelliferyl oleate (4-MUO), which releases fluorescent 4-methylumbelliferone (4-MU) upon decomposition by lipase.

[0032] [Test Method] Using oleate ester of fluorescent 4-MU (4-MUO), the fluorescence of 4-MU produced by the degradation reaction by lipase was measured to confirm its effect on lipase enzyme activity. The specific test method is as follows. Lipase was prepared using Sigma-14 Lipase (derived from porcine spleen: Type II, >=125 units / protein) and diluted to 2 mg / mL using 13 mM Tris-HCl buffer (pH 8.0). After sufficient suspension, suspended solids were observed, so the mixture was centrifuged at 10,000 rpm for 10 minutes at 4°C, and the supernatant was used. The substrate, 4-MUO, was prepared as a 0.1 M stock solution using dimethyl sulfoxide (DMSO) and stored at -30°C. Immediately before use, it was diluted to 0.1 mM using 13 mM Tris-HCl buffer, incubated at 37°C for 5 minutes, and then sonicated at 37°C for 5 minutes. The mineral sample of the present invention was the same mineral used in Example 2 and diluted to 0.001-1% using 13 mM Tris-HCl buffer. Furthermore, as a control sample, orlistat, a lipase inhibitor, was prepared to a concentration of 0.01 mM using 13 mM Tris-HCl buffer. Using these prepared solutions, the decomposition reaction was carried out according to the following procedure.

[0033] First, 25 μL of the sample (mineral or inhibitor of the present invention) was dispensed into 96-well microplates (black). 25 μL of 13 mM Tris-HCl buffer was dispensed into each of the 0% mineral condition and the negative control (triple measurement). Next, 50 μL of 4-MUO was added to each and mixed by stirring. Then, 25 μL of lipase was immediately added to each of the sample solution and the 0% mineral condition. For the negative control, 25 μL of 13 mM Tris-HCl buffer was added instead of lipase. After stirring, the mixtures were incubated in the dark at 37°C for 30 minutes. Subsequently, 100 μL of 0.1 M citrate buffer (pH 4.2) was added to each to stop the reaction, and measurements were performed using a multimode microplate reader (Tecan, Infinite 200 PRO M Plex) at an excitation wavelength of 350 nm and a measurement wavelength of 450 nm. From the obtained measurements, the enzyme activity rate (%) of lipase was calculated using the following formula. Lipase enzyme activity rate (%) = (Measured value of sample - Measured value of negative control) / (Measured value under 0% conditions - Measured value of negative control) × 100

[0034] The results are shown in Figure 3. These results indicate that both the conventional BIE mineral and the newly formulated mineral have a concentration-dependent effect in promoting lipase enzyme activity. Therefore, it has been confirmed that the mineral of the present invention acts as a lipase enzyme activity enhancer. [Examples]

[0035] <Confirmation of the effects on the enzymatic activity of cholesterol esterase, cholesterol oxidase, and peroxidase> Since the above-mentioned test showed an effect of promoting enzyme activity against lipase, we investigated the effect of the minerals of the present invention on the enzyme activity of other enzymes with lipid-soluble substrates, such as cholesterol esterase, in order to confirm this effect. For measuring the enzyme activity of cholesterol esterase, we used the Amplex Red Cholesterol Assay Kit (Invitrogen). This kit contains three types of enzymes (cholesterol esterase, cholesterol oxidase, and horseradish peroxidase (HRP)). Esterified cholesterol is converted to free cholesterol by the action of cholesterol esterase. Free cholesterol is then oxidatively decomposed by cholesterol oxidase to produce hydrogen peroxide (H2O2). By reacting this hydrogen peroxide, a byproduct of this cholesterol oxidation reaction, with Amplex Red, a colorless substrate reagent, a fluorescent substance called resorphine is produced. The activity of each enzyme can be evaluated by comparing the fluorescence values ​​of the produced resorphines. The enzyme reaction solutions used in the experiment had compositions shown in Table 2, depending on the target substance to be measured. A reaction solution without any enzymes was used as a negative control. The sample-substrate mixture was prepared using 40 mM Tris-HCl buffer (pH 7.5) to a sample concentration of 0-1%. The mineral of the present invention used as the sample is the same mineral used in Example 2. The substrate added was as shown in Table 2, depending on the substance to be measured.

