Method for producing equol

Abstract

To provide fermentation extracts of lactic acid bacteria comprising products produced by lactic acid bacteria, which exert in the body bactericidal effect against so-called bad bacteria as well as anti-aging effect without using chemicals (synthetic chemical substances) by adding a mixture to the extract.SOLUTION: Provided is a method of producing equal using a lactic acid bacteria fermentation extract comprising lactobacillus acidophilus products, including: a step of producing a mixture by mixing lactic acid bacteria fermentation extract derived from brown sugar, okara (bean curd), or soybeans, more than 22% by weight of plum extract, and soy isoflavones including daidzein or daidzein under a predetermined condition; and a step of fermenting the mixture with hydrogen and deep-sea water.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition containing a lactic acid bacterium-produced substance using a lactic acid bacterium fermentation extract composed of a lactic acid bacterium-produced substance, a method for producing equol, and a method for producing a composition containing a lactic acid bacterium-produced substance.

Background Art

[0002] Conventionally, the effects of lactic acid bacteria have been known, and for example, they are used to solve problems in the oral cavity. Patent Document 1 discloses a technique for causing coaggregation with major oral bacteria that form biofilms in the oral cavity by containing lactic acid bacteria of the genus Leuconostoc in an oral composition. In this technique, the lactic acid bacteria adhere to the oral mucosa and colonize in the oral cavity, thereby efficiently inhibiting the growth of pathogenic bacteria present in the oral cavity, aiming to prevent and treat oral diseases.

[0003] Further, Patent Document 2 discloses a technique related to a microorganism or a fragment thereof as an oral care agent for preventing dental calculus. This technique relates to a composition containing a microorganism or a fragment thereof for reducing Streptococcus mutans. Such a composition can be used, for example, in an oral care composition for preventing dental caries or for preventing dental calculus or bad breath.

[0004] Further, Patent Document 3 discloses an inducer of a safe and side-effect-free antibacterial peptide. In this inducer, the lactic acid bacteria may be ingested as intact cells as live bacteria, or may be ingested in the form of a live bacteria-containing material obtained by fermenting the lactic acid bacteria. By ingesting this in vivo secretion inducer of the antibacterial peptide, it is expected that the antibacterial peptide is induced in the body, and an improvement in immunity due to various infection defenses, anti-aging, and an extension of a healthy life span can be expected.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] As described above, the effects of lactic acid bacteria are known. However, regarding how a lactic acid bacteria fermentation extract composed of lactic acid bacteria-produced substances is formulated with other substances to exhibit a bactericidal effect against so-called harmful bacteria or to decompose substances that may cause health problems, it has not been fully elucidated.

[0007] The present invention has been made in view of such circumstances. By additionally formulating a mixture with a lactic acid bacteria fermentation extract, without using chemical substances (synthetic chemical substances), it is possible to provide a lactic acid bacteria-produced substance-containing composition that can exhibit a bactericidal effect against so-called "harmful bacteria" (referring to mutans bacteria, gingivalis bacteria, pasteurella bacteria, pylori bacteria, staphylococcus aureus, welchii bacteria, candida bacteria, etc. The same shall apply hereinafter) in the living body and an anti-aging effect (anti-glycation, anti-oxidation, skin care). Although candida bacteria are generally known as opportunistic bacteria that are neither beneficial nor harmful, once the body's immunity declines, they rapidly increase in the oral cavity and intestines, causing oral candidiasis and intestinal leakage (leaky gut syndrome), and it is said that it is not easy to suppress their growth. [Means for Solving the Problems]

[0008] (1) To achieve the above object, the present invention has taken the following means. That is, the composition containing lactic acid bacteria-produced substances of the present invention is a composition containing lactic acid bacteria-produced substances using a lactic acid bacteria fermentation extract composed of lactic acid bacteria-produced substances, which contains a lactic acid bacteria fermentation extract derived from brown sugar, okara, or soybeans, and is characterized by containing ume extract, which is a Rosaceae plant, at 22% or more by weight ratio.

[0009] (2) Further, in the composition containing lactic acid bacteria-produced substances of the present invention, the lactic acid bacteria fermentation extract contains peptides at 377 (mg / L) to 566 (mg / L).

[0010] (3) Further, the composition containing lactic acid bacteria-produced substances of the present invention further contains at least one of deep sea water, water-soluble silicon, ceramide, hyaluronic acid, eggshell membrane peptide, nacre pearl powder, equol, or daidzein-containing soy isoflavones.

[0011] (4) Further, the composition containing lactic acid bacteria-produced substances of the present invention is characterized by further containing at least one or any plurality of Rosaceae plant extracts, hibiscus extracts, cranberry extracts, pomegranate extracts, olive extracts (leaves, fruits, flowers), camellia extracts (leaves, fruits, flowers), perilla seed extracts, tea seed extracts, or soybean extracts at 0.03% to 5% by weight ratio.

[0012] (5) Further, the composition containing lactic acid bacteria-produced substances of the present invention is characterized by further containing ionized apatite containing at least natural biogenic or mineral-derived hydroxyapatite and citric acid.

[0013] (6) Further, the composition containing lactic acid bacteria-produced substances of the present invention is characterized by further containing silk hydrogen pearl powder, which is a combination of silk powder, hydroxyapatite, hydrogen, and deep sea water.

[0014] (7) Further, the composition containing lactic acid bacteria-produced substances of the present invention is characterized by further containing curcumin and pea sprout extract.

[0015] (8) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing at least one of rose petal powder, grape seed powder, hibiscus powder, and cranberry extract.

[0016] (9) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing barley lactic acid fermentation extract and passion flower extract.

[0017] (10) Further, the composition containing the lactic acid bacteria-produced substance of the present invention contains at least fulvic acid among fulvic acid or humic acid, and at least reduced deep-sea water mineral or reduced fermentation fulvic acid containing lactic acid bacteria among deep-sea water mineral or Great Salt Lake mineral reduced by hydrogen.

[0018] (11) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing coconut shell activated carbon.

[0019] (12) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing a combination of red orange extract, pineapple enzyme, papaya enzyme, hydrogen, and deep-sea water.

[0020] (13) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing hydrocharcoal which is a combination of at least one of activated carbon or edible charcoal, inulin, hydrogen, and deep-sea water.

[0021] (14) Further, in the composition containing the lactic acid bacteria-produced substance of the present invention, the hydrocharcoal is characterized by further containing Ecklonia cava powder and citric acid. In addition to Ecklonia cava, seaweed-derived materials such as Undaria pinnatifida, sea mustard, sea lettuce, and dulce may be used.

[0022] (15) Further, the composition containing the lactic acid bacteria-produced substance of the present invention is characterized by further containing chlorogenic acid.

[0023] (16) Further, the composition containing the lactic acid bacterium-produced substance of the present invention is characterized by further containing a plurality of types of oligosaccharides, lactic acid bacteria, and green papaya extract.

[0024] (17) Further, the composition containing the lactic acid bacterium-produced substance of the present invention is characterized by further containing at least one of astaxanthin, shiitake mushroom extract, coprinus, tamogitake, and benikisnokitake with respect to the red pine bark extract powder.

[0025] (18) Further, in the composition containing the lactic acid bacterium-produced substance of the present invention, the water-soluble silicon is composed of at least one silicon selected from plant-derived silicon, mineral-derived silicon, and water-soluble silicon (meta-silicic acid, or hydrous silica or hydrous silicic acid such as ortho-silicic acid) derived from hot springs, spring water, lava, and rocks in the Fuji, Hakone, Kirishima, and Sakurajima regions.

[0026] (19) Further, the method for producing equol of the present invention is characterized by adding soybean isoflavone containing daidzin or daizein to the composition containing the lactic acid bacterium-produced substance described in (1) above under predetermined conditions.

[0027] (20) Further, the method for producing the composition containing the lactic acid bacterium-produced substance of the present invention is a method for producing the composition containing the lactic acid bacterium-produced substance using a lactic acid bacterium fermentation extract composed of a lactic acid bacterium-produced substance, and includes at least a step of mixing a lactic acid bacterium fermentation extract derived from brown sugar, okara, or soybeans with ume extract, which is a Rosaceae plant, at a weight ratio of 22% or more to produce a mixture, and a step of fermenting the mixture using hydrogen and deep ocean water.

Advantages of the Invention

[0028] According to the present invention, it becomes possible to obtain an effective bactericidal effect and anti-aging effect against so-called "bad bacteria" in the living body without using chemical substances (synthetic chemical substances).

Brief Description of the Drawings

[0029]

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Mode for Carrying Out the Invention

[0030] The present inventors focused on the efficacy of lactic acid bacteria fermentation extracts derived from brown sugar, okara, or soybeans, and by blending ume extract, hydrogen and deep ocean water, and other highly functional raw materials, they found that remarkable bactericidal effects and anti-aging effects can be obtained, thus arriving at the present invention.

[0031] That is, the composition containing lactic acid bacteria-produced substances of the present invention is a composition containing lactic acid bacteria-produced substances using a lactic acid bacteria fermentation extract composed of lactic acid bacteria-produced substances, and contains a lactic acid bacteria fermentation extract derived from brown sugar, okara, or soybeans and deep ocean water, and is characterized by containing ume extract, which is a Rosaceae plant, at 22% or more by weight ratio.

[0032] Thereby, the present inventors were able to provide a composition containing lactic acid bacteria-produced substances that can exhibit a bactericidal effect and an anti-aging effect against so-called "bad bacteria" in the living body without using chemical substances (synthetic chemical substances). Hereinafter, embodiments of the present invention will be specifically described.

[0033] In this embodiment, the "composition containing lactic acid bacteria-produced substances" according to the present invention is referred to as "reduced-fermentation lactic acid bacteria". This reduced-fermentation lactic acid bacteria contains a lactic acid bacteria fermentation extract derived from brown sugar, okara, or soybeans. Here, the "lactic acid bacteria fermentation extract derived from brown sugar, okara, or soybeans" means a lactic acid bacteria-produced substance cultured using brown sugar, okara, or soybeans as food for a complex lactic acid bacteria group. That is, the reduced-fermentation lactic acid bacteria according to this embodiment contains "a lactic acid bacteria-produced substance cultured using brown sugar as food (medium) for a complex lactic acid bacteria group", "a lactic acid bacteria-produced substance cultured using okara as food (medium) for a complex lactic acid bacteria group", or "a lactic acid bacteria-produced substance cultured using soybeans as food (medium) for a complex lactic acid bacteria group". This lactic acid bacteria fermentation extract is not particularly limited in its preferred state and may be liquid, powdery, or granular. Also, the lactic acid bacteria may be live bacteria or dead bacteria. And the reduced-fermentation lactic acid bacteria according to this embodiment contains deep ocean water and ume extract, which is a Rosaceae plant, at 22% or more by weight ratio. Ume extract takes various forms such as powder, fruit juice, and concentrated fruit juice, but in the present invention, it is assumed to be in the form of a stock solution (or a form equivalent to the stock solution) regardless of the form.

[0034] Although reduced-fermentation lactic acid bacteria are acidic, when they are present in the oral cavity, they produce a umami flavor with a sour and sweet taste, rapidly secreting saliva and neutralizing the acidity. As a result, it becomes a training for promoting the secretion of the salivary glands, and it is expected to have an effect of promoting the remineralization of teeth by adding calcium and phosphate ions contained in saliva. Thereby, it is possible to expect a periodontal disease inhibitory effect and a halitosis inhibitory effect. Furthermore, as described later, by adding deep-sea water minerals reduced and ionized by hydrogen, a positive effect is expected.

[0035] [Mechanism of Reduced-Fermentation Lactic Acid Bacteria] Figure 4 is a diagram showing an overview of the mechanism of reduced-fermentation lactic acid bacteria. As shown in Figure 4, when reduced-fermentation lactic acid bacteria are subjected to aging fermentation by mixing hydrogen and deep-sea water at an optimal concentration during the co-culture of multiple types (for example, specific 8 types or 12 types) of lactic acid bacteria, antibacterial peptides are produced (indicated by the symbol "P" in Figure 4). This antibacterial peptide P is secreted by the lactic acid bacteria in the co-culture competing with each other to protect themselves from other bacteria. And in the state where this antibacterial peptide P is produced, ume extract is added. As a result, an antibacterial peptide with high antibacterial power (indicated by the symbol "P+" in Figure 4) is produced. This antibacterial peptide P+ has high antibacterial power and can be utilized for oral care and gastrointestinal care by adjusting the concentration. That is, it is expected to act on suppressing harmful bacteria present in the body and to improve the internal environment. In this way, reduced-fermentation lactic acid bacteria are developed for the purpose of suppressing harmful bacteria, based on the inventors' knowledge and basic concepts of health, through research on combining multiple materials to enhance the synergistic effect.

[0036] Conventionally, in appealing to the intestinal flora, the main focus has been on growing beneficial bacteria by ingesting lactic acid bacteria, bifidobacteria, oligosaccharides, etc. That is, the goal has been to improve the intestinal environment by relatively reducing the ratio of harmful bacteria as the number of beneficial bacteria increases. However, in addition to increasing beneficial bacteria, it is necessary to pursue suppressing harmful bacteria themselves. From the perspective of "internal beauty", the inventors have found an approach to reducing harmful bacteria in the intestine and oral cavity in addition to the approach of increasing beneficial bacteria. Figure 5A is a diagram showing the logic of the inventors, and Figure 5B is a diagram showing the functions of lactic acid bacteria fermentation extract (BioGenics). According to the reducing fermented lactic acid bacteria according to this embodiment, the number of beneficial bacteria increases and the intestinal environment is improved by the actions of "lactic acid bacteria (live bacteria and dead bacteria)" and "lactic acid bacteria fermentation extract (BioGenics)", and a decrease in harmful bacteria can be expected by the action of antibacterial peptide P+. That is, it becomes possible to expect "antioxidation based on the action of reducing harmful bacteria", which is said to generate 90% of reactive oxygen in the intestine, and "antiglycation based on the action of suppressing the production of AGEs", which causes aging and diseases when accumulated. Based on the countermeasures against "oxidation and glycation", which are the two major factors of aging and diseases, and by further having additional functionality, the reducing fermented lactic acid bacteria according to this embodiment have been realized.

[0037] The lactic acid bacteria fermentation extract contains peptides at a concentration of 377 (mg / L) to 566 (mg / L). That is, the lactic acid bacteria fermentation extract filtered through a 0.45 μm filter was used as a measurement sample, and for this measurement sample, total peptide quantification was performed using "Pierce Quantitative Colorimetric Peptide Assay" (manufactured by Thermo Fisher Scientific). As a result, it was found that the lactic acid bacteria fermentation extract contains 472 (mg / L) of peptides. When a range of ±20% centered on this value is defined as the effective range, it is preferable that the peptides are contained at a concentration of 377 (mg / L) to 566 (mg / L). Also, it may be contained as lactic acid bacteria cells. Since lactic acid bacteria secrete peptides with antibacterial properties to protect themselves, in this embodiment, this is used as a bactericidal function. Also, the lactic acid bacteria cells include either live bacteria or dead bacteria. This is because the same effect can be obtained with either. In this embodiment, it is preferable that the peptides are contained at a concentration of 377 (mg / L) to 566 (mg / L). However, according to research by the inventors, there is a possibility of achieving a peptide concentration of about 1000 (mg / L). Therefore, the invention of the present application is not limited to this numerical range. Also, when actually producing the reduced-fermentation lactic acid bacteria according to this embodiment, it is also possible to use a KS melt lactic acid bacteria production substance derived from brown sugar. However, the invention of the present application is not limited to this, and it is also possible to use a lactic acid bacteria production substance derived from okara or soybeans.

[0038] Regarding lactic acid bacteria, within a range not conflicting with legal regulations, for example, the following types can be used. Also, many other existing lactic acid bacteria and bifidobacteria may be additionally blended in the complex culture.

Table 1

[0039] In addition, the reduced fermented lactic acid bacteria (powder) according to this embodiment can be adjusted, for example, to "100 billion lactic acid bacteria per gram" by co-culturing 12 types of lactic acid bacteria. Also, for example, it can be adjusted to "50 billion lactic acid bacteria per gram" by co-culturing 8 types of lactic acid bacteria. Thus, the reduced fermented lactic acid bacteria (powder) according to this embodiment contains about "30 to 150 billion" lactic acid bacteria per gram. Assuming that one commercially available yogurt is 100 mL and calculating based on the number of lactic acid bacteria (1 billion) specified in the fermented milk ingredient standards of the Milk etc. Ordinance, by ingesting "1 g" of the reduced fermented lactic acid bacteria powder, it becomes possible to ingest the lactic acid bacteria equivalent to 30 to 150 commercially available yogurts. Note that these numerical values are an example for practical implementation, and the present invention is not limited thereto.

[0040] Deep ocean water is generally understood to be seawater at a depth of 200 m or deeper, and has characteristics such as cleanliness, rich in inorganic nutrients, and low-temperature stability compared to surface water. That is, since deep ocean water is not affected by river water polluted by human sewage, it is not contaminated by chemical substances, and since sunlight does not reach and plankton etc. do not grow, harmful bacteria etc. are also less than one-thousandth of surface water. Also, compared to surface water, it is rich in inorganic nutrients necessary for the growth of phytoplankton, and further has the characteristics that the water temperature and contained components are difficult to change and the water quality is stable. The reduced fermented lactic acid bacteria according to this embodiment contains 0.03 to 5% of deep ocean water reduced by hydrogen. By performing reduction treatment using hydrogen, it is considered that hydrogen is in a state of being occluded and contributes to promoting the ionization of minerals such as calcium and magnesium when dissolved in water.

[0041] In addition, in this embodiment, an example is shown in which deep ocean water is subjected to reduction treatment with hydrogen and then mixed with lactic acid bacteria fermentation extract or the like. However, the present invention is not limited to this, and it is also possible to perform reduction treatment with hydrogen after the oral composition is completed. Note that the present invention does not necessarily require reduction treatment of deep ocean water with hydrogen. Also, deep ocean water may be handled in a powder state for convenience, but the present invention is not limited to powders. Further, deep ocean water in which minerals are concentrated using an ion exchange technique with activated carbon may be added.

[0042] The present inventors conducted tests on the function of "deep ocean water subjected to reduction processing with hydrogen" (sometimes referred to as "reduced processed mineral super-concentrated solution") to eliminate alcohol and acetaldehyde. The equipment used was "Gas Chromatograph GC-2010AF / AOC: manufactured by Shimadzu Corporation", and the reagents used were "ethanol 95% (manufactured by Wako Pure Chemical Industries, Ltd.)", "acetaldehyde (manufactured by Wako Pure Chemical Industries, Ltd.)", and "whiskey (Suntory Kakubin)". The test method is as follows. [Regarding alcohol] (a) Three drops of the sample were added to 100 mL of whiskey stock solution (40% alcohol), stirred, and after standing for 2 hours, the alcohol was measured. (b) Six drops of the sample were added to 100 mL of whiskey stock solution (40% alcohol), stirred, and after standing for 2 hours, the alcohol was measured. [Regarding acetaldehyde] Acetaldehyde (90%) was diluted 100-fold to prepare a 0.9% solution. (A) Three drops of the sample were added to 100 mL of the above (a), stirred, and the acetaldehyde concentration was measured after 30 minutes. (B) Six drops of the sample were added to 100 mL of the above (a), stirred, and the acetaldehyde concentration was measured after 30 minutes. The results are as shown in the following table.

Table 2

[0043] As described above, by subjecting deep ocean water to hydrogen processing, it contains essential and trace minerals such as calcium, magnesium, and silicon, and can efficiently generate both hydrogen gas and hydrogen ions with electrons, and can also dissolve them in water continuously for a long time. Regarding this "hydrogen ion with electrons", while a general hydrogen ion is "H+" lacking one electron, there exists a "negative hydrogen ion H-" with two electrons charged, and there are reports that research towards practical application is being advanced at Kyoto University and Tokyo Institute of Technology. If the "deep ocean water subjected to reduction processing with hydrogen" according to this embodiment is consumed, it can be expected to efficiently deliver hydrogen into the gastrointestinal tract, liver, and blood. Also, if it is utilized as a cosmetic applied to the skin and scalp such as a hydrogen gel pack or hydrogen treatment, it can be expected to efficiently deliver hydrogen from the surface such as the skin and scalp to the endothelium against reactive oxygen species generated by damage caused by ultraviolet rays, and eliminate the reactive oxygen species.

[0044] Next, silicon and its related substances will be described. Here, based on the International Atomic Weight Table (2010), the atomic weights are set as "Si 28.0855", "H 1.00794", and "O 15.9994", and the third decimal place is rounded. Silicon is represented by "Si" and has an atomic weight of 28.09. The amount of silicon required by the human body per day is "10 - 40 mg", and the recommended intake amount is used as the reference for silicon intake. Next, silica is also called silicic acid, silicic anhydride, and silicon dioxide, represented by "SiO2", and its molecular weight is 60.09 (28.09 + 16.00 × 2 = 60.09). Although it is anhydrous and not a water-soluble silicon that is easily absorbed by the body, due to the pleasant sound of the word "silica", it is sometimes called "silica" as an alias for water-soluble silicon. However, water-soluble silicon (metasilicic acid, or hydrated silica / hydrated silicic acid such as orthosilicic acid) and silica (silicic anhydride) are different substances as shown in the following molecular formulas and molecular weights. Silica (silicic acid / silicic anhydride) hydrates and one molecule of H2O is added to form metasilicic acid, and further hydrates with one more molecule of H2O added to form orthosilicic acid, which is more easily taken up by the body and effectively utilized. Water-soluble silicon is hydrated silica (hydrated silicic acid), referring to substances in the form of metasilicic acid or orthosilicic acid. Insoluble silica (silica / silicic acid / silicic anhydride) that makes up mountains and rocks combines with H2O and dissolves out, changing into metasilicic acid. Further hydration progresses (with the addition of H2O), changing into orthosilicic acid, which is more easily absorbed by the body, reaching the ocean, being taken up by "phytoplankton diatoms" at the bottom of the food chain, and progressing in utilization within the body. Although it is repetitive, the "drinking silica derived from hot springs and spring water" utilized in mineral water is "water-soluble silicon", referring to "metasilicic acid (hydrated silica, hydrated silicic acid)" or "orthosilicic acid", which dissolves out from rocks (silica / silicic acid / silicic anhydride) and has been loved by drinking springs for many years as a beauty bath ingredient. As a result, "silicon / silica" = "28.09 / 60.09" = "0.47" times. Also, "silica / silicon" = "60.09 / 28.09" = "2.14" times. From these facts, silicic acid (silica, SiO2) = silicon (Si) × 1 / 0.47 = silicon (Si) × 2.14 (based on the conversion values used by the Japan Food Analysis Center).

[0045] Next, metasilicic acid is represented by "H2SiO3" and has a molecular weight of 78.1 (1×2 + 28.09 + 16.00×3 = 30.09 + 48.0 = 78.09). From this, it can be said that metasilicic acid is the result of silica (silicic acid, silicon dioxide) hydrating and changing to "H2O + SiO2 = H2SiO3". Also, the following relationships are found. "Silicon / Metasilicic acid" = "28.09 / 78.09" = "0.36" times "Metasilicic acid / Silicon" = "78.09 / 28.09" = "2.78" times "Silica / Metasilicic acid" = "60.09 / 78.09" = "0.77" times "Metasilicic acid / Silica" = "78.09 / 60.09" = "1.30" times

[0046] Next, orthosilicic acid is represented by "H4SiO4" and has a molecular weight of 78.1 (1×4 + 28.09 + 16.00×4 = 32.09 + 64.0 = 96.09). Compared with the molecular formula of the above metasilicic acid, it takes a form with an additional "H2O" hydrated. Silicic acid mainly exists in the form of "orthosilicic acid (H4SiO4)", and its biogeochemical cycle is controlled by diatoms. This "orthosilicic acid (H4SiO4)" is "Si(OH)4", and with Si as the center, it has a "beautiful molecular structure where 4 OH groups hold hands" and is excellent in absorbency. Also, the following relationships are found. "Silicon / Orthosilicic acid" = "28.09 / 96.09" = "0.29" times "Orthosilicic acid / Silicon" = "96.09 / 28.09" = "3.42" times "Silica / Orthosilicic acid" = "60.09 / 96.09" = "0.63" times "Orthosilicic acid / Silica" = "96.09 / 60.09" = "1.60" times

[0047] Here, the recommended daily intake is described. As mentioned above, "silica / silicon" = "60.09 / 28.09" = "2.14" times, "metasilicic acid / silicon" = "78.09 / 28.09" = "2.78" times, "orthosilicic acid / silicon" = "96.09 / 28.09" = "3.42" times. Since the recommended daily intake of elemental silicon is "10 - 40 mg", the recommended daily intake when converted to silica, metasilicic acid, and orthosilicic acid is considered to be within the respective conversion value ranges from the silicon amount. That is, as silicon, it is "10 - 40 mg", as silica, it is "21.4 - 85.6 mg (10 - 40 mg × 2.14 times)", as metasilicic acid, it is "27.8 - 111.2 mg (10 - 40 mg × 2.78 times)", and as orthosilicic acid, it is "34.2 - 136.8 mg (10 - 40 mg × 3.42 times)".

[0048] According to the literature "Biochemistry of Silicon and Related Problems (Nobel Foundation Symposia)" edited by "Gerd Bendz", it is stated that "the silicon content in the human aorta changes with age". As shown in Figure 16, for water-soluble silicon (hydrated silica / hydrated silicic acid such as metasilicic acid or orthosilicic acid), if the value in the body at birth is taken as 100, ignoring individual differences, it will decrease by about half by the age of 40. Since humans cannot produce the necessary water-soluble silicon in their own bodies, it is important to actively intake water-soluble silicon to maintain beauty and health.

[0049] Water-soluble silicon (hydrated silica or hydrated silicic acid such as metasilicic acid or orthosilicic acid) is also contained in the human body and exists in hair, nails, blood vessels, bones, joints, cell walls, etc. Silicon in the living body has the effect of binding collagen and is useful for the regeneration, reinforcement, and maintenance of bones, hair, nails, and collagen. It also affects skin moisturization. In addition, silicon is contained in the skin (dermis), hair, nails, etc., and has the function of binding collagen, ceramide, elastin, hyaluronic acid, chondroitin, etc., maintaining the firmness and elasticity of the skin, and binding tissues to make them strong. Furthermore, it is known that excellent beauty and health effects can be obtained by combining ceramide or hyaluronic acid, lactic acid bacteria, and silicon. That is, ceramide has the function of enhancing the barrier function of the skin (especially the moisturizing effect) and suppressing the evaporation of moisture from the epidermis. Hyaluronic acid enhances the water retention function of the skin to prevent dryness. Lactic acid bacteria have the function of increasing immunity and anti-allergic function along with intestinal regulation. Moreover, silicon promotes the synthesis of collagen in the skin and has the function of being responsible for the adhesion and keratinization between collagen layers. Therefore, it is expected that beauty and health effects will be exerted by combining these. Furthermore, silicon is absorbed from the intestinal wall and has the effect of solubilizing deposits inside blood vessels when passing through the blood vessels, and is also effective in preventing arteriosclerosis. In addition, it has the function of promoting plant growth or strengthening the stems.

[0050] Thus, since a synergistic effect on beauty can be expected, in addition to the reduced-fermentation lactic acid bacteria according to this embodiment, lactic acid bacteria, lactic acid bacteria fermentation extract, oligosaccharides, and various polyphenols (ume extract, rose petal extract, pomegranate extract, camellia seed extract, camellia flower extract, etc.), plant enzymes, and citric acid may be added. Also, collagen, ceramide, elastin, hyaluronic acid, chondroitin, proteoglycan, swiftlet nest extract, GABA (gamma-aminobutyric acid), and "equol" derived from soybean germ isoflavones may be added.

[0051] In the Framingham Offspring Study in the United States, it was found that there is a close relationship between the intake of silicon (contained in water-soluble silicon) and bone mineral density (BMD). In this study, 2,846 men and women aged from their 30s to 80s were divided into four groups according to the measurement results of "silicon intake" and compared in their eating habits. As a result, it was found that in men and pre-menopausal women, the higher the silicon intake, the higher the bone density of the femoral neck. Thus, the effect of silicon in preventing osteoporosis is expected. Since the importance of silicon has become clear in this way, in Europe and the United States, health supplements and foods containing water-soluble silicon (hydrated silica or hydrated silicic acid such as metasilicic acid or orthosilicic acid), which is easily absorbed by the body, have been attracting attention for quite some time, and the market for silicon products in Europe and the United States has already reached an extremely large scale. Human bones are composed of about 70% hydroxyapatite and about 30% collagen, and water-soluble silicon runs through them to make the tissue strong. Therefore, by containing silicon, hydroxyapatite, and collagen, a synergistic effect due to the collaboration of these three components is expected. In addition, in this embodiment, "sakuran" may be blended. Sakuran belongs to the same "polysaccharide" as "hyaluronic acid", which is essential as a moisturizer in cosmetics, and "chondroitin sulfate", which is used in health foods. It is a Japanese indigenous edible cyanobacterium and is produced from the agar-like substance of the freshwater photosynthetic microorganism Aphanothece sacrum (commonly known as Mizusasa nori, scientific name: Aphanothece sacrum). Sakuran exhibits high viscosity and has high moisturizing, anti-inflammatory, and film-forming functions.

[0052] Alternatively, instead of the equol derived from the above-mentioned soybean germ isoflavones, a sweet potato extract containing diosgenin may be blended. This sweet potato extract containing diosgenin has estrogenic activity and thus can be used as an alternative raw material for equol. Furthermore, it may contain "natural sweet potato extract powder" which is the most nutritious and strengthening among sweet potatoes. Also, Coprinus tamogitake rich in ergothioneine, an antioxidant amino acid, may be added, and mushroom extract powders such as shiitake mushroom and Grifola frondosa, which are expected to have excellent immunostimulatory effects and antitumor effects by the "β-glucan-protein complex", may be added. Additionally, excipients such as water-soluble dietary fibers such as inulin, acacia dietary fiber, indigestible dextrin, cellulose, dextrin, starch, and insoluble dietary fibers may be added. Furthermore, excellent antioxidant and anti-glycation raw materials such as Japanese red pine bark extract powder, Japanese larch bark extract powder, pine bark (picnogenol), astaxanthin, coenzyme Q10, and fullerene may be added, and raw materials expected to have anti-glycation, hair growth, and diet effects such as Salacia, fermented soybean isoflavones (hydroxylated isoflavones), fucoidan, and kudzu flower isoflavones may be added. Also, SENSEPUR (registered trademark), a "rice germ and soybean fermented extract" containing yucca extract and kiraiya extract, may be blended to enhance the deodorizing effect.

[0053] In particular, the combination of Japanese red pine bark extract powder and astaxanthin is expected to have excellent antioxidant and anti-glycation functions. Furthermore, by blending hydrogen, water-soluble silicon, deep ocean water, or adding red or orange colors, the attractiveness of the product can be enhanced, and the realization of a collaborative product with the reduced fermented lactic acid bacteria according to this embodiment is expected. Also, in the collaboration between Japanese red pine bark extract powder and shiitake mushroom extract powder or "β-glucan of mushrooms" such as Grifola frondosa, enhancement of the immunostimulatory effect is expected, and in the collaboration with Coprinus tamogitake rich in ergothioneine, an antioxidant amino acid, enhancement of the antioxidant effect can be expected.

[0054] In addition, the inventors of the present invention verified the α-glucosidase inhibitory activity of the Pinus densiflora bark extract powder through a third-party institution. Carbohydrates contained in food are decomposed from polysaccharides to monosaccharides by digestive enzymes and absorbed from the intestinal wall. The enzyme that decomposes disaccharides into monosaccharides at the end of the decomposition process is α-glucosidase. Inhibiting α-glucosidase can prevent the decomposition of disaccharides into monosaccharides and suppress the absorption of sugar from the small intestine. As a result, an effect of suppressing a rapid increase in blood glucose level after a meal can be expected. The test method is as follows. That is, the provided sample was dissolved in 50% DMSO at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain a stock solution. For the stock solution and the solutions diluted 10-fold and 100-fold with 50% DMSO, the α-glucosidase inhibitory activity was measured. A solution obtained by dissolving "7 mM p-nitrophenyl-α-D-glucopyranoside" in 50 mM phosphate buffer (pH 7.0) was used as a substrate solution, and a solution obtained by dissolving α-glucosidase at 0.9 U / mL in 50 mM phosphate buffer (pH 7.0) was used as an enzyme solution. Using a microtube, 10 μL of the sample solution and 40 μL of the enzyme solution were placed, incubated at 37°C for 5 minutes, then 950 μL of the substrate solution was added, and incubated at 37°C for 15 minutes. Thereafter, 1000 μL of 0.5 M Tirs solution was added to stop the reaction. The absorbance at 405 nm of p-nitrophenol released by this reaction was measured. The inhibition rate of the production of p-nitrophenol when the sample was added was calculated from the decrease from the absorbance value without the addition of the sample, and was used as the α-glucosidase inhibitory activity. The results are as shown in the following table, indicating that the higher the α-glucosidase inhibitory activity, the higher the effect of suppressing the increase in blood glucose level.

Table 3

[0055] In addition, the inventors of the present invention verified the inhibitory activity of the Amadori compound formation of the Pinus densiflora bark extract powder through a third-party institution. The Amadori compound is an important intermediate in the early stage of the glycation reaction, and by suppressing the formation of the Amadori compound, it becomes possible to suppress the formation of AGEs, which are the late-stage products of the glycation reaction. The test method is as follows. That is, the provided sample was dissolved in 50% DMSO at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with 50% DMSO were used as measurement samples, and the inhibitory effect on Amadori compound formation was measured. For the measurement, the measurement sample, glucose solution, and bovine serum albumin solution were mixed and incubated at 60 °C for 48 hours to prepare a glycation reaction solution. 300 μL of a 0.25 mM nitroblue tetrazolium solution (pH 9.0) was added to 40 μL of the glycation reaction solution, incubated at 37 °C for 30 minutes, allowed to stand at room temperature for 16 hours, and then the absorbance at 540 nm was measured. The inhibitory activity of Amadori compound formation when the sample was added was calculated from the change in absorbance and used as the inhibitory activity of Amadori compound formation. The results are as shown in the following table, indicating that the higher the inhibitory activity, the higher the anti-glycation activity.

Table 4

[0056] In addition, the inventors of the present invention verified the AGEs cross-link cleavage activity of the Pinus densiflora bark extract powder through a third-party institution. α-Dicarbonyl compounds are known to be intermediates generated in the glycation reaction process of proteins in vivo, such as 3-deoxyglucosone and glyoxal. AGEs generated by the non-enzymatic reaction (Maillard reaction) between the α-dicarbonyl compounds generated in vivo and the amino groups of proteins are, as described above, factors for various diseases. It is expected to prevent protein glycation by a component having the activity of decomposing α-dicarbonyl compounds, that is, AGEs cross-link decomposition activity.

[0057] The test method for the AGEs cross-link cleavage test is as follows. That is, it was carried out by measuring the amount of benzoic acid decomposed and generated by the cleavage of the dicarbonyl bond of the model compound "1-phenyl-1,2-propanedione" having a dicarbonyl bond. The provided sample was dissolved in a 50% DMSO aqueous solution at a concentration of 1 mg / mL, and the filtered solution through a 0.45 μm filter was used as the stock solution. Diluted 10-fold and 100-fold with the stock solution and 50% DMSO aqueous solution, the diluted solutions were used as measurement samples. The measurement sample, "1-phenyl-1,2-propanedione solution" and methanol were mixed to form a reaction solution. After incubating the reaction solution at 37 °C for 24 hours, hydrochloric acid was added to stop the reaction. The quantification of the decomposed benzoic acid in the reaction solution was carried out using a high performance liquid chromatograph. The AGEs cross-link cleavage rate was calculated with 100 (%) when all of the "1-phenyl-1,2-propanedione" in the reaction solution was decomposed into benzoic acid. The results are as shown in the following table.

Table 5

[0058] The inventors of the present invention verified the "fluorescent AGEs generation inhibitory activity" and "non-fluorescent AGEs (CML) generation inhibitory activity" of the Pinus densiflora bark extract powder through a third-party institution. AGEs (Advanced Glycation End products) are substances generated by the non-enzymatic reaction (Maillard reaction) of reducing sugars such as glucose with the amino group of proteins. When AGEs accumulate in blood vessels, it causes myocardial infarction and cerebral infarction, and when it accumulates in bones, it causes osteoporosis, and when it accumulates in the eyes, it causes cataracts. AGEs are roughly classified into fluorescent AGEs with fluorescence and non-fluorescent AGEs without fluorescence.

[0059] The test method for the inhibitory activity of fluorescent AGEs production is as follows. That is, the provided sample was dissolved in 50% DMSO at a concentration of 1 mg / mL and filtered through a 0.45 μm filter, and the resulting solution was used as the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with 50% DMSO were subjected to measurement of the inhibitory activity of fluorescent AGEs production. Glucose and bovine serum albumin were mixed using test tubes, incubated at 60 °C for 40 hours, and the generated fluorescent AGEs were measured with a fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The inhibition rate of fluorescent AGEs production when the sample was added was calculated from the decrease in fluorescence intensity and taken as the inhibitory activity of fluorescent AGEs production. The results are as shown in the following table, indicating that the higher the inhibition rate, the higher the anti-glycation activity.

Table 6

[0060] Next, the test method for the inhibitory activity of non-fluorescent AGEs (CML) production will be described. CML (carboxymethyl lysine) is a representative non-fluorescent AGE and is produced by glycation modification of the lysine residue of proteins. It is expected that components with an inhibitory effect on CML production can prevent protein glycation. The test method is as follows. That is, the sample was dissolved in a 50% aqueous DMSO solution at a concentration of 1 mg / mL and filtered through a 0.45 μm filter, and the resulting solution was used as the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with a 50% aqueous DMSO solution were used as the measurement samples. The measurement samples, glucose, and bovine serum albumin were mixed using microtubes, incubated at 60 °C for 40 hours, then a portion was taken, a protease solution was added, and incubation was carried out at 37 °C for 24 hours for enzymatic degradation. Thereafter, substances with a molecular weight of 10,000 or more were removed by ultrafiltration, and CML was analyzed by liquid chromatography-mass spectrometry. The inhibition rate of CML production when the sample was added was calculated from the change in CML concentration and taken as the inhibitory activity of non-fluorescent AGEs (CML) production. The higher the inhibitory activity, the higher the inhibitory effect on non-fluorescent AGEs production. The results are as follows.

Table 7

[0061] In addition, the present inventors verified the hyaluronidase inhibitory activity of the Pinus densiflora bark extract powder through a third-party institution. Hyaluronic acid exists around cells and between cells in the living body, maintains the moisture of the skin to maintain healthy skin, and is involved in the transport of nutrients and waste products. The amount of hyaluronic acid in the living body decreases with aging, causing arthritis, wrinkle formation, etc. On the other hand, hyaluronidase is an enzyme that decomposes hyaluronic acid and is widely present in the living body. It is considered to be activated during inflammation, destroy the tissue structure, and promote inflammation. Inhibiting hyaluronidase can prevent the decomposition of hyaluronic acid, and the effects of maintaining skin moisture and suppressing inflammation can be expected.

[0062] The test method for hyaluronidase inhibitory activity is as follows. That is, the provided sample was dissolved in a 50% DMSO aqueous solution at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain a stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with the 50% DMSO aqueous solution were measured for hyaluronidase inhibitory activity. Bovine-derived hyaluronidase dissolved in 0.1 M acetate buffer (pH 4.0) at 400 units / mL was used as the enzyme solution. Potassium hyaluronate dissolved in acetate buffer at 1 mg / mL was used as the substrate solution. Compund48 / 80 dissolved in acetate buffer at 0.1 mg / mL was used as the enzyme activator. p-dimethylaminobenzaldehyde (p-DABA) dissolved in a hydrochloric acid-acetate mixed solution at 100 mg / mL was used as the color-developing solution, and it was diluted 10-fold with acetic acid immediately before use.

[0063] For the measurement, 12 μL of the sample solution and 12 μL of the enzyme solution were placed in a microtube and incubated at 40°C for 20 minutes. Then, 12 μL of the enzyme activator was added and incubated at 40°C for 20 minutes. Further, 12 μL of the substrate solution was added and incubated at 40°C for 40 minutes. Thereafter, 12 μL of 0.4 N NaOH aqueous solution was added to stop the reaction, immediately ice-cooled for 5 minutes, 12 μL of 0.8 M boric acid buffer (pH 9.0) was added, boiled for 3 minutes, and then further ice-cooled for 10 minutes. 180 μL of the coloring solution was added thereto, incubated at 40°C for 30 minutes, and then the absorbance at 585 nm was measured. When the activity of hyaluronidase is inhibited upon addition of the sample, N-acetylglucosamine, which is a degradation product of hyaluronic acid, decreases, and the absorbance due to p-DABA becomes low. It was calculated from the change amount of the absorbance and used as the hyaluronidase inhibitory activity. The results are as shown in the following table.

Table 8

[0064] In addition, the present inventors verified the collagenase inhibitory activity of the Pinus densiflora bark extract powder through a third-party institution. Collagen accounts for about 1 / 3 of the total body proteins and is contained in the skin, bones, etc., and is greatly involved in maintaining its structure. Collagenase is an enzyme that decomposes collagen. When collagenase is activated by aging, ultraviolet rays, etc., collagen is decomposed, making it difficult to maintain the structure of the skin, leading to the occurrence of wrinkles and sagging. By adding a component that inhibits collagenase to cosmetics, an anti-aging effect against wrinkles and sagging can be expected.

[0065] The test method is as follows. That is, the provided sample was dissolved in a 50% DMSO aqueous solution at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with the 50% DMSO aqueous solution were subjected to collagenase inhibitory activity measurement. Collagenase dissolved in pure water at 0.1 mg / mL was used as the enzyme solution, and Pz-peptide dissolved in 0.1 M Tris buffer (pH 7.1, 20 mM CaCl2) at 0.39 mg / mL was used as the substrate solution. For the measurement, 12.5 μL of the sample solution, 12.5 μL of the enzyme solution, and 100 μL of the substrate solution were placed in a glass test tube, incubated at 37°C for 30 minutes, and then 250 μL of 25 mM citric acid was added to stop the reaction. In this reaction, the degradation product of Pz-peptide by collagenase was extracted by shaking with 1250 μL of ethyl acetate, centrifuged at 3000 rpm for 10 minutes, and the upper layer (ethyl acetate layer) was recovered, and the absorbance at 320 nm was measured. The inhibition rate of the formation of the Pz-peptide degradation product when the sample was added was calculated from the decrease from the absorbance value without the sample addition, and was taken as the collagenase inhibitory activity. The results are as shown in the following table.

Table 9

[0066] In addition, the present inventors verified the elastase inhibitory activity of the Pinus densiflora bark extract powder through a third-party institution. Elastase is an enzyme that degrades elastin, an elastic fiber that determines the mechanical properties of connective tissue together with collagen. When elastase is activated by aging, ultraviolet rays, etc., elastin is degraded, making it difficult to maintain the structure of the skin, leading to the formation of wrinkles and sagging. By adding a component that inhibits elastase to cosmetics, an anti-aging effect against wrinkles and sagging can be expected.

[0067] The test method is as follows. That is, the provided sample was dissolved in a 50% DMSO aqueous solution at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with the 50% DMSO aqueous solution were subjected to elastase inhibitory activity measurement. STANA (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) dissolved in Tris buffer (pH 7.4) at 1 mM was used as the substrate solution, and porcine pancreatic elastase dissolved in Tris buffer (pH 7.4) at 0.5 units / mL was used as the enzyme solution. For the measurement, 50 μL of the sample solution, 50 μL of the enzyme solution, and 100 μL of the substrate solution were placed in a microtube and incubated at 37 °C for 30 minutes. In this reaction, nitroaniline, which is a substrate degradation product by elastase, was measured at an absorbance of 405 nm. The inhibition rate of nitroaniline production when the sample was added was calculated from the decrease from the absorbance value without the sample addition, and was taken as the elastase inhibitory activity. The results are as shown in the following table.

Table 10

[0068] Both silicon derived from plants and silicon derived from minerals can be processed under the same conditions using temperature, pressure, etc. to obtain water-soluble silicon (hydrated silica or hydrated silicic acid such as metasilicic acid or orthosilicic acid). However, since the mineral balances contained in each are different, if one wishes to enjoy both the bounty of the earth and the bounty of plants, a synergistic effect can be expected by mixing concentrated solutions of plant-derived water-soluble silicon and mineral-derived water-soluble silicon. Regarding "water-soluble silicon", any silicon can be used, whether it is "plant-derived silicon" such as bamboo-derived, or from sugarcane, kuma-sasa, susuki, rice husk, or inawara, which are also Poaceae plants like bamboo, or "mineral-derived silicon" such as quartz or crystal. Water-soluble silicon derived from natural water or hot spring water in Kirishima (Kirishima mountain range) or Hakone can also be blended. Additionally, by utilizing the water-soluble silicon eluted from Mount Fuji lava marimo powder or combining it with the water-soluble silicon abundantly contained in the spring water or hot spring water of Mount Fuji, a further synergistic effect of minerals can be expected. The "bounty of minerals in Mount Fuji" and the "bounty of deep ocean water minerals" can be said to be precisely the bounty of high and low minerals and the bounty of the balance of yin and yang minerals. Note that the spring water, hot spring water, lava, etc. of Mount Fuji contain "vanadium", and it is known that "vanadium" can be expected to have an effect on diabetes. It is known that the rain and snow that fall on the ground of Mount Fuji become natural water that has taken in a rich balance of minerals including vanadium while permeating through the thick basalt layer over a long period of time. Also in this embodiment, by containing "vanadium", an effect on diabetes can be expected.