[0036] [Table 2]

[0037] The measurement procedure was as follows: 50 μL of each concentration of sample-substrate mixture was dispensed into a 96-well (black) microplate. In addition, 50 μL of 0% sample-substrate mixture was dispensed as a negative control (triple measurement). When the substrate was cholesterol-linoleic acid or cholesterol, the mixture was allowed to stand at room temperature for 10 minutes after dispensing into the microplate. Next, 50 μL of each enzyme reaction solution or the negative control reaction solution was added, and the mixture was reacted at 37°C for 30 minutes in the dark. After that, measurements were performed using a multimode microplate reader (Tecan, Infinite 200 PRO M Plex) at an excitation wavelength of 550 nm and a measurement wavelength of 590 nm. The activity rate (%) of each enzyme was calculated using the following formula. Enzyme activity rate (%) = (Measured value of sample-substrate mixture at each concentration - Measured value of negative control) / (Measured value of 0% sample-substrate mixture - Measured value of negative control) × 100

[0038] The measurement results are shown in Figure 4. (A) shows the results of measuring the activity of cholesterol esterase using cholesterol linoleic acid as a substrate. (B) shows the results of measuring the activity of cholesterol oxidase using cholesterol as a substrate. (C) shows the results of measuring the enzyme activity of peroxidase using hydrogen peroxide as a substrate. From the results shown in (A) and (B), it can be seen that the minerals of the present invention, both the conventionally formulated minerals and the newly formulated minerals, have the effect of promoting the enzyme activity of cholesterol esterase and cholesterol oxidase in a concentration-dependent manner. Since the effect of promoting the activity of cholesterol oxidase, an enzyme that works downstream in the reaction, was confirmed, it is expected that the effect of promoting the activity of cholesterol esterase, an enzyme that works upstream in the reaction, is secondary. Furthermore, it is also expected that the decomposition is promoted not so much because the enzyme promotes the hydrolysis of ester bonds, but because the oil particles become smaller due to the action of the minerals, making it easier for the enzyme to act. From the results shown in (C), it was confirmed that the enzyme activity of peroxidase was suppressed in a concentration-dependent manner, especially with the newly formulated minerals, which is presumed to be due to the antioxidant power of the minerals of the present invention. Based on the above, it has been confirmed that the minerals of the present invention act as enzyme activity enhancers for cholesterol esterase or cholesterol oxidase. [Examples]

[0039] <Confirmation of the oil droplet micronization effect> In the previous test, it was considered that the mineral of the present invention may have the effect of breaking down oily substances into smaller particles, so this was confirmed by the following test. [Test Method] Canola oil was added to DMSO to a concentration of 32%, dispersed using a micropipette, and then an equal volume of 100 mM Tris-HCl buffer (pH 7.5) was added. The mixture was then sonicated while inverting and mixing until the oil solution became cloudy. The mineral sample of the present invention (the same mineral used in Example 2) was prepared at concentrations of 5% and 10% in 50 mM Tris-HCl buffer (pH 7.5). Equal volumes of the cloudy canola oil solution and the sample were added to a 1.5 mL Eppendorf tube and allowed to stand for 30 minutes. In the 0% plant mineral condition, a 50 mM Tris-HCl buffer without added minerals was used, and in the surfactant condition, a solution of Triton X-100 prepared to a concentration of 2% in 50 mM Tris-HCl buffer was used. After pipetting, the mixture was transferred to a 96-well microplate (black glass bottom), and images were acquired under bright-field conditions using an all-in-one fluorescence microscope (Keyence Corporation, BZ-X810) for comparison. The obtained images are shown in Figure 5. From these results, it can be seen that both the conventionally formulated minerals and the newly formulated minerals have a concentration-dependent effect in promoting the micronization of oil. Therefore, it has been confirmed that the minerals of the present invention act as an oil droplet micronizing agent. [Examples]