[0069] In addition, the production areas of mineral water rich in water-soluble silicon are mainly distributed in the Fuji-Hakone region and the Kyushu region. The Kyushu region has some of the world's largest volcanic and hot spring groups, such as Aso, Unzen, Kirishima, Kusatsu, Sakurajima, and Beppu. The strata in this area are known to contain a large amount of silica (silicic acid, silicic anhydride), which has been dissolved in water over a long period of time (changing into hydrous silica or hydrous silicic acid such as metasilicic acid or orthosilicic acid). Also, regarding the hot spring water in Kirishima, which is rich in water-soluble silicon, there is a myth that "a long time ago, Izanagi no Mikoto and Izanami no Mikoto put the weak-kneed Hiruko no Mikoto on a boat and had him undergo hot spring treatment at the 'Valley of Sighs'." In this embodiment as well, it may contain water-soluble silicon (hydrous silica or hydrous silicic acid such as metasilicic acid or orthosilicic acid) derived from the spring water or natural water in Kirishima or Sakurajima.

[0070] In addition, the mineral water produced in Sakurajima may contain natural germanium. Germanium has been found to bind with oxygen and perform the function of transporting oxygen, and to activate interferon in the body. Therefore, in this embodiment as well, by using the mineral water produced in Sakurajima, it may be possible to obtain the advantages of natural germanium. Interestingly, carbon (atomic number 6), silicon (atomic number 14), and germanium (atomic number 32) belong to the same Group 14 elements in the periodic table, and while having an electron structure with "s 2 p 2 " of 4 valence electrons, each has characteristics that can be expected to have unique health effects.

[0071] Thus, it is meaningful to add collagen, ceramide, and silicon to the reducing fermented lactic acid bacteria. However, the inventors verified the synergistic effect on collagen production based on the research results of research institutions. The test substances were (a) collagen, (b) a water-soluble ionized silicon super-concentrate solution, and (c) ceramide (1% ceramide solution), and the test method was as follows. That is, normal human dermal fibroblasts were cultured and transferred to the logarithmic growth phase. At the same time, the required number of cells was ensured, and normal human dermal fibroblasts were seeded in a "96-well micro plate" at a concentration of "1×10 4 cells / 100μL / well". Next, pre-culture was performed for 24 hours, the culture medium was removed, and the prepared test sample solution was added to each well, followed by culturing for 48 hours. Next, the culture supernatant was collected, and this culture supernatant was measured using an ELISA collagen kit and expressed as a value corrected with cell protein. The results are as shown in the following table.

Table 11

[0072] Thus, with the combination of "Collagen 31.25 (μg / mL)" and "Ceramide (1% Ceramide solution) 125 (μg / mL)", the growth rate was 128.8% (an increase of 28.8%), and with the combination of "Collagen 31.25 (μg / mL)", "Ceramide (1% Ceramide solution) 125 (μg / mL)", and "Water-soluble Silicon 125 (μg / mL)", the growth rate was 148.3% (an increase of 48.3%). This result is shown in Figure 17. Also, with the combination of "Collagen 125 (μg / mL)" and "Ceramide (1% Ceramide solution) 125 (μg / mL)", the growth rate was 165.2% (an increase of 65.2%), and with the combination of "Collagen 125 (μg / mL)", "Ceramide (1% Ceramide solution) 125 (μg / mL)", and "Water-soluble Silicon 125 (μg / mL)", the growth rate was 208.8% (an increase of 108.8%). This result is shown in Figure 18. Thus, with the addition of "Water-soluble Silicon", a high synergistic effect was confirmed at a statistically significant level for the two-component mixture of Collagen and Ceramide (Significance test (Student's t-test) vs. two-component mixture: p-value < 0.001).

[0073] In addition, the inventors verified the synergistic effect on hyaluronic acid production based on the research results of research institutions. That is, a hyaluronic acid production effect test using normal human dermal fibroblasts was carried out for the test substances "Collagen", "Silicon RICH ultra-concentrated solution", and "Ceramide (1% Ceramide solution)" alone and in combinations of each test substance. The test method is as follows. (A) Normal human dermal fibroblasts were cultured to ensure the required number of cells. (B) Normal human dermal fibroblasts were seeded on each plate at a concentration of "1×10 4 cells / 100μL / well". (C) Pre-culture was carried out for 24 hours. (D) After pre-culture, the culture supernatant was removed, and the adjusted test substance solution was added to each well, followed by 48 hours of culture. (E) After the culture was completed, the culture supernatant was collected, and the amount of hyaluronic acid produced in the culture supernatant was measured using an ELISA hyaluronic acid kit.

[0074] The calculation method of the test results is as follows. (A) The hyaluronic acid production amount was divided by the protein amount proportional to the number of cells to calculate the hyaluronic acid production amount per protein amount. (B) As a relative evaluation, the hyaluronic acid production amount of the Control group was converted to 100%, and the hyaluronic acid production rate of the test substance was calculated. (C) The cell viability, hyaluronic acid production amount, and hyaluronic acid production rate were tested for significant differences by the t-test of Control and Student. The data of each test group were independently tested 3 times, and a significant difference test was performed. The significance level was set at p < 0.05 for two-sided tests. The test results of the hyaluronic acid production effect by the test substance alone and its combination are as shown in the following table.

Table 12

Table 13

Table 14

[0075] In the case of the test substances alone, a hyaluronic acid production-promoting effect was observed for each test substance. Collagen and ceramide showed high hyaluronic acid production as single components, and a significant production-promoting effect was observed in all test groups compared to the non-additive group. For water-soluble silicon, a significant production-promoting effect was observed in the 125 μg / mL test group compared to the non-additive group. Also, in the combinations of two substances, in the combination of collagen and water-soluble silicon, hyaluronic acid production was significantly promoted in all test groups compared to collagen alone. In the combination of ceramide and water-soluble silicon, hyaluronic acid production was promoted by the combination with water-soluble silicon in the concentration groups where the ceramide concentration was 31.25 μg / mL or higher, and a hyaluronic acid production-promoting effect was observed compared to ceramide alone. In the combination of two substances, collagen and ceramide, in the combinations where the collagen was 31.25 μg / mL or higher, hyaluronic acid production was promoted compared to collagen alone, and a synergistic effect of the combination was observed.

[0076] Furthermore, in the three-component mixture with the addition of water-soluble silicon to the combination of collagen and ceramide, in the test groups where the collagen was 31.25 μg / mL or higher, the hyaluronic acid production amount was significantly promoted compared to the two-component combination, and a synergistic effect of water-soluble silicon was observed. This is shown in FIGS. 25A to 25C.

[0077] Silicon is also known to have a significant impact on the growth and carapace formation of "sakura shrimp," which are called the jewels of the sea. The globally precious sakura shrimp can only be caught in two locations: Taiwan and Suruga Bay. Almost 100% of the domestic catch in Japan comes from Suruga Bay in Shizuoka Prefecture. As shown in Figure 14, rivers such as the Fuji spring water flowing into Suruga Bay are rich in "water-soluble silicon (hydrated silica or hydrated silicic acid such as metasilicic acid or orthosilicic acid)," which is an important factor in the skeletal formation of the phytoplankton "diatoms," which serve as food for juvenile sakura shrimp. The diatoms incorporated into the sakura shrimp play the role of an essential mineral for biological growth as "intracellular silicon" through the food chain. That is, it reaches the sea (Suruga Bay) through rivers from water and hot springs, becomes an important component of the body of the phytoplankton "diatoms," and through the rich ecosystem and food chain of Suruga Bay, including the sakura shrimp, it plays a role in strengthening the bones and tissues of the Japanese people's bodies. The process of "transforming from mineral silicon to plant silicon to intracellular silicon, returning to feces and carcasses through the food chain, and then changing back to mineral silicon over a long period of time" is the "silicon cycle on Earth." Thus, silicon circulates on Earth over a long period of time, moving through minerals, plants, and the body, and can be said to be an "essential mineral for beauty" for the human body. The lactic acid bacteria-produced substance-containing composition according to this embodiment effectively utilizes such advantages of silicon.

[0078] The inventors of the present invention conducted a skin permeability test of the water-soluble silicon according to this embodiment through a third-party institution. In this test, the skin permeability was evaluated by comparing the water-soluble silicon according to this embodiment with commercially available silicon water. That is, the test specimens were the water-soluble silicon according to this embodiment and commercially available silicon water, the subject was a 51-year-old male, and the test site was the inner side of the right forearm. The test method was as follows: First, in order to make the permeability of the test specimens visible, a predetermined amount of fluorescent agent was added to each test specimen. The subject was acclimated for about 10 minutes in a test room environment set to certain conditions (room temperature: 25°C, humidity 50%) with the test site exposed and in a seated position. After the acclimation ended, two 3 cm × 3 cm test areas were set on the test site, one being the "water-soluble silicon according to this embodiment" test area and the other being the "commercially available silicon water" test area. 20 μL of the specimen was dropped onto each test area and spread evenly within the test area. Next, after spraying and spreading distilled water over the entire area of both test areas, a black light was immediately irradiated, and the presence or absence of fluorescence emission was observed.

[0079] The results were that in the test area of the "water-soluble silicon according to this embodiment", no color development of the fluorescent agent was observed, and fluorescence color development was only observed in the test area of the "commercially available silicon water". From this, since the fluorescent agent remained on the skin in the case of the "commercially available silicon water", it is considered that no skin penetration phenomenon occurred. On the other hand, since there was no remaining color development of the fluorescent agent in the "water-soluble silicon according to this embodiment", it is considered that penetration into the skin occurred. Fig. 26 is a diagram showing the function of water-soluble ionized silicon. As shown in Fig. 26, silicon binds to collagen etc. and plays a role of filling the gaps in the skin and maintaining elasticity. However, since silicon itself is difficult to penetrate into the skin, in this embodiment, "water-soluble ionized silicon" with excellent penetration rate was realized by special processing. As a result, the penetration rate becomes high, and further effects such as restoring the vitality of the skin and enabling the reconstruction of the skin can be expected.

[0080] In addition, it may contain eggshell membrane peptides or nacre pearl powder. Eggshell membrane contains proteins such as type I collagen, elastin, and keratin, glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, and keratan sulfate, and hexosamines such as glucosamine and hexose, and is known to have anti-inflammatory, analgesic, and cartilage protection functions.

[0081] In addition, nacre pearl powder is composed of multiple types of amino acids, contains conchiolin, which is said to be a beauty ingredient, and mineral components such as calcium, and is known to have functions of reducing visceral fat and supplementing minerals. This "nacre" refers to the mother-of-pearl-colored porcelain layer that covers the inner surface of the shell and is secreted from the entire surface of the mollusk's mantle. In particular, there is a beautiful nacre layer inside the Akoya oyster and the black-lip pearl oyster, and it is known that this is reflected in the color of the pearl. Pearls are known to be formed by the shellfish wrapping a foreign object that it has taken into its body and cannot spit out with its own secretions in order to protect itself. From this production process of "eventually turning pain into a beautiful gemstone", pearls are regarded as "representing the character of a beautiful woman who can overcome difficulties", and there are anecdotes that Yang Guifei and Cleopatra loved to drink it, etc., and they have been loved by people all over the world as a symbol of health, longevity, whitening, and wealth. By containing the above "eggshell membrane peptides" or "nacre pearl powder", beauty effects such as beautiful skin, beautiful nails, and beautiful hair are expected.

[0082] In addition, keratin may be contained alone. Keratin is a protein that is abundant in nails and hair and gives them flexibility and strength. Keratin in the skin is made up of an amino acid combination that is almost the same as that of the epidermis, and the amino acid combination of nails and hair (referred to as the amino acid composition). By containing keratin, beauty effects such as beautiful skin, beautiful nails, and beautiful hair are expected.

[0083] In addition, the reducing fermented lactic acid bacteria according to this embodiment may contain soy isoflavones including equol, daidzin or daizein. In this case, soy isoflavones including daidzin or daizein may be added to the reducing fermented lactic acid bacteria according to this embodiment under predetermined conditions. When soy isoflavone glycosides are ingested, in the large intestine, due to the action of enzymes of intestinal bacteria, the sugar moiety of the soy isoflavone glycosides is separated, and substances not bound to sugar, that is, three substances, namely daizein, genistein, and glycitein, are produced. These are collectively referred to as soy isoflavone aglycones (sometimes referred to as "aglycone-type isoflavones"). Among these, daizein is changed to equol by the influence of intestinal bacteria. When women reach menopause, the secretion of female hormones decreases, and the risks of menopausal disorders and osteoporosis increase. However, equol exhibits a function similar to that of "estrogen," a female hormone directly related to women's beauty, and is known to be effective against osteoporosis and menopausal disorders. It has also been found to be effective in preventing prostate enlargement and AGA (alopecia) in men. In addition, research results showing that equol reduces skin wrinkles and research results showing that it suppresses the onset of cancer have also been reported. Such equol is produced by intestinal bacteria, but not everyone has these bacteria in their intestines. It is said that 20-30% of Westerners and about 40%-50% of Japanese people have them. However, the intestinal environment is not uniformly and genetically determined, but changes depending on eating habits and the like. Even a person who does not possess equol up to a certain point may become a person who possesses equol thereafter.

[0084] As an example, there is a report that when Westerners were made to ingest aglycone-type isoflavones (soybean isoflavone aglycone) containing daidzein, the rate of equol producers became about 70%. Also, there is a report that by drinking aglycone-type isoflavones rich in daidzein for three weeks, the calculated amount of equol increased compared to drinking glycoside isoflavones. Furthermore, in a large-scale human clinical trial, it has been reported that the blood equol concentration increased in a dose-dependent manner by drinking aglycone-type isoflavones rich in daidzein. The reduced fermented lactic acid bacteria powder according to this embodiment can adjust the intestinal environment, improve and maintain health, and obtain an anti-aging effect by containing equol or daidzein. In addition, it may contain "fermented soybean extract containing hydroxylated isoflavones (fermented isoflavones)" for which the inhibitory effect on the production of AGEs which is in vivo glycation and the inhibitory effect on glycation hair loss have been published. Note that "lactobionic acid", a functional carbohydrate produced from lactose, has an action of promoting calcium absorption and an action of promoting the production of equol, and is expected as an anti-aging material for the prevention and alleviation of osteoporosis and lifestyle-related diseases. Therefore, by blending "lactobionic acid", a synergistic effect on equol is expected. Also, in addition to lactobionic acid produced from "lactose", "maltobionic acid", which is the same oligosaccharide acid and has the same characteristics as lactobionic acid such as an action of promoting calcium absorption, has the merit of being a non-allergenic substance derived from "corn starch". The blending of maltobionic acid is expected to be effective for equol production.

[0085] In addition, the reduced fermented lactic acid bacteria according to this embodiment further contains at least one or any plurality of extracts of plants of the Rosaceae family, hibiscus extract, cranberry extract, pomegranate extract, olive extract (leaves, fruits, flowers), camellia extract (leaves, fruits, flowers) or perilla seed extract in a weight ratio of 0.03 to 5%. Also, by further mixing various polyphenols such as mangosteen extract, bay leaf extract, quercetin, and eucalyptus, the taste becomes good, it becomes effective for oral care, and the functionality is improved, so it is possible to enhance the attractiveness of the product.

[0086] Moreover, it may contain tea seed extract. The tea seed extract inhibits the absorption of starch and especially sugar by inhibiting the α-glucosidase activity necessary for the absorption of sugar and starch in the digestive tract. This function can reduce the energy intake from carbohydrates and balance the intake calories and consumption calories. By containing the tea seed extract, it becomes possible to sufficiently intake essential nutrients for the human body, such as proteins, vitamins, and minerals, without excessive dietary restrictions, and at the same time, it is possible to only reduce the intake calories derived from sugar. Furthermore, by ingesting it during meals, it is possible to lower the postprandial blood glucose level.

[0087] In addition, it is also possible to blend the soybean fiber enzyme decomposition product. This soybean fiber enzyme decomposition product is a water-soluble dietary fiber and an insoluble dietary fiber obtained by partially decomposing cellulose by cellulase treatment after removing "protein and lipid" from okara, which is a by-product of tofu production, and is, for example, a powder of "1:2". This soybean fiber enzyme decomposition product is used as a constipation countermeasure material. Furthermore, by blending the tea seed extract into the powder, in addition to the constipation countermeasure, it becomes possible to have a function of inhibiting the absorption of sugar.

[0088] Also, it may be blended with "charcoal or activated carbon with high adsorption performance carbonized by utilizing pressure, reduced water, etc. in addition to temperature (hereinafter referred to as 'functional edible charcoal (registered trademark)')". By adsorbing food additives, heavy metals, malodorous substances such as indole and skatole produced by bad bacteria, and substances "AGEs" (advanced glycation end products) produced by heating proteins and sugars contained in food, etc., it is possible to reduce the bad substances that cause oxidation and glycation and expect a synergistic effect with reducing and fermenting lactic acid bacteria. Note that the "functional coconut shell activated carbon" according to this embodiment meets the medicinal charcoal standard and has passed the test of the Japan Food Analysis Center.

[0089] The inventors conducted indole and skatole adsorption tests on "functional coconut shell activated carbon powder (the figure of coconut shell activated carbon / coconut tree is a TM)" and "Ina red pine excellent charcoal (registered trademark) powder". The analysis samples were "functional coconut shell activated carbon powder" and "Ina red pine excellent charcoal powder", the equipment used was "gas chromatograph mass spectrometer (manufactured by Shimadzu Corporation: QP5050A)", and the reagents used were "indole reagent (manufactured by Wako Pure Chemical Industries, Ltd.)" and "skatole primary reagent (manufactured by Wako Pure Chemical Industries, Ltd.)". The test method is as follows. Dissolve the above indole and skatole in water and adjust them to 20 ppm each. Next, add 1 g of the reagent to 100 mL of the 20 ppm solution, stir for 10 minutes, and then filter the mixture, which was analyzed using a gas chromatograph mass spectrometer. The results are as shown in the following table.

Table 15

[0090] In addition, the inventors conducted indole and skatole adsorption tests on "Kishu Binchotan activated carbon powder" and "Kamakura Kishitake charcoal (registered trademark) powder". The analysis samples were "Kishu Binchotan activated carbon powder" and "Kamakura Kishitake charcoal powder", the equipment used was "gas chromatograph mass spectrometer (manufactured by Shimadzu Corporation: QP5050A)", and the reagents used were "indole reagent (manufactured by Wako Pure Chemical Industries, Ltd.)" and "skatole reagent (manufactured by Wako Pure Chemical Industries, Ltd.)". The test method is as follows. Dissolve the above indole and skatole in water and adjust them to 20 ppm each. Next, add 1 g of the reagent to 100 mL of the 20 ppm solution, stir for 10 minutes, and then filter the mixture, which was analyzed using a gas chromatograph mass spectrometer. The results are as shown in the following table.

Table 16

[0091] Here, since the inventors found that the functional edible charcoal is characterized in that, in addition to temperature and pressure, it is carbonized and pulverized into a "fluffy and powdery" state using reduced water, a test was conducted to prove that the polishing action that damages teeth is low. Since the glass surface is softer than the enamel of teeth, a polishing action test using an electric toothbrush was performed on the glass surface using "Ina Akamatsu Myo Coal · Coconut Shell Activated Carbon Granules (Dispersion Processed)". As a result, it was found that the glass surface was not damaged at all even after being polished with an electric toothbrush using charcoal for 1 hour. From this, it was found that there is no problem with the polishing action of the functional edible charcoal on teeth. In this embodiment, an example of carbonization and pulverization using reduced water was shown, but the present invention is not limited to this. Carbonization and pulverization may be performed using reduced water, particularly deep ocean water subjected to reduction processing with hydrogen, or water containing water-soluble silicon.

[0092] In addition, within the EU region, the effects and efficacy of activated carbon are recognized. Specifically, in the category of "reduction of excessive intestinal gas accumulation", in the "EU Health Claim (a label suggesting a beneficial effect on health)", it is stated that "activated carbon contributes to the reduction of excessive abdominal distension after meals", and as the "labeling condition", it is stated that "it can be used only for foods containing 1 g of activated carbon per meal portion". And it is recognized that "it is necessary to provide consumers with information that a beneficial effect can be obtained by ingesting 1 g of activated carbon more than 30 minutes before a meal or immediately after a meal".

[0093] In addition, an article on new uses of charcoal is published on the homepage of the Forestry Agency of Japan. For example, by putting charcoal into a bathtub, the bathwater can be made alkaline, providing a bathing effect similar to that of a hot spring, and the purification of the water itself can also be expected. Also, by putting charcoal into rivers or lakes, the adsorption action of charcoal can remove pollutants, and the decomposition of the pollutants themselves can be expected by the microorganisms that inhabit the pores of the charcoal, thus water purification can be expected. As is clear from such examples, by applying "functional edible charcoal" to various life scenes such as beverages, bath products, and cosmetics, it can ultimately flow into wastewater and is expected to contribute to the environment in the same way as charcoal.

[0094] In addition, the reducing fermented lactic acid bacteria according to this embodiment may be powdered in consideration of convenience in storage, transportation, user convenience, etc. That is, after mixing the liquid at the above-mentioned blending ratio, the liquid is adsorbed to water-soluble dietary fibers such as "inulin", "acacia dietary fiber" or "dextrin", "resistant dextrin" by methods such as spray drying or freeze drying, and it is possible to powder the reducing fermented lactic acid bacteria. Here, spray drying is inexpensive, but since it is heated at a high temperature during production, there is a risk of inactivation of raw material properties such as enzymes and polyphenols, and since it becomes a powder with a low concentration, freeze drying is considered to be suitable. It should be noted that the present invention is not limited to these descriptions, and generally known "water-soluble dietary fibers such as pectin and alginic acid", "insoluble dietary fibers such as cellulose and lignin", and "oligosaccharides such as raffinose and fructooligosaccharide" can also be applied during powdering.

[0095] The reducing fermented lactic acid bacteria according to this embodiment are composed by blending the above substances, and as described below, it is possible to obtain a bactericidal effect against so-called "bad bacteria" in the living body and an anti-aging effect. In addition, it is effective not only for oral use but also for application to the outer skin of the human body, and it is useful for hair care, face washing, laundry use, etc. by mixing it into shampoo, facial wash, or detergent.

[0096] [Verification Example on Bactericidal Effect against Mutans Bacteria] As the sample, a 5-fold dilution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. After inoculating the test solution into this sample, the viable cell count in the test solution was measured after a predetermined time had elapsed. Also, a preliminary test (confirmation of neutralization conditions) was performed in advance to confirm the conditions under which the viable cell count could be measured without being affected by the sample. The test conditions are as shown in the following table.

[0097]

Table 17

[0098] Next, the test results are shown.

Table 18

[0099] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, since viable bacteria were not detected after 5 minutes, it became clear that there is a sufficient bactericidal effect against mutans bacteria. As a result, it is expected that the number of mutans bacteria, which are the causative bacteria of dental caries in the oral cavity, will be reduced, the oral environment will be improved, and it can be expected to be utilized for oral care.

[0100] [Verification Example Regarding Bactericidal Effect Against Streptococcus mutans] As the sample, a 5-fold dilution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. After inoculating the test solution into this sample, the viable cell count in the test solution was measured after a predetermined time had elapsed. Also, a preliminary test (confirmation of neutralization conditions) was performed in advance to confirm the conditions under which the viable cell count could be measured without being affected by the sample. The test conditions are as shown in the following table.

[0101]

Table 19

[0102] Next, the test results are shown.

Table 20

[0103] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, since viable bacteria were not detected after 1 minute, it was revealed that there is a sufficient bactericidal effect against Porphyromonas gingivalis. As a result, it is expected that the number of Porphyromonas gingivalis, which is a causative bacterium of periodontal disease in the oral cavity, can be reduced, the oral environment can be improved, and it can be expected to be utilized for oral care.

[0104] [Verification example of bactericidal effect against Pasteurella bacteria] As a specimen, a 5-fold diluted solution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. A test solution was added to and mixed with this specimen. Then, immediately after mixing and after reacting at room temperature for a certain period of time, the number of remaining viable bacteria was measured. The test conditions are as shown in the following table.

[0105]

Table 21

[0106] Next, the test results are shown.

Table 22

[0107] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, since viable bacteria were not detected after 1 minute, it was revealed that there is a sufficient bactericidal effect against Pasteurella bacteria. As a result, it is expected that the number of Pasteurella bacteria, which are oral bacteria that infect humans from dogs and cats, can be reduced, the oral environment can be improved, and it can be expected to be utilized for oral care.

[0108] [Verification example of bactericidal effect against Helicobacter pylori] As the sample, a 5-fold diluted solution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. After inoculating the sample with a test bacterial solution (hereinafter referred to as "test solution"), the viable cell count in the test solution was measured after a predetermined time had elapsed. Also, a preliminary test (confirmation of neutralization conditions) was conducted in advance to confirm the conditions under which the viable cell count could be measured without being affected by the sample. The test conditions are as shown in the following table.

[0109]

Table 23

[0110] Next, the test results are shown.

Table 24

[0111] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, the viable cell count was 1 / 350 or less of the control after 1 minute, and was not detected after 5 minutes or later, indicating that it has a sufficient bactericidal effect against Helicobacter pylori. As a result, by being used for drinking, it can be expected to reduce Helicobacter pylori, which is a cause of gastric cancer and gastric ulcers, and improve the gastric environment.

[0112] [Verification Example Regarding Bactericidal Effect Against Staphylococcus aureus] As the sample, a 5-fold diluted solution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. After inoculating the sample with the test solution, the viable cell count in the test solution was measured after a predetermined time had elapsed. Also, a preliminary test (confirmation of neutralization conditions) was conducted in advance to confirm the conditions under which the viable cell count could be measured without being affected by the sample. The test conditions are as shown in the following table.

[0113]

Table 25

[0114] Next, the test results are shown.

Table 26

[0115] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, since viable bacteria were not detected after 5 minutes, it was clarified that there is a sufficient bactericidal effect against Staphylococcus aureus. As a result, it is expected to reduce the harmful bacteria in the intestine that increase with aging, that is, Staphylococcus aureus, which is one of the two major harmful bacteria in the large intestine, and lead to a beneficial bacteria-dominant intestinal flora. In addition, Staphylococcus aureus is said to be the causative bacterium of atopic dermatitis on the skin and scalp. By mixing the reduced fermented lactic acid bacteria powder according to this embodiment into hair shampoos, hair treatments, facial cleansers, etc., it is expected to be used for skin care and scalp care. That is, it can be used for face washing, oral care, scalp care, or added to skin gel packs, bath agents, etc. By mixing and using the reduced fermented lactic acid bacteria powder according to this embodiment in shampoos and facial cleansers, it is possible to easily supply hydrogen and minerals to the scalp and skin, and reduce the skin to be clean and suppress oxidation problems. Thus, since the bactericidal effect of the reduced fermented lactic acid bacteria powder according to this embodiment is high, its effect on acne bacteria and Malassezia bacteria is also inferred, and it is considered that it can also be expected to be used for acne treatment and folliculitis (inflammation of hair follicles).

[0116] [Verification Example on Bactericidal Effect against Bacillus welchii] As a specimen, a 5-fold dilution of the reduced fermented lactic acid bacteria powder according to this embodiment was used. A test solution was added to this specimen and mixed. After a predetermined time had passed, the number of viable bacteria in the test solution was measured. The test conditions are as shown in the following table.

[0117]

Table 27

[0118] Next, the test results are shown.

Table 28

[0119] Thus, in the reduced fermented lactic acid bacteria powder according to this embodiment, since viable bacteria were not detected after 1 minute, it became clear that it has a sufficient bactericidal effect against Clostridium welchii. In the human intestine, changes characterized by a decrease in Bifidobacterium and an increase in Clostridium welchii occur after middle age. Clostridium welchii is one of the putrefactive bacteria and produces harmful substances such as ammonia, amines, phenols, and indoles by putrefying proteins. These harmful substances include carcinogens, and most of them are decomposed in the liver, but if they exceed the processing capacity of the liver, they will affect the whole body. Indole is converted to indoxyl sulfate in the liver. Although healthy people with normal kidneys can excrete indoxyl sulfate in urine and there is no problem, for chronic kidney disease patients, about 13 million in Japan, one in eight Japanese people, who cannot excrete indoxyl sulfate in urine, research is underway on the grounds that indoxyl sulfate flowing into the blood is a causative substance that causes uremia and heart disease. This decrease in Bifidobacterium and relative increase in Clostridium welchii can be considered as intestinal aging. According to the reduced fermented lactic acid bacteria powder according to this embodiment, it is expected to have the effect of reducing Clostridium welchii, which is one of the two major harmful bacteria in the intestine that increases with aging, and leading to a beneficial bacteria-dominant intestinal flora.

[0120] [Verification example of the "bactericidal effect on oral bacteria" of a 70-year-old male with pointed periodontal disease] As the sample, the reduced fermented lactic acid bacteria powder according to this embodiment was used. Also, "saliva of a 70-year-old man with a diagnosis of periodontal disease" was used as the test solution. As a control, gels containing only the KS melt lactic acid bacteria product substance with and without peppermint were used. That is, "KS melt gel with peppermint" and "KS melt gel without peppermint". Furthermore, a commercially available toothpaste "control sample 1" was used. The test conditions are as shown in the following table. That is, (1) the test solution was diluted 100-fold with physiological saline and then filtered through Advantec filter paper No. 2, and (2) 1 g of the sample was added to 10 mL of the liquid obtained in (1) above, mixed for 3 minutes, and then inoculated into the diluted medium. As the medium, Nissui Compact Dry TC manufactured by Nissui Pharmaceutical Co., Ltd. was used. The results are as follows.

[0121] [Table 29] Note that in the table showing the test results, "<10" indicates that it was not detected. Also, ">99" indicates that it is almost close to 100%.

[0122] Thus, in "KS melt gel with peppermint", "KS melt gel without peppermint" and control sample 1, although a decrease in the number of bacteria was observed, a considerable proportion of bacteria remained, whereas in the reduced fermented lactic acid bacteria powder according to this embodiment, no bacteria were detected, indicating that bacteria can be almost completely eliminated. As a result, it was revealed that the reduced fermented lactic acid bacteria powder according to this embodiment has a sufficient bactericidal effect against oral bacteria.

[0123] [Verification Example for Existing Viable Bacteria] As the samples, "KS melt gel with peppermint", "KS melt gel without peppermint" and the reduced fermented lactic acid bacteria powder according to this embodiment were used. A mixture obtained by adding physiological saline to 1 g of each of these samples to make 5 g was inoculated into the medium. As the medium, Nissui Compact Dry TC manufactured by Nissui Pharmaceutical Co., Ltd. was used. The results are as follows.

[0124]

Table 30

[0125] In this way, it was confirmed that there are no viable bacteria in "KS Melt Gel with Hacka", "KS Melt Gel without Hacka", and the reduced fermented lactic acid bacteria powder according to this embodiment.

[0126] [Verification Example of Antioxidant Activity (SOD-Like Activity)] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter, and the resulting solution was used as the stock solution. For this stock solution and the solutions diluted 10-fold and 100-fold with the buffer solution attached to the SOD Assay Kit-WST (manufactured by Dojindo Laboratories), the inhibition rate of superoxide generation was calculated as SOD-like activity (%) using the SOD Assay Kit-WST according to a predetermined method. The results are as follows, indicating that the higher the SOD-like activity, the higher the antioxidant activity.

[0127]

Table 31

[0128] In this way, the reduced fermented lactic acid bacteria powder according to this embodiment has a high SOD-like activity in the stock solution. Also, even when diluted 10-fold and 100-fold, it has a sufficiently high SOD-like activity. Therefore, it is expected that a sufficient effect can be obtained even when diluted, and it was found that a sufficiently high antioxidant activity (SOD-like activity) can be obtained from a practical point of view. As a result, an antioxidant effect on the body by active oxygen can be expected.

[0129] [Verification Example of α-Glucosidase Inhibitory Activity] The α-glucosidase inhibitory activity was verified (measurement of α-glucosidase inhibitory activity). α-Glucosidase creates a state of high blood glucose levels in the living body. Therefore, if the α-glucosidase inhibitory activity is high, it is possible to inhibit the absorption of sugar in the living body and avoid a state of high blood glucose levels, and as a result, the generation of AGEs is suppressed.

[0130] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45-μm filter, and the resulting solution was used as the stock solution. This stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples. After adding 10 μL of the sample solution to each microtube and stirring, 40 μL of the enzyme solution and 40 μL of 50 mM sodium phosphate buffer were added, and after pre-incubating at 37°C for 5 minutes, 950 μL of the substrate solution was added and stirred, followed by incubation at 37°C for 15 minutes. Then, 1000 μL of 0.5 M Tris aqueous solution was added to stop the reaction, and the absorbance at 405 nm was measured. The α-glucosidase inhibitory activity (%) when the sample was added was calculated from the change in absorbance. The results are as follows.

Table 32

[0131] Thus, it was found that the reduced fermented lactic acid bacteria powder according to this embodiment has extremely high α-glucosidase inhibitory activity. Thereby, in vivo, it is considered that by inhibiting α-glucosidase activity, sugar absorption can be inhibited and a state with a high blood glucose level can be avoided. If a state with a high blood glucose level can be avoided, it becomes possible to make the state in which the early AGEs generation reactants are hardly generated.

[0132] [Verification Example for Inhibitory Activity of Amadori Compound Generation] In the glycation reaction in vivo, Amadori compounds are generated as "early AGEs generation reactants". Therefore, if the generation of Amadori compounds is suppressed, the generation of the following intermediate reactants and subsequent late AGEs generation reactants will be suppressed, and as a result, the generation of AGEs will be suppressed. When the blood glucose level rises, "hemoglobin, a protein present in red blood cells" undergoes glycation and changes to "glycated hemoglobin". "Glycated hemoglobin" is a type of Amadori compound, and the test item for examining this is "hemoglobin A1c", which is used as a diabetes judgment criterion.

[0133] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter to obtain a stock solution. This stock solution and solutions obtained by diluting the stock solution 10-fold and 100-fold with pure water were used as measurement samples. For the measurement of the inhibitory activity of Amadori compound formation, the measurement sample, glucose solution, and bovine serum albumin solution were mixed and incubated at 60°C for 48 hours to prepare a glycation reaction solution. To 40 μL of the glycation reaction solution, 300 μL of a 0.25 mM nitroblue tetrazolium solution (pH 9.0) was added, and after incubation at 37°C for 30 minutes and then standing at room temperature for 16 hours, the absorbance at 540 nm was measured. The inhibitory activity of Amadori compound formation when the measurement sample was added was calculated from the change in absorbance. The results are as follows.

[0134]

Table 33

[0135] Thus, the reduced fermented lactic acid bacteria powder according to this embodiment has a high inhibition rate in the stock solution. Accordingly, since it can be said that the reduced fermented lactic acid bacteria powder according to this embodiment has the inhibitory activity of Amadori compound formation, a glycation inhibitory effect of lowering the value of "hemoglobin A1c" by drinking can be expected.

[0136] [Verification Example Regarding AGEs Crosslink Cleavage Function] Verification of the AGEs crosslink cleavage function was performed by measuring the amount of benzoic acid decomposed and generated by cleavage of the dicarbonyl bond of the model compound 1-phenyl-1,2-propanedione having a dicarbonyl bond. The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter to obtain a stock solution. This stock solution and solutions obtained by diluting the stock solution 10-fold and 100-fold with pure water were used as measurement samples.

[0137] A measurement sample, a 1-phenyl-1,2-propanedione solution, and methanol were mixed to obtain a reaction solution. After incubating the reaction solution at 37 °C for 24 hours, hydrochloric acid was added to stop the reaction. The quantification of benzoic acid decomposed in the reaction solution was performed using high performance liquid chromatography. The AGEs crosslink cleavage rate was calculated with 100 (%) when all of the 1-phenyl-1,2-propanedione in the reaction solution was decomposed into benzoic acid. The results are as follows.

[0138]

Table 34

[0139] Accordingly, the reduced fermented lactic acid bacteria powder according to this embodiment has an AGEs crosslink cleavage function, decomposes the intermediate reaction product "AGEs crosslink" once formed, and is expected to have an effect of suppressing glycation.

[0140] [Verification Example for Fluorescent AGEs Generation Suppression Activity] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter to obtain a stock solution. This stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples. For the measurement of the fluorescent AGEs generation suppression activity, a measurement sample, a glucose solution, and a bovine serum albumin solution were mixed and incubated at 60 °C for 40 minutes, and then the generated fluorescent AGEs were measured with a fluorescence spectrophotometer at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The generation suppression activity (%) of fluorescent AGEs when the measurement sample was added was calculated as the suppression rate from the decrease in fluorescence intensity. The results are as follows.

[0141]

Table 35

[0142] Thus, the reduced fermented lactic acid bacteria powder according to this embodiment has a high inhibition rate in the undiluted solution. Also, even when diluted 10-fold or 100-fold, it still has a sufficiently high inhibition rate. Therefore, even when diluted, a sufficient effect is expected, and it has been found that it has a sufficiently high inhibitory effect on the generation of late-stage AGEs "fluorescent AGEs" from a practical perspective. As a result, a glycation inhibitory effect by drinking can be expected.

[0143] [Verification Example Regarding Tyrosinase Inhibitory Activity] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter to obtain an undiluted solution. This undiluted solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples. For the measurement, a solution prepared by dissolving mushroom-derived tyrosinase at 300 units / mL in 0.1 M phosphate buffer (pH 6.7) was used as the enzyme solution, and a solution prepared by dissolving L-DOPA at 1 mM in 0.1 M phosphate buffer (pH 6.7) was used as the substrate solution.

[0144] Using a 96-well microplate, 25 μL of the sample solution and 100 μL of the enzyme solution were added, incubated at 37°C for 10 minutes, then 125 μL of the substrate solution was added, incubated at 37°C for 30 minutes, and the absorbance at 490 nm was measured. The tyrosinase inhibitory activity (%) when the sample was added was calculated from the change in absorbance. The results are as follows.

[0145]

Table 36

[0146] Tyrosinase is known to produce melanin in the body. Therefore, when there is a large amount of it, the skin will have more freckles. The reduced fermented lactic acid bacteria powder according to this embodiment has a high tyrosinase inhibitory activity in the undiluted solution, but showed a higher value when diluted 10-fold. As a result, even when diluted, a sufficient effect is expected, and it has been found that a sufficiently high tyrosinase inhibitory activity function can be obtained from a practical perspective. As a result, it is expected that melanin will be less likely to be synthesized and the skin freckles will be prevented.

[0147] [Verification Example of Hyaluronidase Inhibitory Activity] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter, and the resulting solution was used as the stock solution. This stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples. For the measurement, a solution prepared by dissolving hyaluronidase at 400 units / mL in 0.1 M acetic acid buffer (pH 4.0) was used as the enzyme solution, a solution prepared by dissolving potassium hyaluronate at 1 mg / mL in acetic acid buffer (pH 4.0) was used as the substrate solution, a solution prepared by dissolving Compound 48 / 80 at 0.1 mg / mL in acetic acid buffer (pH 4.0) was used as the enzyme activator, and a solution prepared by dissolving dimethylaminobenzaldehyde at 10 mg / mL in a hydrochloric acid-acetic acid mixed solution was used as the coloring solution.

[0148] 12 μL of the sample solution and 12 μL of the enzyme solution were placed in a microtube, incubated at 40 °C for 20 minutes, then 12 μL of the enzyme activator was added and incubated at 40 °C for 20 minutes. Further, 12 μL of the substrate solution was added and incubated at 40 °C for 40 minutes, then 12 μL of 0.4 N NaOH aqueous solution was added and ice-cooled. Thereafter, 12 μL of 0.8 M borate buffer (pH 9.0) was added, boiled for 3 minutes and then ice-cooled, 180 μL of the coloring solution was added, incubated at 40 °C for 30 minutes, and the absorbance at 595 nm was measured. The hyaluronidase inhibitory activity (%) when the sample was added was calculated from the change in absorbance. The results are as follows.

[0149]

Table 37

[0150] Hyaluronic acid is abundantly present between cells and is said to protect cells by its water retention function and buffering function. On the other hand, hyaluronidase is an "enzyme that decomposes hyaluronic acid" that increases due to aging, ultraviolet stimulation, etc. If this is present in large amounts, it may become impossible to maintain the moisture retention and viscoelasticity of the skin, and wrinkles and sagging of the skin may occur. The reduced fermented lactic acid bacteria powder according to this embodiment has a high hyaluronidase inhibitory activity in the stock solution. Also, even when diluted 10-fold or 100-fold, it has a sufficiently high hyaluronidase inhibitory activity. Therefore, even when diluted, sufficient effects are expected, and it has been found that a sufficiently high hyaluronidase inhibitory activity function can be obtained from the viewpoint of practicality. As a result, it is expected that hyaluronic acid will be less likely to be decomposed, the moisture retention and viscoelasticity of the skin will be maintained, and wrinkles and sagging of the skin will be prevented.

[0151] [Verification Example on Collagenase Inhibitory Activity] Collagenase is a proteolytic enzyme that hydrolyzes collagen. The inventors conducted a test on the "collagenase inhibitory activity" of the reduced fermented lactic acid bacteria powder according to this embodiment. The test method was as follows: The reduced fermented lactic acid bacteria powder was dissolved in pure water and prepared to be 10 mg / mL, and the filtrate through a 0.45 μm filter was used as the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples.

[0152] For the measurement, a substrate solution was prepared by dissolving Pz-peptide at 0.39 mg / mL in 0.1 M Tris buffer (pH 7.1), and an enzyme solution was prepared by dissolving collagenase TypIV at 0.1 mg / mL in pure water. 25 μL of the sample solution, 25 μL of the enzyme solution, and 200 μL of the substrate solution were placed in a glass test tube, incubated at 37 °C for 30 minutes, then 250 μL of 25 mM citric acid was added to stop the reaction. Then, 2500 μL of ethyl acetate was added and shaken for extraction, followed by centrifugation (1500 rpm, 5 minutes), and the upper layer (ethyl acetate layer) was recovered, and the absorbance at 320 nm was measured. The collagenase inhibitory activity when the sample was added was calculated from the change in absorbance and used as the collagenase inhibitory activity. The test results are as shown in the following table.

Table 38

[0153] Thus, it was revealed that the reduced fermented lactic acid bacteria powder according to this embodiment has the function of inhibiting collagenase that decomposes collagen. Accordingly, it is expected that by ingesting or applying the reduced fermented lactic acid bacteria according to this embodiment, the collagen in the skin will be less likely to be decomposed.

[0154] [Verification Example Regarding Elastase Inhibitory Activity] Elastase is an enzyme that decomposes elastin of elastic fibers that determine the mechanical properties of connective tissues together with collagen. The inventors of the present invention conducted a test on the "elastase inhibitory activity" of the reduced fermented lactic acid bacteria powder according to this embodiment. The test method was to dissolve the reduced fermented lactic acid bacteria powder in pure water and prepare it to 10 mg / mL, and use the filtrate through a 0.45 μm filter as the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples.

[0155] For the measurement, STANA (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) dissolved in 50 mM Tris buffer (pH 8.8) at 1 mM was used as the substrate solution, and porcine pancreatic elastase dissolved in 50 mM Tris buffer (pH 8.8) at 0.5 units / mL was used as the enzyme solution. Using a 96-well microplate, 50 μL of the sample solution, 50 μL of the enzyme solution, and 100 μL of the substrate solution were added, incubated at 37 °C for 30 minutes, and then the absorbance at 405 nm was measured. The elastase inhibitory activity when the sample was added was calculated from the change amount of the absorbance and used as the elastase inhibitory activity. The test results are as shown in the following table.

Table 39

[0156] Thus, it has been clarified that the reduced fermented lactic acid bacteria powder according to this embodiment has the function of inhibiting elastase that decomposes elastin. Accordingly, it is expected that by ingesting or applying the reduced fermented lactic acid bacteria according to this embodiment, the elastin in the skin will be less likely to be decomposed.

[0157] [Verification Example Regarding Lipase Inhibitory Activity] The reduced fermented lactic acid bacteria powder according to this embodiment was diluted 100-fold with pure water and filtered through a 0.45 μm filter, and the resulting solution was used as the stock solution. This stock solution and the solutions diluted 10-fold and 100-fold with pure water were used as measurement samples. The measurement was carried out using a lipase kit S (DS Pharma Biomedical) with some modifications to the operation procedure. The measurement samples, lipase solution, substrate solution, and coloring solution were mixed, incubated at 30 °C for 30 minutes, then a stop solution was added, and the absorbance at 405 nm was measured. The lipase inhibitory activity (%) when the sample was added was calculated from the change in absorbance. The results are as follows.

[0158]

Table 40

[0159] Lipase is an enzyme that plays a role in decomposing and emulsifying lipids among the three major nutrients and is mainly secreted in the stomach. The emulsified oil moves to the small intestine and is finely decomposed by the action of bile acids and absorbed by the body. Here, if the decomposition (emulsification) of oil in food by lipase can be suppressed, it will move to the large intestine without being absorbed by the body and be excreted with feces, so a dieting effect is expected. The reduced fermented lactic acid bacteria powder according to this embodiment exhibits a lipase inhibitory activity of 18.1% in the stock solution, inhibits lipase, which is an enzyme that plays a role in decomposing lipids, and can be expected to have a dieting effect in making it difficult for the body to absorb fat.

[0160] [Verification Example Regarding the Influence on Lactic Acid Bacteria] As described above, the reducing fermented lactic acid bacteria powder according to this embodiment has a high bactericidal ability against harmful bacteria, and thus is effective in removing so-called "harmful bacteria". At the same time, it was verified whether it would also affect lactic acid bacteria (Lactobacillus plantarum), which are so-called "good bacteria".

[0161] The reducing fermented lactic acid bacteria powder according to this embodiment was used as the "specimen", and a sample diluted to 1% with sterilized water was used as the test sample. The test bacteria (lactic acid bacteria: Lactobacillus plantarum) were inoculated on BCP agar medium and cultured at 30 °C for 48 hours. After culturing, the number of bacteria was adjusted to "10 8 / mL" with sterilized physiological saline to obtain the test bacterial solution. Then, 0.1 mL of the test bacterial solution was inoculated into 10 mL of the test sample and cultured at 25 °C. At 5 minutes and 15 minutes after inoculation, a 10-fold dilution series of the test sample diluted with sterilized physiological saline was used as the test solution. These test solutions were inoculated on BCP agar medium and cultured at 30 °C for 48 to 72 hours. After culturing, the formed colonies were counted and the viable cell count was calculated. In addition, sterilized phosphate buffered saline was used as a control and the test was conducted in the same manner. The results are as follows.