[0040] <Confirmation of the effect of preventing browning of food> As an application of the antioxidant effect of the minerals of this invention, we confirmed their effect in preventing discoloration of food. (1) Preventive effect against browning of avocados Small pieces of avocado were mixed with water or a mineral solution (for example, 1 mL of mineral solution per 20 g of avocado) to a concentration of 5% of the avocado's weight. The mixture was then mashed into a paste and left at room temperature for 5 hours. The resulting state was then compared. The minerals used were either the conventionally formulated mineral (BIE) or the newly formulated mineral (the same mineral used in Example 2). The mineral concentrations of the aqueous solutions were set to 0.016%, 0.08%, 0.4%, 2%, and 10%, respectively. The results are shown in Figure 6(A). From these results, it was confirmed that both the conventionally formulated and newly formulated minerals had a concentration-dependent effect in suppressing avocado browning. In the comparison between the conventionally formulated and newly formulated minerals, as shown in Figure 6(A), at low mineral concentrations (0.4% or less), the newly formulated minerals showed a browning suppression effect equivalent to or better than the conventionally formulated minerals. At high concentrations (2%, 10%), the newly formulated minerals showed a superior browning suppression effect compared to the conventionally formulated minerals.

[0041] (2) Browning inhibitory effect on apple juice The state of the fruit juice obtained by adding water or a mineral solution (for example, 2 mL of mineral solution for 40 g of apple) to small pieces of apple, then blending and filtering the mixture was compared. The minerals used were either the conventionally formulated mineral solution (BIE) or the newly formulated mineral solution (the same mineral used in Example 2), with mineral solution concentrations of 0.25%, 0.5%, 1%, and 2%, respectively. The results are shown in Figure 6(B). Each sample juice was stored on ice until the images were taken. For the sample with 0% mineral addition (water only), browning of the juice was observed immediately after obtaining the juice. In samples with mineral solution concentrations of 0.5% or less, while some suppression of browning was observed compared to the unadded sample, the suppression effect was not significant. Furthermore, no significant difference in suppression effect was observed between the conventionally formulated mineral solution and the newly formulated mineral solution. However, in samples with mineral solution concentrations of 1% or more, a clear browning suppression effect was observed, as shown in the figure. Furthermore, when compared at the same concentration, the newly formulated minerals exhibit a greater browning-inhibiting effect than the conventionally formulated minerals. Therefore, it can be concluded that the minerals of the present invention have an effect in suppressing browning of food. [Industrial applicability]

[0042] The minerals of the present invention provide previously unknown effects of minerals, such as promoting the enzymatic activity of lipase, promoting the enzymatic activity of cholesterol esterase or cholesterol oxidase, and promoting the micronization of oil droplets of oily substances. Furthermore, the new formulation provides a superior inhibitory effect on tyrosinase enzymatic activity compared to conventionally formulated minerals. Therefore, it is expected to have applications in promoting the breakdown of neutral fats, promoting cholesterol breakdown, suppressing melanin production, and promoting the emulsification of oily substances, and is expected to have not only cosmetic effects but also medical effects. Moreover, the minerals of the present invention are expected to have a wide range of applications in the food manufacturing field. For example, because they have an effect of preventing browning of food, they can be used as a browning inhibitor for foods such as fruits and vegetables, fresh fish, and crustaceans where browning over time is a problem. In addition, because they have an enzymatic activity promoting effect on cholesterol-related enzymes and lipase, as well as an oil and fat micronization effect, they can also be used as a quality improver for oils and fats. Furthermore, the lipase enzymatic activity promoting effect of the minerals of the present invention can also be used to promote the tenderization of meat.

Claims

1. The raw material is one of the plant ashes selected from wood ash, grass ash, and seaweed ash obtained by burning trees, grasses, and seaweed, respectively. Reheat seaweed ash, or a mixed ash obtained by mixing 50% or more by weight of seaweed ash with at least one of wood ash and grass ash. Minerals obtained by extracting water from the reheated ash.

2. A lipase enzyme activity enhancer characterized by containing the mineral described in claim 1.

3. A cholesterol esterase or cholesterol oxidase enzyme activity enhancer characterized by containing the mineral described in claim 1.

4. An oil droplet micronizer characterized by containing the mineral described in claim 1.

5. The mineral according to claim 1, characterized in that the mixed ash is a mixed ash obtained by mixing only wood ash with 50% by weight or more of seaweed ash.

6. An antioxidant characterized by containing the mineral described in claim 5.

7. An enzyme activity inhibitor of tyrosinase, characterized by containing the mineral described in claim 5.

8. The process involves selecting either wood ash or seaweed ash, obtained by burning trees and seaweed respectively, as the raw material, and A process of reheating seaweed ash, or a mixed ash obtained by mixing only wood ash with seaweed ash containing 50% or more by weight, A method for producing minerals according to claim 1, comprising the steps of: and extracting the ash after reheating with water.

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