Table 41

[0162] Thus, it was found that the viable cell count of lactic acid bacteria did not decrease even after 15 minutes for the reducing fermented lactic acid bacteria powder according to this embodiment. As a result, it became clear that the reducing fermented lactic acid bacteria powder according to this embodiment exhibits an effective bactericidal function against harmful bacteria while having no effect on good bacteria. The reducing fermented lactic acid bacteria powder according to this embodiment is made from ume extract, deep ocean water, and hydrogen based on good bacteria (viable cells) and their secretions. From the above test results, it is considered that it is less likely to have an adverse effect on good bacteria (viable cells), that is, bacteria beneficial to the body such as Bifidobacterium and lactic acid bacteria.

[0163] On the one hand, periodontal disease is a disease that is less likely to cause pain compared to tooth decay and progresses slowly. It is characteristic in that anyone can develop it, with 80% affected by the age of 30 and 90% by the age of 50. In the oral cavity, Porphyromonas gingivalis, which is the main cause of periodontal disease, spreads, creating a situation that can be described as an environment dominated by harmful bacteria. Therefore, in order to improve the oral environment, chewables, gums, etc. containing beneficial bacteria (live bacteria) have been successively launched on the market. It is important to introduce beneficial bacteria (live bacteria) into the oral cavity. Although the image is good, the colonization rate of beneficial bacteria (live bacteria) in the oral cavity is not high. For this reason, it is also important to directly act to reduce harmful bacteria such as Porphyromonas gingivalis and Streptococcus mutans, which are dominant. In that sense, the collaboration between beneficial bacteria (live bacteria) and lactic acid bacteria of the reductive fermentative type is expected to have a synergistic effect on the approach to oral care.

[0164] As lactic acid bacteria with abundant evidence for oral care, "Lactobacillus KT-11 (heat-killed bacteria)" is well-known, and oral chewables and supplements containing "Lactobacillus KT-11" have been launched on the market. "Lactobacillus KT-11 (heat-killed bacteria)" is the same as "Bacteroides forsythus", which is abundant in the birth canal of women and is the first lactic acid bacteria that a baby receives at birth. It has been confirmed to have the function of increasing the components that protect the intestinal mucosa. However, it does not act directly on the bacteria in the oral cavity. When ingested, the intestinal immune activity increases, and as an indirect effect, the "IgA antibody" in saliva increases, enhancing the bactericidal power of saliva, and it has been reported that this leads to the mechanism of reducing Porphyromonas gingivalis, which is a harmful bacterium in the oral cavity. For this reason, oral chewables and supplements of beneficial bacteria are thought to enhance intestinal immunity by ingestion, whether live or heat-killed bacteria, and as a result, increase the "IgA antibody" in saliva, indirectly having a good effect on the oral environment. The lactic acid bacteria powder for reductive fermentation according to this embodiment is a lactic acid bacteria fermentation extract based on the "KS Melt lactic acid bacteria product substance" that has been utilized in a long-term dental research society and has sufficient evidence for oral care. At the same time, it contains "30 billion to 150 billion cells / g" as good bacteria (dead cells). In addition to the direct action on harmful bacteria in the oral cavity, an increase in "IgA antibodies" in saliva through the improvement of intestinal immunity can also be expected. Furthermore, the above-mentioned "KT-11 lactic acid bacteria" has a patent for enhancing the synergistic effect with other good bacteria (Patent No. 4942831). By simultaneously blending the reductive fermentation lactic acid bacteria and "KT-11 lactic acid bacteria", a further enhancement of the synergistic effect can be expected. Fig. 15 is a diagram showing the test results of the synergistic effect between the "KS Melt lactic acid bacteria product substance (raw material for the base of reductive fermentation lactic acid bacteria)" and "KT-11 lactic acid bacteria HP". This test uses the "KS Melt lactic acid bacteria product substance: manufactured by KS Nippon Co., Ltd.", which is the raw material for the base of reductive fermentation lactic acid bacteria, and "KT-11 lactic acid bacteria HP: manufactured by Kitty Co., Ltd." as samples, and evaluates the ability to induce the production of interleukin 12 (IL-12) using animal immune cells. This "KT-11 lactic acid bacteria HP: manufactured by Kitty Co., Ltd." is composed of a mixed raw material of "20% KT-11 lactic acid bacteria (dead bacteria) and 80% dextrin" and is known to have the function of immune activation (biogenics) by intestinal stimulation. The test method is as follows. That is, macrophages-like cell lines (immune cells) were used as cells. After suspending the cultured cells in the medium, they were dispensed into microplates, and the amount of IL-12 in the culture supernatant produced by adding the test substance to the final concentration was measured by the ELISA method. The test results are shown in Fig. 15. As shown in Fig. 15, the "immune activity IL-12 value" when the same amount of "KT-11 lactic acid bacteria HP" was added to the "KS Melt lactic acid bacteria product substance (raw material for the base of reductive fermentation lactic acid bacteria)" was "2515 (pg / mL)", which is higher than the "immune activity IL-12 value" of "1847 (pg / mL)" for "KT-11 lactic acid bacteria HP" alone, showing a synergistic effect of 36% or more.

[0165] Conventionally, verification results regarding the influence of deep ocean water according to this embodiment on bacteria such as lactic acid bacteria have been known. The inventors of the present invention confirmed the function of the reduced fermented lactic acid bacteria powder according to this embodiment with reference to these verification results.

[0166] [Verification example of the growth of bacteria in commercially available yogurt] The inventors of the present invention focused on the influence of deep ocean water according to this embodiment on the growth of bacteria in commercially available yogurt and confirmed the test results by a third party. The test method was as follows: (a) 1 mL of commercially available yogurt A and B diluted 100-fold was added to 12 mL of MRS medium or MRS medium supplemented with 1% deep ocean water powder, and (b) the mixture was cultured at 37°C, and the turbidity was measured over time. This "turbidity" indicates the degree of turbidity of the liquid, and the degree of turbidity when 1 mg of kaolin is contained in 1 L of distilled water is defined as 1 degree (or 1 ppm). FIG. 1 shows the effect of deep ocean water powder on the growth of bacteria in commercially available yogurt A, and FIG. 2 shows the effect of deep ocean water powder on the growth of bacteria in commercially available yogurt B. As shown in FIGS. 1 and 2, it has been clarified that the addition of deep ocean water powder promotes the growth of bacteria in yogurt.

[0167] [Verification example of the growth of "Lactobacillus gasseri JCM1131" bacteria] Verification results are known regarding the effect of deep ocean water on the growth of the bacterium "Lactobacillus gasseri JCM1131", which is known as an intestinal lactic acid bacterium. The inventors of the present invention confirmed the function of the reduced fermented lactic acid bacterium powder according to this embodiment with reference to this verification result. This test method was as follows: (a) 1 mL of the "Lactobacillus gasseri JCM1131" strain with a turbidity (OD600) value of 1.0 (MRS medium) was added to 12 mL of modified MRS medium or modified MRS medium supplemented with 1% or 2% of deep ocean water powder, and (b) cultured at 37°C, and the turbidity was measured over time. Figure 3 shows the effect of the addition of deep ocean water powder on the growth of "Lactobacillus gasseri JCM1131". As shown in Figure 3, it has been clarified that the addition of deep ocean water powder promotes the growth of the "Lactobacillus gasseri JCM1131" strain.

[0168] Thus, by blending deep ocean water into the lactic acid bacterium fermentation extract, the usefulness of the reduced fermented lactic acid bacterium powder when ingested for intestinal lactic acid bacteria can be expected.

[0169] [Verification Example of Total Peptide Quantification of Reduced Fermented Lactic Acid Bacterium Powder] The inventors of the present invention conducted the following test regarding the total peptide quantification of the reduced fermented lactic acid bacterium powder. The test method was to dissolve the reduced fermented lactic acid bacterium powder in pure water, adjust it to 5 mg / mL, and use the filtered product through a 0.45 μm filter as the measurement sample. For the measurement sample, total peptide quantification was performed using "Pierce Quantitative Colorimetric Peptide Assay (manufactured by Thermo Fisher Scientific)". The results are as shown in the following table.

[0170]

Table 42

[0171] [Verification Example on Deodorizing Property] The inventors of the present invention conducted a test on the deodorizing property of the reduced-fermentation lactic acid bacteria powder according to this embodiment. In this deodorizing test, the deodorizing property of the reduced-fermentation lactic acid bacteria powder according to this embodiment was verified for "ammonia" which causes malodors such as sweat odor, aging odor, excrement odor, tobacco odor, garbage odor, and ammonia odor, and "trimethylamine" which emits the odor of spoiled fish. The specimen was the reduced-fermentation lactic acid bacteria powder according to this embodiment, but the addition amounts of hydrogen and deep seawater during the reduction fermentation with hydrogen were increased by more than twice compared to the reduced-fermentation lactic acid bacteria powder used in the verification examples such as the above-mentioned Mutans and Zingibariscus bacteria, and a 5-fold dilution of the produced reduced-fermentation lactic acid bacteria powder was used. The test method was to dilute the sample 5-fold with distilled water, use ammonia (100 ppm) and "trimethylamine (28 ppm)" as odor components, set the sample amount to 0.5 mL, and set the standing times to 30 minutes and 120 minutes. The test results are as shown in the following table.

[0172]

Table 43

[0173] It was found that even water alone has a deodorizing effect on the blank. In contrast, the reducing fermented lactic acid bacteria powder SP1 and the reducing fermented lactic acid bacteria powder SP2 according to this embodiment were found to exhibit a high reduction rate (deodorizing rate) of 98% or more for both "ammonia" and "trimethylamine". In addition, the reducing fermented lactic acid bacteria powder SP1 and the reducing fermented lactic acid bacteria powder SP2 according to this embodiment have a higher deodorizing function for "ammonia" than "Ina Akamatsu Myo Coal·Coconut Shell Activated Carbon Granules (Dispersion Processed)", which is generally said to have a high deodorizing effect, and also have a deodorizing function that is not inferior to that of "Persimmon Tannin Extract", which is famous for its high deodorizing effect. From the above, it is expected to reduce the malodor itself generated in the oral cavity, intestines, etc., and improve the oral environment and gastrointestinal environment, and it can be expected to be used for oral care and gastrointestinal care. It should be noted that "Helicobacter pylori" is known to produce an enzyme called urease to create alkaline ammonia around itself, neutralize gastric acid, and exist in the stomach. As described above, a total approach to Helicobacter pylori, which is the cause of gastric cancer, can be expected through the collaboration of reducing fermented lactic acid bacteria that eliminate Helicobacter pylori and functional edible charcoal such as Ina Akamatsu Myo Coal·Coconut Shell Activated Carbon Granules (Dispersion Processed) that has a high adsorption activity for "ammonia" produced by Helicobacter pylori through the urease enzyme.

[0174] [Verification Example of Bactericidal Effect against Candida] "Candida" is a "opportunistic fungus" that is neither a good nor a bad bacterium and is a type of mold. When the body's immunity declines, it rapidly proliferates, causing oral candidiasis or breeding in the intestines and leading to intestinal leakage (leaky gut syndrome). The inventors of the present invention conducted tests on the bactericidal effect of the reduced fermented lactic acid bacteria powder according to this embodiment against Candida. The sample was the reduced fermented lactic acid bacteria powder according to this embodiment, but a 5-fold dilution of the "reduced fermented lactic acid bacteria powder produced by increasing the addition amount of hydrogen and deep sea water to more than twice that of the reduced fermented lactic acid bacteria powder used in the verification examples of the above-mentioned mutans and zingibarris bacteria during the reduction fermentation by hydrogen" was used. This is hereinafter referred to as "reduced fermented lactic acid bacteria powder SP2". The test bacteria were Candida albicans: NBRC1594. The test method is as follows. That is, the sample was diluted to 1%, 10%, and 20% using sterilized water, and the diluted samples were used as test samples. The test bacteria were inoculated on a potato dextrose agar medium and cultured at 25°C for 48 hours. After culturing, the number of bacteria was adjusted to 10 8 / mL using sterilized physiological saline to obtain a test bacterial solution. Next, 0.1 mL of the test bacterial solution was inoculated into 10 mL of the test sample and cultured at 25°C. For the 1% solution, 10-fold dilution series of the test sample were prepared using sterilized physiological saline at 30 minutes and 1 hour after inoculation. For the 10% and 20% solutions, 10-fold dilution series of the test sample were prepared using sterilized physiological saline at 5 minutes, 15 minutes, 30 minutes, and 1 hour after inoculation. These test solutions were inoculated onto a sub-ro dextrose agar medium with a neutralizing agent added and cultured at 25°C for 48 to 72 hours. After culturing, the formed colonies were counted and the viable cell count was calculated. In addition, sterilized phosphate buffered saline was used as a control and the test was conducted in the same manner. The test results are as shown in the following table.

[0175]

Table 44

[0176] [Formulation of Ionized Apatite] The reduced fermented lactic acid bacteria powder according to this embodiment may contain "hydroxyapatite" and an oral composition containing citric acid (hereinafter referred to as "ionized apatite").

[0177] Regarding hydroxyapatite, it is desirable to use hydroxyapatite derived from scallop shells (bioapatite: registered trademark, which is defined as being derived from natural organisms and having low crystallinity). Hydroxyapatite accounts for about 97% of human tooth enamel and about 65% of bone, and is composed of phosphoric acid and calcium. It is said to have high biocompatibility, be neutral to weakly alkaline, and be safe for the human body. And because hydroxyapatite has almost the same components as tooth enamel, it has the function of remineralizing and repairing the damage formed on the tooth surface and the initial dental caries due to demineralization when a person eats or brushes their teeth. Note that mineral-derived hydroxyapatite may be used instead of natural organism-derived hydroxyapatite.

[0178] In addition, hydroxyapatite has 10 calcium atoms in one molecule, and these calcium atoms are replaced by various metals such as cadmium and lead through ion exchange. Due to such ion exchangeability, it has the function of adsorbing harmful substances such as heavy metals. Also, hydroxyapatite has a complex crystal structure and its surface is charged, so it has the function of adsorbing bacteria, viruses, pollen, etc. Furthermore, hydroxyapatite has the function of adsorbing pigments and the function of adsorbing lipid peroxides.

[0179] Generally, most of the commercially available hydroxyapatite is synthesized using minerals (apatite) as raw materials. However, in the ionized apatite according to this embodiment, natural scallop shells are used as raw materials. However, the present invention is not limited to hydroxyapatite derived from scallop shells, and it is also possible to use natural biomaterial-derived hydroxyapatite using corals, fish scales and bones, mammalian bones such as cow bones, seaweeds, eggshells, sea urchin shells, clam shells, oyster shells, pearls, dolomite, weathered shells (Kamionishiki shells), etc. as raw materials. It is also possible to use hydroxyapatite derived from shells utilized in pearl mother bodies. In this case, for example, shells such as Akoya oysters and black-winged pearl oysters are suitable.

[0180] Natural biomaterial-derived hydroxyapatite is said to have high biocompatibility in that it contains natural trace minerals such as magnesium and sodium in the apatite crystal structure, unlike mineral-derived hydroxyapatite. Furthermore, by adding deep ocean water, it becomes possible to additionally supply natural ionized minerals other than calcium.

[0181] Also, in this embodiment, it is possible to use weathered shells (Kamionishiki shells), fossil coral powder, and dolomite instead of hydroxyapatite. Weathered shells (Kamionishiki shells), fossil coral powder, and dolomite are not hydroxyapatite, but they possess minerals other than high-quality calcium, and are expected to be effective in terms of the synergistic effect with calcium supplementation for teeth and bones.

[0182] [Verification Example Using Immune Cells of Bioapatite (Registered Trademark: Derived from Natural Organism and Low Crystalline) and Lactobacillus KT-11] The inventors conducted an evaluation test on the ability to induce the production of interleukin 12 (IL-12) using immune cells. "IL-12" is a cytokine produced by immune cells that leads to immune activation and anti-allergic effects. Also, "IL-12" is known to activate "natural killer cells" that control immune cells and enhance immunity. Furthermore, "IL-12" activates "Th1 cells" that lead to anti-allergic effects and is useful for alleviating allergic symptoms. In this test, macrophages (immune cells) were used as the cells. Macrophages were dispensed into microplates, samples were added and cultured, and then the amount of IL-12 produced was measured by ELISA. As shown in Figure 6, when bioapatite and Lactobacillus KT-11 were used in combination (1% or 10% of Lactobacillus KT-11 was added to 100% of hydroxyapatite), a "substantial improvement in IL-12 production" was confirmed compared to when each was used alone. Also, the "amount of IL-12 produced" was confirmed even when bioapatite was used alone.

[0183] [Verification Example Regarding Anti-Aging Effect Using Nematodes (in the Case of Granules)] The inventors focused on the effect of the ionized apatite according to this embodiment on the average lifespan of nematodes and conducted an examination based on the test results by a third party. That is, it is said that by ingesting foods containing antioxidant components, it is possible to suppress the decline in the ability to remove active oxygen in the body that occurs with aging, and it is expected to prevent lifestyle-related diseases due to aging. However, the functional evaluation of foods is mainly carried out "in vitro". In order to demonstrate the effect in vivo, a "in vivo" biological test is required. In this "in vivo" test, as a substitute for organisms such as mice, there is a type of nematode called "Caenorhabditis elegans". This "C.elegans" is a non-parasitic nematode with a lifespan of about three weeks and inhabits soil of about 1 mm. All of its genes have been elucidated, and it is used in tests around the world as a model organism for experiments analyzing various life phenomena.

[0184] The test method in this embodiment is as follows. That is, the Escherichia coli OP-50 strain used as food was suspended in "S-medium" shown in the following table to prepare a culture solution of nematodes (fer-15 mutant strain). Using this culture solution, the nematodes were cultured (at 20 °C, 100 rpm) until they became adults. Thereafter, the nematodes were collected and washed with "S-buffer" shown in the following table, and then the bodies of the nematodes were lysed using an NaOH solution and Hiter (registered trademark) to recover eggs from inside the bodies. The recovered eggs were cultured overnight at 20 °C for hatching. The hatched L1 larvae were synchronously cultured at 26.5 °C using heat-treated Escherichia coli OP-50 strain (dead cells) as food in a 24-well microplate. At this time, the number of nematodes per well of the microplate was adjusted to be about 20. A solution prepared by dissolving ionized apatite (granules) in pure water at a concentration of 100 mg / mL was used as the stock solution. After egg recovery, on the 5th day, the stock solution was filter-sterilized with a 0.22-μm filter, and added to each well of the microplate so that the final concentration in the culture solution was 10-fold, 100-fold, and 1000-fold dilutions of the stock solution. Thereafter, the survival rate of the nematodes was examined under a microscope every few days. Taking the number of survivors on the 5th day as 100%, a survival rate curve was drawn by the "Kaplan Meier method", and a p-value was calculated by the log-rank test. Also, the average lifespan was calculated from the survival days. The results are as follows.

Table 45

Table 46

Table 47

[0185] For the ionized apatite (granules) according to this embodiment, a statistically significant lifespan extension was observed for all treatment groups compared to the control (p-value < 0.05). In particular, at 10-fold dilution, the lifespan extended by 16.6% compared to the control, and also at 1000-fold dilution, a statistically significant effect of extending the average lifespan was confirmed. Thus, it was found that even when diluted, a high anti-aging effect can be expected in practical applications.

[0186] [Verification Example of Anti-Aging Effect Using Nematodes (in the Case of Powder)] The inventors focused on the effect of the ionized apatite according to this embodiment on the average lifespan of nematodes and conducted an examination based on the test results by a third party. The test method is as follows. That is, Escherichia coli OP-50 strain as food was suspended in "S-medium" to prepare a culture solution of nematodes (fer-15 mutant strain). Using this culture solution, the nematodes were cultured (at 20 °C, 100 rpm) until they became adults. Thereafter, the nematodes were collected with "S-buffer", washed, and then the bodies of the nematodes were dissolved using an NaOH solution and Hiter (registered trademark) to recover eggs from inside the body. The collected eggs were cultured overnight at 20 °C and hatched. The hatched L1 larvae were synchronized and cultured at 26.5 °C using heat-treated Escherichia coli OP-50 strain (dead cells) as food in a 24-well microplate. At this time, the number of nematodes per well of the microplate was adjusted to about 20. A solution prepared by dissolving ionized apatite (powder) in pure water at a concentration of 100 mg / mL was used as the stock solution. After egg collection, on the 5th day, the stock solution was filter-sterilized with a 0.22 μm filter, and added to each well of the microplate so that the final concentration in the culture solution was 10-fold, 100-fold, and 1000-fold dilutions of the stock solution. Thereafter, the survival rate of the nematodes was examined under a microscope every few days. Taking the number of survivors on the 5th day as 100%, a survival rate curve was drawn by the "Kaplan Meier method", and a p-value was calculated by the log-rank test. Also, the average lifespan was calculated from the number of survival days. The results are as follows.

Table 48

[0187] In all treatment groups of the ionized apatite (powder) according to this embodiment, a statistically significant lifespan extension was observed compared to the control (p-value < 0.05). In particular, the extension effect was highest at 1000-fold dilution, which was further diluted compared to 10-fold and 100-fold dilutions, and the lifespan extended by 15.86% compared to the control, obtaining remarkable numerical values. From this, it was found that a high anti-aging effect can be expected in practical applications even when diluted.

[0188] Furthermore, comparative examples will be described. Mineral-derived hydroxyapatite is utilized in many oral care products. The inventors examined the "anti-aging effect" of mineral-derived hydroxyapatite based on the test results by a third party. The test method is as follows. That is, Escherichia coli OP-50 strain used as food was suspended in "S-medium" to prepare a culture solution for nematodes (fer-15 mutant strain). Using this culture solution, the nematodes were cultured until they became adults (20 °C, 100 rpm). After that, the nematodes were collected and washed with "S-buffer", and then the bodies of the nematodes were dissolved using a NaOH solution and Hiter (registered trademark) to recover eggs from inside the body. The recovered eggs were cultured overnight at 20 °C and hatched. The hatched L1 larvae were synchronously cultured at 26.5 °C using heat-treated Escherichia coli OP-50 strain (dead cells) as food in a 24-well microplate. At this time, the number of nematodes per well of the microplate was adjusted to be about 20. A solution prepared by dissolving mineral-derived hydroxyapatite in pure water at a concentration of 100 mg / mL was used as the stock solution. After egg recovery, on the 5th day, the stock solution was filter-sterilized with a 0.22-μm filter, and added to each well of the microplate so that the final concentration in the culture solution was diluted 10-fold, 100-fold, and 1000-fold of the stock solution. Thereafter, the survival rate of the nematodes was examined under a microscope every few days. Taking the number of survivors on the 5th day as 100%, a survival rate curve was drawn by the "Kaplan Meier method", and the p-value was calculated by the log-rank test. Also, the average lifespan was calculated from the survival days. The results are as follows.

Table 49

Table 50

Table 51

[0189] Thus, in all treatment groups, a shortening of the average lifespan was observed for the mineral-derived hydroxyapatite compared to the control. In particular, the mineral-derived hydroxyapatite at 100-fold dilution had a statistically significantly shorter average lifespan compared to the control (p-value < 0.05).

[0190] [Verification Example of Ionization Rate] The inventors verified the ionization rate of the ionized apatite according to this embodiment. As the specimen, the ionized apatite according to this embodiment was used, and as Comparative Example 1, a dispersion liquid obtained by pulverizing and nanopowderizing hydroxyapatite derived from scallop shells and hydroxyapatite derived from scallop shells was used as the specimen. 50 mL of water was added to 5 g of each specimen, and after shaking for 30 minutes, the contents of phosphorus and calcium in the liquid filtered through a 0.45 μm membrane filter were measured by ICP emission spectrometry. However, it was calculated from the specimen weight. Also, for pH, a 1% aqueous solution was measured by the glass electrode method. The measurement results are shown in the following table.

[0191]

Table 52

[0192] In order not to progress from dental demineralization to dental caries, in addition to using sugar alcohols such as xylitol that do not produce the acid causing demineralization as a substitute for sugar, it is known that it is important to supply calcium that is easily soluble in saliva. Human saliva contains sufficient phosphate ions necessary for remineralization, but not sufficient calcium ions. That is, the phosphate ion concentration is sufficient, but the calcium ion concentration is less than half of that. As described above, the ionized apatite according to the present embodiment can supply a large amount of calcium ions together with phosphate ions, so it is possible to dissolve sufficient calcium ions in saliva to replenish the minerals lost in the initial "tooth decay". Hydroxyapatite promotes tooth remineralization by supplying calcium ions and phosphate ions to the teeth. Moreover, in saliva, since calcium ions are relatively insufficient compared to phosphate ions, ionized apatite that can supply a large amount of calcium ions can be expected to have a more useful effect.

[0193] [Blending of Silk Hydrogen Pearl Powder] The reducing fermented lactic acid bacteria according to this embodiment may contain silk hydrogen pearl powder in which hydrogen is carried on "silk powder impregnated with deep ocean water" and "hydroxyapatite derived from pearl oyster". Thereby, it becomes possible to synergistically obtain the effects of silk, hydrogen, and hydroxyapatite derived from pearl oyster. In this embodiment, "hydroxyapatite derived from pearl oyster" is used for explanation. Pearl oysters grow in a mille-feuille-like layer, and the hydroxyapatite using pearl oysters has the lowest bulk specific gravity among bioapatites, is suitable for compatibility with hydrogen loading, and when used for oral care, it can be expected to have the highest affinity with teeth in a more low-crystalline form. Thus, by combining "silk powder impregnated with deep ocean water" and "hydroxyapatite derived from pearl oyster", it becomes possible to obtain a "hybrid" hydrogen raw material (for food, acidic) that generates both hydrogen gas and hydrogen ions. This combination has a redox potential of "-270 mV (25 °C)" and a pH of "5.4 (22 °C)". However, the present invention is not limited to these, and the redox potential can be made even lower, it can be made alkaline, and furthermore, it is also possible to use "hydroxyapatite" that is not "derived from pearl oyster". Note that silk hydrogen pearl powder is a material that can generate both hydrogen gas and hydrogen ions (hydrogen ions with electrons, sometimes also called negative hydrogen ions) simultaneously. The amount of hydrogen gas generated can be examined by a gas chromatography test of the Japan Food Analysis Center, and the amount of hydrogen ions generated can be measured by reacting "NAD+ reagent" with silk hydrogen pearl powder and examining the number of generated NADH.

[0194] The inventors measured the amount of hydrogen generated from "Silk Hydrogen Pearl Powder" by gas chromatography. The test method is as follows. That is, after pulverizing the specimen (Silk Hydrogen Pearl Powder) in a mortar, 0.2 g and 0.1 g were collected and added to a 125 mL vial. 25 mL of purified water was added thereto, the lid was quickly closed, and ultrasonic extraction was performed for 30 minutes. After standing at room temperature for 48 hours or more, 0.5 mL of the headspace gas in the vial was injected into the gas chromatograph to measure hydrogen. As a result, "12 mL per 1 g" of hydrogen gas was detected for the Silk Hydrogen Pearl Powder. Usually, when hydrogen gas is dissolved in water, under normal temperature and pressure (15 - 25 °C, 1 atm), the dissolved hydrogen concentration of 1.6 ppm (a state where 1.6 mg of hydrogen is dissolved in 1 L of water) is said to be the limit (saturated hydrogen water), and it is calculated that 0.018 mL of hydrogen gas is dissolved in 1 mL of water, that is, 1.8 mL of hydrogen gas is dissolved in 100 mL of water. In contrast, according to 1 g of Silk Hydrogen Pearl Powder, the same amount of hydrogen gas can be ingested as when "drinking 6 cups of 100 mL of saturated hydrogen water (one cup)". Furthermore, since hydrogen ions with electrons are generated by the synergistic action with nacre-derived hydroxyapatite, it leads to the result of producing the effect of the dissolved hydrogen amount, which is usually said to "halve every 3 hours", lasting for a long time.

[0195] Also, the inventor measured the amount of hydrogen ions in the Silk Hydrogen Pearl Powder. The reagents used are "Reduced NADH Standard Solution: manufactured by Wako Pure Chemical Industries, Ltd.", "Potassium Pyrophosphate Buffer (F-Kit)", and "NAD Tablets (F-Kit)". The measurement principle is "NAD + +H + +2e - →NADH", and the NADH generated by the generation of hydrogen is measured to calculate the amount of hydrogen. The test method is as follows. First, 10 mL of distilled water was added to 1 g of the reagent, stirred, and after standing for 1 hour, the supernatant was analyzed. The molar concentration of NADH was calculated and regarded as the amount of hydrogen. The results are as shown in the following table.

Table 53

[0196] The inventors measured the dissolved hydrogen content of a "1% aqueous solution in which 0.5 g of silk hydrogen pearl powder was dissolved and stirred" with respect to "50 mL of Yokohama City tap water" using "Trustrex ENH-1000". As a result, it was "1035 ppb" 10 minutes after the addition, but "1106 ppb" was measured 12 hours after the addition. Furthermore, "1127 ppb" was measured 25 hours after the addition, "1239 ppb" was measured 60 hours after the addition, "1265 ppb" was measured 72 hours after the addition, and "1286 ppb" was measured 96 hours after the addition. Furthermore, "1274 ppb" was measured 108 hours after the addition, and "1034 ppb" was measured 132 hours after the addition.

[0197] Thus, even 12 hours after the addition, instead of decreasing, the numerical value increased, which is a remarkable result. Even more surprisingly, 25 hours, 60 hours, 72 hours, and 96 hours after the addition, the numerical value further increased, and a "high dissolved hydrogen content of 1000 ppb or more" was confirmed. The fact that the dissolved hydrogen content is "1000 ppb" is one of the standard levels for high-concentration hydrogen water. However, silk hydrogen pearl powder can achieve a dissolved hydrogen content exceeding that standard level. Furthermore, since a "high dissolved hydrogen content of 1000 ppb or more" was maintained even 108 hours and 132 hours after the addition, it was found that "silk hydrogen pearl powder" can maintain a state in which hydrogen is dissolved in water for a long time. Thus, it can be said that a high dissolved hydrogen content is achieved by the synergistic effect of hydrogen gas and hydrogen ions with electrons.

[0198] By drinking hydrogen water, it is expected that harmful substances and harmful bacteria will react, and sufficient hydrogen can be supplied to the intestine, where the most active oxygen is produced in the body, and the liver, which is the largest detoxifying organ in the body and fights against active oxygen (hydrogen absorbed from the intestinal tract is carried directly to the liver through the portal vein). According to the announcement of the Tokyo Metropolitan Institute of Gerontology, it has been confirmed that continuous drinking of high-concentration hydrogen water suppresses blood vessel aging in mice, and as shown in Fig. 13, it is known that hydrogen water has various effects.

[0199] In addition, the present inventors conducted a test on the effect of a hydrogen bath, in which hydrogen bath powder was dissolved in warm water, on dry skin via the Nagano University of Nursing. Dry skin is known to be caused by aging, ultraviolet rays, etc. and is one of the main causes of skin problems. If an improvement effect by a hydrogen bath using a hydrogen bath agent is recognized, it is expected as one of effective skin care methods. The test method was to anesthetize hairless rats before bathing and measure the skin keratin water content of the dry skin-treated area and the untreated area. After photographing with a digital camera, the rats were bathed for 15 minutes. This was performed 10 times during the 28-day test period. The hydrogen warm bath was carried out by adding 20 g of hydrogen bath agent to warm water at 42 degrees, and the initial dissolved hydrogen concentration was about 1000 ppb.

[0200] As shown in Fig. 19, the results showed that in all trials, the hydrogen water group increased the "keratin water content" more than the warm water group. As shown in Fig. 20, the effect was confirmed in a short period (6 bath times) and became even greater in the long term (12 times). Also, as shown in Fig. 21, an effect was recognized on the water loss due to dry skin. Skin problems are considered to be caused in part by oxidative stress, and it is thought that using substances with antioxidant effects is effective. These test results suggest that the reducing action of hydrogen worked on the oxidative stress in the living body, and it can be said that they show the possibility of hydrogen bath for skin care.

[0201] Since ancient times, silk has been highly valued as a high-class clothing material. Silk has been mostly used as a fiber, but in recent years, edible silk peptides have been developed using silk as a raw material. Silk peptides are functional peptides obtained by enzymatic decomposition of silk, and it is known that they have various physiological activities when the molecular weight is between 300 and 5,000. By providing such silk peptides for food, it is said to have the effect of restoring the water evaporation amount and elasticity of the skin, improving weight gain and fat increase, and preventing obesity.

[0202] According to the publicly available information, in the "stress-induced skin damage test" using mice, it has been confirmed that silk peptides can improve skin elasticity and skin moisture retention. Also, in the "antioxidant test", peroxidase was added to silk peptides and the chemiluminescence inhibition rate was calculated. As a result, a strong antioxidant effect of silk peptides has been confirmed.

[0203] In addition, as an "immune activation test" using mice, a test has been published in which sheep red blood cell suspension was administered intraperitoneally to mice, the spleen was removed 4 days later, a spleen cell suspension was prepared, silk peptides were added to and cultured in this suspension, and the amount of antibodies against sheep red blood cells produced in the culture solution was measured. As a result of this test, it has been confirmed that silk peptides have the effect of increasing the amount of antibody production, and the consideration that they exhibit an immune activation effect has been obtained.

[0204] Also, in the "cytokine production test" using mice, it has been confirmed that silk peptides specifically and strongly enhance the production of IL-2, IL-4, and IFN-γ by the stimulation of "Concanavalin A". As a result, activation of lymphocyte cells in vivo, prevention of viral and bacterial infections, and antitumor effects are expected. Furthermore, it is known that IL-4 and IFN-γ of cytokines have the function of suppressing the differentiation of osteoclasts, and prevention of osteoporosis is also expected by these.

[0205] Furthermore, silk peptides have been confirmed to have the effects of improving obesity, preventing and improving diabetes, and suppressing cancer development caused by carcinogens. It has also been confirmed that there is no acute toxicity in the acute toxicity test and no subacute toxicity in the subacute toxicity test.

[0206] Regarding the structure of silk fibroin, for example, in silk fibroin with a diameter of about 20 μm, it is known that about 1000 fibrils are contained therein, and about 900 microfibrils are contained in one fibril. And one microfibril contains about 350 fibroin molecules, and one fibroin molecule is composed of about 4000 amino acid bonds. Silk fibroin having such a porous structure has a function of adsorbing food oils and fats and discharging them outside the body, and thus has attracted attention as a diet material.

[0207] On the other hand, reactive oxygen species generated in the human body, from the perspective of beauty, cause aging phenomena that occur on the skin such as spots and wrinkles, and from the perspective of internal medicine, are also said to be the cause of lifestyle-related diseases including diabetes. Such reactive oxygen species are generated just by breathing, and also from stress, excessive exercise, ultraviolet rays, radiation, overeating, smoking, etc. Such reactive oxygen species also have the role of decomposing and sterilizing bacteria and viruses, but if they increase too much in the body, they will attack themselves and oxidize cells, DNA, mitochondria, and blood vessels. In particular, hydroxy radical, a typical bad reactive oxygen species, is known to have particularly strong oxidizing power and cause various diseases, aging, and fatigue.

[0208] In order to remove such reactive oxygen species, "hydrogen water" is being consumed. It is said that hydrogen water filled in commercially available aluminum cans contains 0.16 mg to 0.32 mg (0.4 ppm to 0.8 ppm) of hydrogen per 410 mL at the time of shipment. However, hydrogen is a "light gas" that is very easy to escape and jump, and there are reports that even in the case of filling aluminum cans, the amount of dissolved hydrogen decreases significantly from the time of shipment after several months from production. Recently, although containers with low hydrogen escape have been developed, it is said that it is important to drink it all up within a few hours after opening.

[0209] The hydrogen-producing fermented lactic acid bacteria according to this embodiment may be configured to generate hydrogen by subjecting them to a special treatment using hydrogen and deep ocean water. In this embodiment, for example, silk fibroin may be used as a hydrogen-carrying material, or silk powder composed of silk peptides obtained by hydrolysis with an enzyme may also be used. Although various average molecular weights of the silk powder material have been proposed, in the present invention, it is not particularly limited. In this embodiment, various forms such as fibroin, peptides, and amino acids can be used as the raw material of the silk powder. Further, the silk powder may have a porous structure and has characteristics suitable for hydrogen carrying. By mixing dextrin having a cyclic structure such as cyclodextrin, it becomes easier to carry hydrogen. By adopting the production method of this embodiment, a large amount of hydrogen that has not been seen conventionally can be carried. Furthermore, the silk hydrogen pearl powder according to this embodiment can function, for example, as a "diet material" that adsorbs food fats and oils as silk fibroin, or can exhibit functions such as antioxidant action, skin beautifying action, improvement of immunity, and diet effect by silk peptides being decomposed and absorbed as amino acids.

[0210] As described above, for the pearl oyster-derived hydroxyapatite, it is desirable to use a naturally occurring and low-crystalline hydroxyapatite (Bioapatite: registered trademark). Hydroxyapatite accounts for about 97% of human tooth enamel and about 65% of bone, and is composed of phosphoric acid and calcium. It is said to have high biocompatibility, be neutral to weakly alkaline, and be safe for the human body. And since hydroxyapatite has almost the same components as tooth enamel, it has the function of remineralizing and repairing the damage formed on the tooth surface when a person eats or brushes their teeth, and the initial tooth decay due to demineralization.

[0211] [Verification Example Regarding Remineralization] The inventors conducted tests on remineralization. The teeth used in the tests were "human deciduous teeth that had fallen out", and the samples were (1) "hydroxyapatite containing only calcium raw materials" and (2) "Bioapatite (registered trademark)". The test method was to apply each sample to the surface of "human deciduous teeth that had fallen out", leave it overnight, then rinse it with tap water, brush the surface 10 times with a toothbrush, and wash it again with tap water. After drying, the surface was observed with an electron microscope.

[0212] Figures 7 to 9 are electron micrographs showing the results of the remineralization test. All are magnified 1500 times. Figure 7 shows the state before the test, Figure 8 shows the test results of "hydroxyapatite containing only calcium raw materials", and Figure 9 shows the test results of Bioapatite (registered trademark). As shown in Figure 7, there were fine grooves on the surface before the test. In Figure 8, the fine grooves remain, but in Figure 9, the fine grooves have disappeared. This indicates that Bioapatite (registered trademark) filled the fine grooves on the tooth surface. Thereby, it was confirmed that Bioapatite (registered trademark) is effective for tooth remineralization.

[0213] In addition, hydroxyapatite has 10 calcium atoms in one molecule, and these calcium atoms have adsorption characteristics by ion exchange. Due to such ion exchangeability, it becomes possible to carry a large amount of hydrogen and minerals contained in deep ocean water. Also, due to such adsorption characteristics, it is considered to function to adsorb pigments, bacteria, and lipid peroxides when blended in toothpaste and soap. Also, when ingested, it is expected to adsorb food fats and oils.

[0214] Generally, the commercially available hydroxyapatite is often synthesized from minerals (apatite) as raw materials. However, in this embodiment, it is low-crystalline apatite (Bioapatite: registered trademark) made from natural Akoya oyster (pearl oyster) shells as raw materials. The present invention is not limited to hydroxyapatite derived from Akoya oyster shells, and it is also possible to use hydroxyapatite derived from natural biological sources such as corals, fish scales and bones, mammalian bones such as cow bones, seaweeds, eggshells, sea urchin shells, clam shells, oyster shells, scallop shells, dolomite, weathered shells (Kamionishiki shells), etc. as raw materials. It is also possible to use hydroxyapatite derived from shells utilized in pearl matrices. In this case, for example, shells such as Kuromicho shells are suitable. Further, instead of hydroxyapatite derived from natural biological sources, hydroxyapatite derived from minerals may be used.

[0215] Natural biologically derived hydroxyapatite is said to have high biocompatibility in that it contains natural trace minerals such as magnesium and sodium in its apatite crystal structure, unlike hydroxyapatite derived from minerals. Furthermore, as will be described later, by adding deep ocean water, it becomes possible to carry natural ionized minerals other than calcium. Also, hydroxyapatite, whether it is derived from natural biological sources or from minerals, is considered to be able to supplement trace amounts of minerals to apatite and enhance biocompatibility by combining with reduced deep ocean water.

[0216] Natural bio-derived hydroxyapatite includes hydroxyapatite derived from natural pearl oyster, scallop shell-derived hydroxyapatite, coral-derived hydroxyapatite, eggshell-derived hydroxyapatite, and the like. And the low-crystalline natural bio-derived hydroxyapatite manufactured by the unique manufacturing method of Bioapatite (registered trademark) has all obtained the quasi-drug raw material standard, and is a high-purity product with a high apatite content ratio. For example, regarding eggshell-derived hydroxyapatite, when used once a day for one month during toothbrushing, it has been reported that the scale-like irregularities called "apatite columns" on the tooth surface were neatly filled with eggshell-derived hydroxyapatite, and the width of the cracks formed on the tooth surface also became narrower.

[0217] In addition, in this embodiment, natural-derived hydroxyapatite has been used for explanation, but in addition to this, it is also possible to use materials that can supply a large amount of calcium ions and phosphate ions. For example, weathered shell calcium powder, fossil coral powder, or dolomite powder can supply a large amount of calcium ions, so it is also possible to use any one of weathered shell calcium powder, fossil coral powder, or dolomite powder instead of natural-derived hydroxyapatite.

[0218] Weathered shell calcium is calcium made from weathered shell fossils produced in the Yakumo area of Hokkaido. Since "Nishiki shells" lived in the Yakumo area 15 to 20 million years ago, there is a sediment layer of these weathered shell fossils. These weathered shell fossils are calcium carbonate with a high purity of 95 to 97% and are effective as a source of calcium ions. In addition, fossil coral, for example, is calculated on Yonaguni Island in Okinawa Prefecture and contains about 70 kinds of minerals such as calcium, magnesium, and iron. Dolomite is a mineral derived from organisms formed after organisms such as coral deposited on the seabed and became limestone, and then a part of the calcium was replaced by magnesium in seawater. Such dolomite contains calcium and magnesium in a balance of 2 to 1. In this embodiment, by using any one of weathered shell calcium powder, fossil coral powder, or dolomite powder, the supply of a large amount of calcium ions is realized.

[0219] In this embodiment, for example, "Silk Hydrogen Pearl Powder" is used as the hydrogen generation powder. When this "Silk Hydrogen Pearl Powder" is dissolved in water, "hydrogen molecules" and "hydrogen ions with electrons" are generated. Some of the hydrogen molecules escape as hydrogen gas, but in the aqueous solution, some of the hydrogen molecules ionize, and there exist "positive hydrogen ions" and "negative hydrogen ions" as hydrogen ions with electrons. Since positive hydrogen ions do not have the ability to release electrons, they do not have reducing power, while negative hydrogen ions have the ability to release electrons and thus have reducing power. The "hydrogen ions" learned in junior high school and high school are "H+ (positive hydrogen ions)" in a state where one electron is lacking. This negative hydrogen ion reduces reactive oxygen species to produce harmless water. On the other hand, "antioxidants" such as vitamin C and polyphenols have the ability to remove reactive oxygen species. However, as a pro-oxidant effect, after removing reactive oxygen species, they themselves remain as "oxides" and may turn the human body into an oxidative constitution. On the other hand, hydrogen gas and negative hydrogen ions exhibit a reducing effect. After combining with reactive oxygen species, only harmless water is produced, and they do not remain as oxides themselves and have no pro-oxidant effect at all. Therefore, it can be said that they are the safest antioxidants without danger. That is, hydrogen, as the smallest atom in the universe, is regarded as one of the best antioxidants in the universe that pervades every corner of the human body. Also, it can be expected to have an effect (reducing effect) of recombining with once-oxidized antioxidants and returning to the state before oxidation. Such "hydrogen ions with electrons, also called negative hydrogen" are also being actively studied at the Tokyo Institute of Technology and Kyoto University. It can be expected that hydrogen gas and hydrogen ions with electrons act synergistically to exhibit an antioxidant function.

[0220] The inventors conducted a "human clinical trial" together with the "Japanese Society of Oxidative Therapy Medicine." A hybrid hydrogen supplement was created by mixing and filling "calcined coral calcium hydrogen powder" as a raw material for generating high concentrations of "electron-carrying hydrogen ions" with "marine mineral hydrogen powder" that utilizes deep seawater minerals as a raw material for generating high concentrations of hydrogen gas containing hydrogen ions. With this supplement, a statistically significant decrease was observed in the "average values ​​before and five days after ingestion" of the "8-OHdG creatinine ratio," a marker that indicates genetic damage in urine (oxidative stress in the body), just five days after starting to drink it. This revealed that this supplement can fully exert the "penetration power" of hydrogen gas and "electron-carrying hydrogen ions" in a short period of time.

[0221] In addition, the inventors measured the amount of dissolved hydrogen in the "calcined coral calcium hydrogen powder" over time in a test conducted at the request of the "Japan Hydrogen Water Promotion Association." As a result, the amount of dissolved hydrogen (redox method) was measured as 840 ppb after 10 minutes, 1,096 ppb after 2 hours, 1,085 ppb after 6 hours, and 1,045 ppb after 24 hours. In addition, at the Shiga Prefecture Northeast Industrial Technology Center, an investigation was conducted on the amount of hydrogen ion generation (inspection organization: Tres Bio Engineering LLC) using an ultraviolet-visible-near infrared spectrophotometer. The measurement principle is to measure the amount of hydrogen generated by the presence of hydrogen and calculate the amount of hydrogen. 10 mL of distilled water was added to 1 g of sample, stirred, and left to stand for 1 hour, and the supernatant was analyzed. As a result, it was found that the amount of hydrogen ions produced from 1 gram of powder was 3.55 x 10 to the power of 20 (3.55 sextillion), which is a level that can deliver 5.7 million hydrogen ions to each of the 60 trillion cells in the human body.

[0222] Furthermore, in addition to the confirmed long-term dissolution of hydrogen, the abundant generation of hydrogen ions was also confirmed. Based on these findings, it can be expected that the "fired coral calcium hydrogen powder" according to this embodiment can approach the intestine and the liver. It is said that about 90% of the body's active oxygen is generated in the intestine, and the liver is the largest detoxifying organ. The discovery of the potential for approaching both organs can be regarded as valuable data indicating the potential of hydrogen power. Additionally, the acetaldehyde elimination ability using an ultraviolet-visible near-infrared spectrometer at the Industrial Technology Center in the northeastern part of Shiga Prefecture was also investigated (testing agency: Tres Bio Co., Ltd.). The test was conducted by adding 0.2 g of the sample to 200 mL of shochu, stirring for 15 minutes, allowing it to stand, and then analyzing the supernatant. As a result, the aldehyde concentration decreased from 10 mg / L to 1.9 mg / L, and an 81% elimination rate was confirmed. The fact that acetaldehyde, which is also said to be the cause of hangover, is eliminated not only clarifies its effectiveness in countering hangovers but also is expected to contribute to maintaining liver health.

[0223] In addition, the inventors commissioned a third-party organization (Designer Foods Co., Ltd.) to verify the hydroxyl radical elimination function of the "fired coral calcium hydrogen powder" according to this embodiment. The verification method is as follows. Usually, it is consumed at 420 mg - 820 mg / 100 mL. Therefore, the fired coral calcium hydrogen powder was suspended in distilled water (32.8 mg / mL) and allowed to stand for about 1 hour to obtain a stock solution. For the measurement, (1) a 10-fold diluted solution of the stock solution and (2) the stock solution were prepared and finally added to the active oxygen generation system, resulting in dilutions of 40-fold and 4-fold (drinking concentration), respectively. The "hydroxyl radical generation method" involved generating it by irradiating hydrogen peroxide with ultraviolet light.

[0224] Next, the adjusted samples were collected in an ESR flat cell, and ESR measurements were performed under the following measurement conditions. Center Field: 335mT Modulation Width : 100μT Sweep Width:± 5.0mT Time Constant : 0.1sec Sweep Time : 1min Gain : 50

[0225] Next, as a hydroxyl radical scavenger, DMPO was used, and the hydroxyl radical scavenging activity of the sample was measured using the following protocol. Distilled water (control), solution (1) or (2): 50 μL 5.7 M DMPO: 20 μL 2.5 mM H2O2: 130 μL After irradiating with ultraviolet light for 30 seconds, it was measured by ESR.

[0226] In Fig. 22, from the signal intensity enclosed by the square of the obtained ESR spin adduct, with the control taken as 100%, the values obtained with each sample addition were evaluated as "% of control". The signal intensity of the spin adduct was analyzed from the ratio to the ESR signal of the external standard sample Mn 2+ (at the left end of Fig. 22). And statistical processing was carried out using individual data. The number of data for each condition was 2 and 3, and for the statistical processing, Tukey's analysis of variance was used, with a significance level of 5% or less.

[0227] The results showing the hydroxyl radical scavenging activity are as shown in Figs. 22 to 24. That is, compared with the control (a in Fig. 22), for sample (1) at 0.82 mg / mL (b in Fig. 22), 10.1% was scavenged, and for sample (2) at 8.2 mg / mL (c in Fig. 22), 72.1% was scavenged. Thus, in both cases, significant hydroxyl radical scavenging was observed (p < 0.05, Tukey multiple test).

[0228] In addition, it is considered effective to mix at least one of the above-mentioned "silk hydrogen pearl powder" and "fired coral calcium hydrogen powder" with the reducing fermented lactic acid bacteria according to this embodiment.

[0229] [Blending with Other Raw Materials] (A) Curcumin and mung bean sprout extract (green bean sprout extract) Curcumin is a yellow polyphenol compound contained in turmeric, etc. Mung bean sprout extract (green bean sprout extract) is also sold under the brand name Anagain (registered trademark) and is a water-soluble extract extracted from the new sprouts of mung beans. By blending these into the reduced fermented lactic acid bacteria powder according to this embodiment, hair beautifying effects, skin beautifying effects, and nail beautifying effects are expected. In particular, as described above, by combining with "silicon" and "hydrogen", synergistic effects such as strengthening tissues and increasing antioxidant action are expected. In addition, when commercializing, "coloring" using natural pigments may be used to enhance the product power.

[0230] (B) Rose petal powder, grape seed powder, hibiscus powder, cranberry extract, pomegranate, mangosteen, cherry blossom extract, melon extract Rose petal powder contains, for example, a lot of polyphenols derived from rose petals, has the function of preventing skin aging, suppressing the absorption of fat by diet, and suppressing post-meal blood glucose levels. It also has antioxidant activity exceeding that of vitamin C. Grape seed powder, hibiscus powder, and cranberry extract contain a lot of vitamin C and polyphenols, have antioxidant activity, and have the functions of preventing periodontal disease and gingivitis, anti-calculus action, and heart protection action. Pomegranate, mangosteen, and cherry blossom extract have high functions of suppressing glycation and suppressing oxidation, so these may be added. Melon extract is an antioxidant material that induces three antioxidant enzymes (SOD, glutathione peroxidase, catalase) in the living body and removes excessive reactive oxygen species, and can be expected to enhance the skin's defense ability against ultraviolet rays, so it may be added for the purpose of whitening care. Furthermore, by combining these with citric acid and hydrogen, the antioxidant and anti-glycation actions can be further enhanced. In addition, when commercializing, "coloring" using natural pigments may be used to enhance the product power. In addition, as a raw material that can be expected to enhance the skin's defense ability against ultraviolet rays, a "composite material of citrus and rosemary" may be further blended.

[0231] (C) Barley lactic acid fermentation extract, passion flower extract Barley lactic acid fermentation extract is rich in GABA (gamma-aminobutyric acid), and by blending it with passion flower extract that has the function of regulating biological rhythms, effects such as improving brain function like hormone balance stability, mental relaxation, and sound sleep are expected. Especially when combined with hydrogen, which is "a molecule as small as the universe that can even pass through the brain gate", a high synergistic effect is expected. In addition, when commercializing, the product power can be enhanced by "coloring" using natural pigments such as spirulina, gardenia, and butterfly pea.

[0232] (D) Fulvic acid, humic acid, deep ocean water minerals, Great Salt Lake minerals By combining fulvic acid, humic acid, deep ocean water minerals, Great Salt Lake minerals, iron, and the above-mentioned silicon, hydrogen, citric acid, etc., it is expected that when ingested by the human body, the mineral absorption efficiency will be improved, the tissues will become stronger, and the antioxidant effect will increase. That is, fulvic acid forms complexes with many metals in nature, and the complex with iron becomes iron fulvate, which occupies a large part of the transfer of iron to the ocean and there is much evidence showing the effect of promoting the growth of plants (including phytoplankton) and livestock. Also, fulvic acid is one of the organic acids present in forests and soil, and it plays the role (ion exchange) of promoting the circulation of nutrients such as minerals taken in by humans and animals through the food chain starting from phytoplankton and carried into the body. The Great Salt Lake is a saltwater lake located in the northern part of the state of Utah in the United States of America.

[0233] In recent years, a phenomenon called "rock burning" has occurred along the Sea of Japan coast in western Hokkaido, where the rock surface on the seabed turns completely white. When this phenomenon occurs, "seaweed and phytoplankton", which are at the bottom of the "food chain" shown in Figure 11, decrease. As a result, the coastal fish that feed on them disappear, having a serious impact on the fishing industry. One of the reasons is the relationship between deforestation and fulvic acid iron. Nitrogen is essential for the growth of algae and phytoplankton in the sea, and "iron ions" that act as a catalyst are required for this nitrogen absorption. Iron ions exist only in trace amounts in seawater, and iron ion deficiency occurs when the iron supply through rivers from the forest decreases. When considering the "iron ions" transported to the sea through rivers, "fulvic acid iron" becomes the keyword. In the forest, the fallen leaves and branches on the ground are decomposed by microorganisms, and fulvic acid is produced at that time, which binds to the iron in the humus soil to form "fulvic acid iron". Iron, in its ionic form, will oxidize into "iron particles" when it comes into contact with oxygen during transportation in the river. However, as shown in Figure 12, the iron ions combined with fulvic acid in the forest reach the sea downstream through the river "as iron ions" in the form of "fulvic acid iron". Fulvic acid iron plays an important role in the growth of phytoplankton and seaweed through nitrogen absorption.

[0234] "Fulvic acid" and "humic acid" are called humus substances, which are produced by the decomposition of organic substances, especially plants, and play a role in supplying minerals to plants. Both have the function of transporting minerals and amino acids with their chelating power (binding power) and discharging excessive minerals. Fulvic acid is soluble in acidic solutions and is said to have a very high rarity value. Humic acid is soluble in alkaline aqueous solutions, and dark-colored melanin pigments are concentrated. Thus, the reason why "humic acid" exhibits black color is mainly because plants such as wakame, nori, and tree leaves that grew in the shallows of ancient seas sank to the seabed and were thickly confined by sediments such as volcanic ash, and were decomposed into organic substances over a long period of hundreds of millions of years through aging. Humic acid is characterized by containing minerals that were bound to organic substances and does not deteriorate even when it comes to the surface of the earth.

[0235] In addition, for example, in order to enable the regeneration of Ariake Sea, a demonstration test of tidal flat purification using iron silicate fulvic acid has been conducted. It is also known that fulvic acid functions as a carrier that transports iron and water-soluble silicon from forests and mountains, and tidal flats in lakes and seas can be regenerated by iron silicate fulvic acid. Research on the utilization of such fulvic acid for the recovery of cell functions and skin beautification, improvement of atopic dermatitis and allergic constitution, vision recovery, hair growth, and enhancement of immunity, etc., through the ability to decompose harmful substances, including lifestyle diseases, is being promoted in the fields of agriculture, livestock, human drinking, and cosmetics. In addition, by adding and fermenting "deep sea water minerals, Great Salt Lake minerals, lactic acid bacteria", etc., which are reduced by hydrogen, to fulvic acid (referred to as reduced fermentation fulvic acid), the minerals are ionized in a reduced state, and it can be expected to have the effect of being easily absorbed by the body without being oxidized. When only fulvic acid is used, the color becomes brown granules. Therefore, in commercialization, in order to improve the visual impression and enhance the product power (functionality), for example, functional materials with evidence of diet effects, anti-glycation, anti-oxidation, antibacterial activity, etc., such as "rose petal powder, hibiscus powder, cranberry extract" in a dark pink color, can be blended so that it becomes a pink or wine-red drink when dissolved in water. Also, as a natural tomato lycopene extract containing lycopene in a certain proportion or more, together with natural compounds contained in tomatoes (such as tocopherol, phytoene, phytofluene, β-carotene, phospholipid, phytosterol, etc.), "tomato-derived lycopene extract powder" can be blended so that it becomes a red-orange drink when dissolved in water.

[0236] Generally, fulvic acid and humic acid are said to react with chlorine to produce trihalomethanes. In water treatment plants, there is a process of removing fulvic acid and humic acid using activated carbon. For this reason, raw material manufacturers of fulvic acid and humic acid sometimes prompt caution in product labeling, stating "There is a concern of generating trihalomethanes, so do not mix with tap water for drinking." Under such circumstances, the inventors of the present invention confirmed through research institutions that the reduced-fermented fulvic acid according to this embodiment does not generate trihalomethanes even when mixed with the chlorine concentration in tap water. This test measured how much total trihalomethanes are generated when the "reduced-fermented fulvic acid powder" according to this embodiment is mixed with tap water. The test method is as follows. First, since the chlorine concentration in tap water is about 0.4 ppm, in this test, sodium hypochlorite (commercially available "Hiter") was diluted and adjusted to have a chlorine concentration of 0.5 ppm to obtain chlorine water. Next, 0.2 g of the "reduced-fermented fulvic acid powder" as a sample was dissolved in 200 mL of the above chlorine water, sealed, and various trihalomethanes were measured by the measurement method defined in "6.1" of "JIS S3201 (Test Method for Household Water Purifiers)" (outsourced to an external analysis). The results are as shown in the following table.

Table 54

[0237] In addition, the inventors of the present invention conducted a comparative test between "reduced fermented fulvic acid powder" and "fulvic acid powder" through a research institution. Here, "reduced fermented fulvic acid powder" is a fulvic acid powder containing "deep sea water minerals and Great Salt Lake minerals" and "lactic acid bacteria" that have been reduced and fermented by hydrogen, and "fulvic acid powder" refers to the fulvic acid powder before the reduction process. The reduced fermented fulvic acid powder according to the present embodiment utilizes high-quality fulvic acid powder produced in Canada, but it is also possible to use that produced in the state of Utah, United States of America.

[0238] (D1) Verification example regarding antioxidant property (SOD-like activity) "Reduced fermented fulvic acid powder" and "fulvic acid powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain stock solutions. Using the stock solutions and the dilution buffer attached to the "SOD Assay Kit-WST (Dojindo Laboratories)", measurements were performed on the solutions diluted 10-fold and 100-fold using the "SOD Assay Kit-WST (Dojindo Laboratories)" according to the operation procedure. The measurement method is as follows. That is, using a 96-well microplate, the superoxide generated by the enzyme reaction was colored with the tetrazolium salt WST-1, and the absorbance at 540 nm was measured. Then, the inhibition rate of superoxide generation when the sample was added was calculated from the change in absorbance and taken as the SOD-like activity. The results are as shown in the following table, indicating that the higher the SOD-like activity, the higher the antioxidant property. [Table 55]

[0239] Next, for comparison, the antioxidant properties of fulvic acids manufactured by other companies were measured. Among the provided samples, for the fulvic acid powder manufactured by Company B and the fulvic acid extract manufactured by Company E, those dissolved in pure water and prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those provided and filtered through a 0.45 μm filter were used as the stock solutions. For the stock solutions and the solutions diluted 100-fold and 200-fold with the buffer solution attached to the SOD Assay Kit-WST (manufactured by Dojindo Laboratories), the inhibition rate was calculated according to a predetermined method using the SOD Assay Kit-WST. The results are as follows.

Table 56

[0240] Thus, it was shown that the collaboration between fulvic acid and "deep sea water minerals and Great Salt Lake minerals" reduced and processed by hydrogen enhances the power of reduced fermented fulvic acid compared to mere fulvic acid. That is, the "reduced fermented fulvic acid powder" has a high inhibition rate in the stock solution. Also, even when diluted 10-fold and 100-fold, it has a sufficiently high inhibition rate, so it is expected to have a sufficient effect even when diluted, and it was found that sufficiently high antioxidant properties can be obtained from a practical perspective. In particular, compared with fulvic acid solutions (or powders / extracts) manufactured by other companies, the "reduced fermented fulvic acid powder" according to this embodiment was found to be outstandingly excellent. Thereby, an antioxidant effect on the body by active oxygen can be expected.

[0241] (D2) Verification Example for α-Glucosidase Inhibitory Activity "Reductive fermentation fulvic acid powder" and "fulvic acid powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. Using the stock solution and pure water, solutions diluted 10-fold and 100-fold were measured for α-glucosidase inhibitory activity. The measurement method is as follows. That is, a solution obtained by dissolving "7 mM p-nitrophenyl-α-D-glucopyranoside" in 50 mM phosphate buffer (pH 7.0) was used as the substrate solution, and a solution obtained by dissolving α-glucosidase at 0.9 U / mL in 50 mM phosphate buffer (pH 7.0) was used as the enzyme solution. Using a microtube, 10 μL of the sample solution and 40 μL of the enzyme solution were added, incubated at 37°C for 5 minutes, then 950 μL of the substrate solution was added, and incubated at 37°C for 15 minutes. Then, 1000 μL of 0.5 M Tirs solution was added to stop the reaction. The absorbance at 405 nm of p-nitrophenol released and decomposed by this reaction was measured. And the inhibition rate of p-nitrophenol production when the sample was added was calculated from the decrease from the absorbance value without the sample addition, and was taken as the α-glucosidase inhibitory activity. The results are as shown in the following table, indicating that the higher the α-glucosidase inhibitory activity, the higher the blood glucose level increase inhibitory effect.

Table 57

[0242] Next, for comparison, the α-glucosidase inhibitory activity of fulvic acid manufactured by other companies was measured. For the fulvic acid powder manufactured by Company B and the fulvic acid extract manufactured by Company E, those dissolved in pure water, prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those provided and filtered through a 0.45 μm filter were used as the stock solutions. The stock solutions and the solutions diluted 10-fold and 100-fold with pure water were used as the measurement samples. 7 mM p-nitrophenyl-α-D-glucopyranoside dissolved in 50 mM phosphate buffer (pH 7.0) was used as the substrate solution, and α-glucosidase dissolved at 0.9 U / mL in 50 mM phosphate buffer (A) was used as the enzyme solution. Using a microtube, 10 μL of the measurement sample and 40 μL of the enzyme solution were added, incubated at 37 °C for 5 minutes, then 950 μL of the substrate solution was added, and incubated at 37 °C for 15 minutes. Then, 1000 μL of 0.5 M Tirs solution was added to stop the reaction, and the absorbance at 405 nm was measured. The α-glucosidase inhibitory activity when the sample was added was calculated from the change in absorbance and taken as the α-glucosidase inhibitory activity. The results are as shown in the following table.

Table 58

[0243] Thus, it was shown that the power of reduced fermentation fulvic acid is enhanced compared to mere fulvic acid through the collaboration of fulvic acid and "deep ocean water minerals and Great Salt Lake minerals" reduced by hydrogen. That is, because of its high α-glucosidase inhibitory activity function, it is considered that in vivo, it can inhibit α-glucosidase activity, inhibit sugar absorption, and avoid a state of high blood sugar. If a state of high blood sugar can be avoided, it becomes possible to make it a state where pre-AGEs generation reactants are not easily generated. In particular, compared with fulvic acid solutions (or powders / extracts) manufactured by other companies, the "reduced fermentation fulvic acid powder" according to this embodiment was found to be outstanding.

[0244] (D3) Verification Example for Tyrosinase Inhibitory Activity "Reduction-Fermented Fulvic Acid Powder" and "Fulvic Acid Powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with pure water were measured for tyrosinase inhibitory activity. The measurement method is as follows. That is, mushroom-derived tyrosinase dissolved in 0.1 M phosphate buffer (pH 6.7) at 300 units / mL was used as the enzyme solution, and L-DOPA dissolved in 0.1 M phosphate buffer (pH 6.7) at 1 mM was used as the substrate solution. The measurement method is as follows. That is, using a 96-well microplate, 25 μL of the sample solution and 100 μL of the enzyme solution were added, incubated at 37 °C for 10 minutes, then 125 μL of the substrate solution was added, and incubated at 37 °C for 30 minutes. Then, the absorbance at 490 nm was measured. And the melanin production inhibition rate when the sample was added was calculated from the change in absorbance and used as the tyrosinase inhibitory activity. The results are as shown in the following table.

Table 59

[0245] Next, for comparison, the tyrosinase inhibitory activity of fulvic acid produced by other companies was measured. Among the provided samples, for the fulvic acid powder manufactured by Company B and the fulvic acid extract manufactured by Company E, those dissolved in pure water, prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those filtered through a 0.45 μm filter as provided were used as the stock solutions. The stock solutions and the solutions diluted 10-fold and 100-fold with pure water were used as the measurement samples. Mushroom-derived tyrosinase dissolved in 0.1 M phosphate buffer (pH 6.7) at 300 units / mL was used as the enzyme solution, and L-DOPA dissolved in 0.1 M phosphate buffer (pH 6.7) at 1 mM was used as the substrate solution. Using a 96-well microplate, 25 μL of the measurement sample and 100 μL of the enzyme solution were added and incubated at 37 °C for 10 minutes. Then, 125 μL of the substrate solution was added, incubated at 37 °C for 30 minutes, and the absorbance at 490 nm was measured. The tyrosinase inhibition rate when the sample was added was calculated from the change in absorbance and used as the tyrosinase inhibitory activity. The results are as shown in the following table.

Table 60

[0246] (D4) Verification example for hyaluronidase inhibitory activity "Reductive fermented fulvic acid powder" and "fulvic acid powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with pure water were measured for hyaluronidase inhibitory activity. The measurement method is as follows. Bovine-derived hyaluronidase dissolved in 0.1 M acetate buffer (pH 4.0) at 400 units / mL was used as the enzyme solution. Potassium hyaluronate dissolved in acetate buffer at 1 mg / mL was used as the substrate solution. Compound 48 / 80 dissolved in acetate buffer at 0.1 mg / mL was used as the enzyme activator. p-Dimethylaminobenzaldehyde (p-DABA) dissolved in a hydrochloric acid-acetate mixture at 0.1 g / mL was used as the color-developing solution and diluted 10-fold with acetic acid immediately before use. For the measurement, 12 μL of the sample solution and 12 μL of the enzyme solution were placed in a microtube and incubated at 40 °C for 20 minutes. Then, 12 μL of the enzyme activator was added and incubated at 40 °C for 20 minutes. Further, 12 μL of the substrate solution was added and incubated at 40 °C for 40 minutes. Thereafter, 12 μL of 0.4 N NaOH aqueous solution was added to stop the reaction, immediately ice-cooled for 5 minutes, 12 μL of 0.8 M borate buffer (pH 9.0) was added, boiled for 3 minutes, and then further ice-cooled for 10 minutes. Then, 180 μL of the color-developing solution was added, incubated at 40 °C for 30 minutes, and the absorbance at 585 nm was measured. When the activity of hyaluronidase is inhibited upon addition of the sample, N-acetylglucosamine, which is a degradation product of hyaluronic acid, decreases, and the absorbance due to p-DABA decreases. The change in absorbance was calculated and used as the hyaluronidase inhibitory activity. The results are as shown in the following table.

Table 61

[0247] Next, for comparison, the hyaluronidase inhibitory activity of fullvoic acid manufactured by other companies was measured. Among the provided samples, for the fullvoic acid powder manufactured by Company B and the fullvoic acid extract manufactured by Company E, those dissolved in pure water, prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those filtered through a 0.45 μm filter as provided were used as the stock solutions. The stock solutions and the solutions diluted 10-fold and 100-fold with pure water were used as the measurement samples. Bovine-derived hyaluronidase dissolved in 0.1 M acetate buffer (pH 4.0) at 400 units / mL was used as the enzyme solution. Potassium hyaluronate dissolved in acetate buffer (pH 4.0) at 1 mg / mL was used as the substrate solution. Compund48 / 80 dissolved in acetate buffer (pH 4.0) at 0.1 mg / mL was used as the enzyme activator. p-dimethylaminobenzaldehyde (p-DABA) dissolved in a hydrochloric acid-acetate mixed solution at 0.1 g / mL was used as the color-developing solution, and it was diluted 10-fold with acetic acid immediately before use. 12 μL of the sample solution and 12 μL of the enzyme solution were placed in a microtube and incubated at 40 °C for 20 minutes. Then, 12 μL of the enzyme activator was added and incubated at 40 °C for 20 minutes. Further, 12 μL of the substrate solution was added and incubated at 40 °C for 40 minutes. Thereafter, 12 μL of 0.4 N NaOH aqueous solution was added to stop the reaction, immediately ice-cooled for 5 minutes, 12 μL of 0.8 M borate buffer (pH 9.0) was added, boiled for 3 minutes, and then further ice-cooled for 10 minutes. 180 μL of the color-developing solution was added thereto, incubated at 40 °C for 30 minutes, and then the absorbance at 585 nm was measured. The hyaluronidase inhibitory activity when the sample was added was calculated from the change in absorbance and taken as the hyaluronidase inhibitory activity. The results are as shown in the following table. [Table 62] Note that in the above table, negative numerical values are equivalent to 0.

[0248] Thus, it has been shown that the power of reduced fermented fulvic acid is enhanced compared to mere fulvic acid through the collaboration of fulvic acid and "deep sea water minerals and Great Salt Lake minerals" reduced by hydrogen. Hyaluronic acid is abundant between cells and is said to protect cells through its water retention and buffering functions. However, hyaluronidase is an "enzyme that decomposes hyaluronic acid" that increases due to aging, ultraviolet stimulation, etc. If there is a large amount of this enzyme, it may become impossible to maintain the skin's moisture retention and viscoelasticity, leading to the formation of wrinkles and sagging of the skin. The "reduced fermented fulvic acid powder" according to this embodiment has a high hyaluronidase inhibitory activity in its undiluted solution. Also, even when diluted 10 times or 100 times, it still has a sufficiently high hyaluronidase inhibitory activity. Therefore, even when diluted, sufficient effects are expected, and it has been found that a sufficiently high hyaluronidase inhibitory activity function can be obtained from a practical perspective. In particular, when compared with fulvic acid solutions (or powders / extracts) manufactured by other companies, the "reduced fermented fulvic acid powder" according to this embodiment has been found to be outstandingly excellent. As a result, it is expected that hyaluronic acid will be less likely to be decomposed, the skin's moisture retention and viscoelasticity will be maintained, and wrinkles and sagging of the skin will be prevented.

[0249] (D5) Verification Example Regarding Collagenase Inhibitory Activity "Reductive fermented fulvic acid powder" and "fulvic acid powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. Using the stock solution and pure water, solutions diluted 10-fold and 100-fold were measured for collagenase inhibitory activity. The measurement method is as follows. Pz-peptide dissolved in 0.1 M Tris buffer (pH 7.1) at 0.39 mg / mL was used as the substrate solution, and collagenase Type IV dissolved in pure water at 0.1 mg / mL was used as the enzyme solution. 25 μL of the sample solution, 25 μL of the enzyme solution, and 200 μL of the substrate solution were placed in a glass test tube, incubated at 37 °C for 30 minutes, then 250 μL of 25 mM citric acid was added to stop the reaction. Thereafter, 2500 μL of ethyl acetate was added and shaken for extraction, followed by centrifugation (1500 rpm, 5 minutes), and the upper layer (ethyl acetate layer) was collected and the absorbance at 320 nm was measured. The collagenase inhibitory activity when the sample was added was calculated from the change in absorbance and taken as the collagenase inhibitory activity. The test results are as shown in the following table.

Table 63

[0250] Next, for comparison, the collagenase inhibitory activity of fulvic acid manufactured by other companies was measured. Among the provided samples, for the fulvic acid powder manufactured by Company B and the fulvic acid extract manufactured by Company E, those dissolved in pure water, prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those provided and filtered through a 0.45 μm filter were used as the stock solutions. The stock solutions and the solutions diluted 10-fold and 100-fold with pure water were used as the measurement samples. A solution prepared by dissolving Pz-peptide at 0.39 mg / mL in 0.1 M Tris buffer (pH 7.1) was used as the substrate solution, and a solution prepared by dissolving collagenase Type IV at 0.1 mg / mL in pure water was used as the enzyme solution. 25 μL of the sample solution, 25 μL of the enzyme solution, and 200 μL of the substrate solution were placed in a glass test tube, incubated at 37°C for 30 minutes, and then 250 μL of 25 mM citric acid was added to stop the reaction. Thereafter, 2500 μL of ethyl acetate was added and shaken for extraction, followed by centrifugation (1500 rpm, 5 minutes), the upper layer (ethyl acetate layer) was collected, and the absorbance at 320 nm was measured. The collagenase inhibitory activity when the sample was added was calculated from the change in absorbance and used as the collagenase inhibitory activity. The results are as shown in the following table.

Table 64

[0251] Thus, it was shown that the power of reduced fermentation fulvic acid is enhanced compared to mere fulvic acid through the collaboration of fulvic acid and "deep sea water minerals and Great Salt Lake minerals" reductively processed with hydrogen. That is, it became clear that the "reduced fermentation fulvic acid powder" has the function of inhibiting collagenase that decomposes collagen. In particular, it was found that the "reduced fermentation fulvic acid powder" according to this embodiment is at a comparable level even when compared with fulvic acid solutions (or powders / extracts) manufactured by other companies. As a result, it is expected that by ingesting or applying the "reduced fermentation fulvic acid powder", the collagen in the skin will be less likely to be decomposed.

[0252] (D6) Verification Example of Elastase Inhibitory Activity "Reduced Fermented Fulvic Acid Powder" and "Fulvic Acid Powder" were dissolved in pure water at a concentration of 1 mg / mL and filtered through a 0.45 μm filter to obtain the stock solution. The stock solution and the solutions diluted 10-fold and 100-fold with pure water were measured for elastase inhibitory activity. The measurement method is as follows. That is, STANA (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) dissolved in 50 mM Tris buffer (pH 8.8) at 1 mM was used as the substrate solution, and porcine pancreatic elastase dissolved in 50 mM Tris buffer (pH 8.8) at 0.5 units / mL was used as the enzyme solution. Using a 96-well microplate, 50 μL of the sample solution, 50 μL of the enzyme solution, and 100 μL of the substrate solution were added, incubated at 37 °C for 30 minutes, and then the absorbance at 405 nm was measured. The elastase inhibitory activity when the sample was added was calculated from the change in absorbance and used as the elastase inhibitory activity. The test results are as shown in the following table.

Table 65

[0253] Next, for comparison, the elastase inhibitory activity of fulvic acid produced by other companies was measured. Among the provided samples, for the fulvic acid powder manufactured by Company B and the fulvic acid extract manufactured by Company E, those dissolved in pure water, prepared to 1 mg / mL, and filtered through a 0.45 μm filter were used as the stock solutions. For the other samples, those filtered through a 0.45 μm filter from what was provided were used as the stock solutions. The stock solutions and the solutions diluted 10-fold and 100-fold with pure water were used as the measurement samples. STANA (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) dissolved in 50 mM Tris buffer (pH 8.8) at 1 mM was used as the substrate solution, and porcine pancreatic elastase dissolved in 50 mM Tris buffer (pH 8.8) at 0.5 units / mL was used as the enzyme solution. Using a 96-well microplate, 50 μL of the measurement sample, 50 μL of the enzyme solution, and 100 μL of the substrate solution were added, incubated at 37 °C for 30 minutes, and then the absorbance at 405 nm was measured. The elastase inhibitory activity when the sample was added was calculated from the change in absorbance and used as the elastase inhibitory activity. The results are as shown in the following table.

Table 66

[0254] Thus, it was shown that the power of reduced fermentation fulvic acid is enhanced compared to mere fulvic acid through the collaboration of fulvic acid and "deep ocean water minerals and Great Salt Lake minerals" reduced by hydrogen. That is, it became clear that the "reduced fermentation fulvic acid powder" has the function of inhibiting elastase that decomposes elastin. In particular, it was found that the "reduced fermentation fulvic acid powder" according to this embodiment is remarkably excellent even when compared with fulvic acid solutions (or powders / extracts) manufactured by other companies. Thereby, it is expected that by ingesting or applying the "reduced fermentation fulvic acid powder", the elastin in the skin will be less likely to be decomposed.

[0255] (D7) Verification Example of Anti-Aging Effect Using Nematodes The inventors focused on the effect of the reduced fermented fulvic acid powder according to this embodiment on the average lifespan of nematodes and conducted an examination based on the test results by a third party. The test method in this embodiment is as follows. That is, the Escherichia coli OP-50 strain used as food was suspended in the "S-medium" shown in the following table to obtain a culture solution of nematodes (fer-15 mutant strain). Using this culture solution, the nematodes were cultured (at 20°C, 100 rpm) until they became adults. Then, the nematodes were collected and washed with the "S-buffer" shown in the following table, and the bodies of the nematodes were dissolved using a NaOH solution and Hiter (registered trademark) to recover eggs from inside the body. The recovered eggs were cultured overnight at 20°C and hatched. The hatched L1 larvae were synchronously cultured at 26.5°C using heat-treated Escherichia coli OP-50 strain (dead cells) as food in a 24-well microplate. At this time, the number of nematodes per well of the microplate was adjusted to about 20. A sample stock solution was prepared by dissolving the reduced fermented fulvic acid powder in pure water at a concentration of 10 mg / mL. After egg collection, on the 4th day, the stock solution was filter-sterilized through a 0.22-μm filter, and the sample stock solution was added to each well of the microplate so that the final concentration in the culture solution was "1 mg / mL" for 10-fold dilution, "0.1 mg / mL" for 100-fold dilution, and "0.01 mg / mL" for 1000-fold dilution. Then, the survival rate of the nematodes was examined under a microscope every few days. Using the "Kaplan Meier method" with the number of survivors on the 4th day as 100%, a survival rate curve was drawn, and a p-value was calculated by the log-rank test. Also, the average lifespan was calculated from the number of survival days. The results are as follows.

Table 67

Table 68

Table 69

[0256] (E) Combination of red orange extract, pineapple enzyme, papaya enzyme, hydrogen, and deep ocean water The red orange extract refers to an extract powder obtained by extracting three types of red oranges (Moro, Sanguinello, Tarocco varieties) native to Sicily, Italy. When combined with hydrogen and deep ocean water, it is expected to have anti-glycation, anti-oxidation, protection against ultraviolet rays, and fat-burning effects. Additionally, by further adding proteolytic enzymes such as pineapple enzyme and papaya enzyme, the ability to break down proteins into amino acids is enhanced, and it is expected to "improve the intestinal environment by preventing protein spoilage" and have a dieting effect. For commercialization, it may be possible to enhance the product's appeal by "coloring" using natural pigments.

[0257] Furthermore, for various proteins such as soy protein and pea-derived protein, when combined with proteolytic enzymes such as pineapple enzyme, papaya enzyme, and green papaya enzyme to increase the conversion and absorption efficiency into amino acids, and further combined with functional edible charcoal, reduced fermented lactic acid bacteria, hydrogen, deep ocean water, etc., excellent collaboration can be expected. Along with efficient intake of proteins and amino acids during exercise and dieting, it can adsorb and excrete intestinal harmful substances, intestinal harmful bacteria, reactive oxygen species, AGEs (advanced glycation end products), etc., and promote intestinal cleansing.

[0258] (F) Functional coconut shell activated carbon "Functional coconut shell activated carbon particles (dispersion processing)" (hereinafter referred to as "coconut shell activated carbon") is a water-dispersible and porous preparation, and thus is expected to function as a coating agent for bacteria and the like. The inventors immersed three types of bacteria in dilute hydrochloric acid solution regarded as gastric acid, measured the number of bacteria with and without mixing coconut shell activated carbon, and measured the difference in its coating power. The test method is as follows. That is, based on the colonies of various bacteria, one colony is suspended in sterilized physiological saline to obtain the original bacterial solution. The original bacterial solution is divided into a group with carbon added and a non-added group, and after adding dilute hydrochloric acid to each, it is left standing. For C. butyruicum (butyric acid bacteria) and B. pseudolongum (Bifidobacterium), GAM medium was used, and for L. acidophilus (lactic acid bacteria), after spreading on LGB medium, anaerobic culture was performed and the number of bacteria was measured. The results are as shown in the following table.

[0259]

Table 70

[0260] Thus, in each test group, the number of viable bacteria in the coconut shell activated carbon administration group was detected to be larger compared to the non-added group. This suggests that by suspending dispersible coconut shell activated carbon in water, it has the function as a carrier to maintain the number of viable bacteria even in a pseudo-gastric acid environment. From this, the reduced fermented lactic acid bacteria according to this embodiment may be viable bacteria, and in particular, when containing coconut shell activated carbon, a synergistic effect with yogurt containing viable bacteria of lactic acid bacteria, Bifidobacterium, and butyric acid bacteria can be expected.

[0261] Next, the caffeine adsorption test of "functional coconut shell activated carbon powder" and "Kishu Binchotan activated carbon powder" will be described. The test method is as follows. Caffeine was adjusted to 600 ppm, 1 g of "functional coconut shell activated carbon powder" and "Kishu Binchotan activated carbon powder" were respectively added to 100 mL of a 600 ppm caffeine solution, stirred with a magnetic stirrer for 3 minutes, and then filtered through filter paper (Advantec No. 2). Next, the concentration was measured by liquid chromatography. The results are as shown in the following table.

[0262]

Table 71

[0263] Next, the acrylamide adsorption test of "functional coconut shell activated carbon powder" and "Ina Akamatsu Myo-charcoal powder" will be described. The test method is as follows. Dissolve 30 mg of acrylamide in 200 mL of distilled water, and add 1 g of each sample to 45 mL of the acrylamide aqueous solution. Next, after stirring for 30 minutes, centrifuge (8000 rpm: 10 minutes), filter the supernatant through a filter, and measure the concentration with a TOC meter (TOC-V csn (total organic carbon meter) manufactured by SHIMADZU Corporation). The results are as shown in the following table.

[0264]

Table 72

[0265] Next, the phenol adsorption test of "Kishu Binchotan Activated Carbon Granules (Dispersed Processing)" will be described. The test method is as follows. Phenol was adjusted to 15 ppm, and 100 mL of this was taken. Then, 1 g of the sample (Kishu Binchotan Activated Carbon Granules (Dispersed Processing)) was added, and it was stirred for 10 minutes with a magnetic stirrer. After filtration, analysis was performed. The results are as shown in the following table.

Table 73

[0266] Next, the bile acid adsorption test using functional coconut shell activated carbon will be described. The inventors of the present invention confirmed the bile acid adsorption effect of functional coconut shell activated carbon. In a test conducted by a third-party institution using reagents by the enzyme method, the adsorption rate of functional coconut shell activated carbon was 97%. Bile acids are produced from bad cholesterol in the liver and play a role in further subdividing and absorbing into the body "oil emulsified by the digestive enzyme lipase". Since about 95% of bile acids are absorbed from the intestinal tract together with oil and returned to the liver for reuse (referred to as enterohepatic circulation), when the synthesis of new bile acids in the liver using bad cholesterol does not progress and the concentration of bad cholesterol in the blood increases, a vicious cycle occurs. That is, it can be said that the situation is such that old engine oil circulates in the body. In addition, Mitsuhiro Watanabe, a professor at the Graduate School of Policy and Media Studies, Keio University (also a professor at the Faculty of Environmental Information and a professor at the Faculty of Medicine), and others have jointly elucidated the mechanism by which metabolic syndrome is improved by bile acid regulation with a research group at the Swiss École Polytechnique Fédérale de Lausanne (Professor Johan Auwerx). The results of this study have been published in "PLos ONE" and "Nature Scientific Reports" respectively. The papers report that by eating bile acid adsorbing materials such as konjac and excreting bile acids from feces, the risk of diabetes can be reduced and the blood cholesterol concentration can be decreased. It is said that when suffering from both diseases of hyperglycemia and hypercholesterolemia, the risk of vascular diseases such as cerebral infarction and myocardial infarction increases by about 5 times. The number of diabetes patients in Japan is estimated to be about 8.9 million, the number of people in the pre-diabetes group is about 13.2 million, and the number of people with hypercholesterolemia is about 25 million. Early treatment intervention for diabetes and borderline cases in hypercholesterolemia has become an important issue in the future of the global medical field. In Japan, with the penetration of Western food, colorectal cancer accounts for a large proportion of the causes of death and ranks first among cancer causes of death in women. By consuming Western food rich in oil, a large amount of bile acids are secreted. Although about 95% of them are absorbed into the body together with oil, the remaining about 5% migrates to the large intestine. It reacts with bad bacteria in the large intestine and changes into a powerful carcinogenic substance, "secondary bile acid", which is said to be the main cause of colorectal cancer.Functional edible charcoal, including functional coconut shell activated carbon, can adsorb and excrete bile acids, and reduction-fermentation lactic acid bacteria can guide the elimination of harmful bacteria, so synergistic effects such as reducing bad cholesterol levels, reducing the risk of diabetes, and reducing the risk of colorectal cancer can be expected.

[0267] (G) Chlorogenic acid It is also possible to incorporate chlorogenic acid contained in coffee into the reduction-fermentation lactic acid bacteria according to this embodiment. The health effects of coffee are obtained from the action of chlorogenic acid, and it is known that it is contained in a large amount in lightly roasted coffee beans. In addition, products containing chlorogenic acid have also been provided on the market for the purpose of improving hypertension. Chlorogenic acid is also known to have the functions of "inhibiting the action of sugar-degrading enzymes (digestive enzymes)", "suppressing the transfer of sugar from the digestive tract to the blood", and "promoting the production of the hormone GLP-1 that suppresses the rise in blood sugar levels provided from the digestive tract". However, most commercially available coffees widely spread in the world are dark-roasted to hide differences in coffee bean origins and quality deterioration. As oxidation progresses, chlorogenic acid is greatly reduced, the sour taste of citric acid is lost, while the carcinogenic substance acrylamide is generated and reported to be harmful to health. Although a certain amount of caffeine also activates the body and is considered effective, excessive intake requires caution as it may cause side effects.

[0268] (H) Hydrocharcoal (a carbon-containing composition combining at least one of activated carbon or edible charcoal, inulin, powder of Sargassum thunbergii, and citric acid) It is also possible to incorporate "Hydrochacol", a carbon-containing composition in which at least one of activated carbon or edible charcoal, inulin, kombu powder, and citric acid are combined, into the reducing fermented lactic acid bacteria according to this embodiment. In particular, activated carbon (functional edible charcoal including functional coconut shell activated carbon) has functions of highly adsorbing oral bacteria, synthetic coloring agents "tar dyes", heavy metals, purine substances, AGEs, acrylamide, nicotine, and tar, highly adsorbing malondialdehyde of lipid peroxides and food oils and fats, and highly adsorbing trihalomethane and residual chlorine. Furthermore, by suspending it in water, it has a protective and transport function of delivering useful microorganisms such as lactic acid bacteria alive to the intestine, that is, a function as a carrier for maintaining the viable cell count. In addition to kombu, seaweed-derived materials such as wakame, mekabu, ganashi, and dulce may be used.

[0269] Also, the Hydrochacol according to this embodiment may contain inulin. Inulin is a kind of polysaccharide produced by plants of the Asteraceae family and has been increasingly used in foods in recent years because of its excellent nutritional value. That is, although inulin belongs to saccharides such as sugar and starch, humans do not have an enzyme to decompose inulin, so when ingesting foods containing inulin, it is hardly absorbed and is excreted outside the body. Therefore, inulin is classified as a water-soluble dietary fiber and is known to become fructooligosaccharide when fermented and decomposed in the intestine. Inulin becomes gel-like when it absorbs water in the intestine and has a function of suppressing the absorption of carbohydrates ingested together. In addition, since it serves as food for good bacteria in the intestine, it has an effect of regulating the intestinal environment and is widely used in diet foods and the like. When humans ingest inulin, an improvement in the intestinal environment and an effect of suppressing an increase in blood glucose level and cholesterol are expected.

[0270] In addition, when used on the human body, Ecklonia kurome powder is expected to have a hair growth effect. That is, in recent years, it has been scientifically elucidated that a component called "fucoidan" contained in seaweeds contributes to hair growth. "Fucoidan" can be extracted from multiple different seaweeds such as kelp, wakame, and mozuku, but in particular, it has been found that the fucoidan of Ecklonia kurome exhibits a high hair growth effect. Ecklonia kurome is a type of kelp that can only be harvested in some limited areas of Hokkaido. The Ecklonia kurome fucoidan extracted from this kelp is said to have a much higher hair growth effect than fucoidan that can be extracted from other seaweeds. More specifically, the foreshore of Minamikayaabe in Hakodate, which is the production area of Ecklonia kurome, is shallow and has high illuminance, and the cold and warm ocean currents converge, providing a water temperature suitable for the growth of kelp. In addition, it has a favorable acidic rock terrain rich in silicon, nutrient salts from broad-leaved forests, and mineral fulvic acid iron flowing in from 30 rivers of various sizes, and these favorable conditions are said to cultivate high-quality kelp.

[0271] According to recent research, Ecklonia kurome contains three types of fucoidan, namely "F-fucoidan", "U-fucoidan", and "G-fucoidan", and among them, it has been found that "F-fucoidan" has a particularly strong hair growth effect. Ecklonia kurome fucoidan is said to increase the production of hair growth factors and promote the proliferation of hair matrix cells. This growth factor is a type of protein called "FGF-7", which is said to extend the growth phase of the human hair cycle and prolong the hair growth period. Since hair can grow as long as the growth period is extended, it is possible to grow thick and strong hair. In addition, it also has the effect of making the hair roots that have entered the resting phase shift to the growth phase earlier, and it is further possible to prolong the hair growth period. In addition, the Ecklonia kurome also has a high moisturizing effect unique to seaweeds, which is said to maintain the moisture of the scalp and maintain a good scalp environment.

[0272] In addition to fucoidan, mozuku seaweed is known to contain iodine and silicon. Iodine is abundant in marine sediments and is taken up by seaweeds. Also, due to the growth environment of mozuku seaweed as described above, it has been found that silicon also has a beneficial effect on healthy growth. Silicon exists in human nails, hair, bones, cell membranes, etc., and has the function of strengthening tissues through its core and protecting cells from oxidation and glycation. Also, as will be described later, when storing hydrogen, mozuku seaweed has the effect that hydrogen is difficult to escape from its "gooiness".

[0273] In addition, citric acid is a weak acid with three carboxyl groups and is contained in citrus fruits (such as oranges, limes, lemons, grapefruits, etc.). Also, because of its sour taste, it is widely used as a food additive. In the living body, citric acid is a component of the "citric acid cycle" and is mainly used as a supplement for the purpose of energy production by the citric acid cycle. Also, citric acid enhances the solubility of calcium by its "chelating action", thus promoting the absorption of calcium from the small intestine.

[0274] Citric acid has various effects and is mainly known to have effects such as fatigue recovery, skin beautifying effect, and hair growth effect. For example, "human hair" is weakly acidic, but shampoo is weakly alkaline, so washing hair may disrupt the pH balance. After washing hair with shampoo, the hair may become frizzy, which means that the hair has become alkaline. Citric acid is useful for restoring this state. By neutralizing alkaline hair with citric acid, it is possible to restore the state of the hair. By using citric acid in the rinse after shampooing, it restores the state of the hair, improves the feel, and also has the effect of promoting blood circulation and softening the scalp. Furthermore, it is said to be effective in preventing thinning hair and hair loss and improving split ends and frayed hair.

[0275] In addition, the hydrocharcoal according to this embodiment contains deep ocean water. Deep ocean water is generally understood to be seawater at a depth of 200 m or more, and has the characteristics of cleanliness, rich in inorganic nutrients, and low-temperature stability compared to surface water. That is, since deep ocean water is not affected by river water polluted by human sewage, it is not contaminated by chemical substances, and sunlight does not reach it and plankton does not grow, so harmful bacteria and the like are also less than one-thousandth of surface water. In addition, compared with surface water, it is rich in inorganic nutrients necessary for the growth of phytoplankton, and furthermore, it has the characteristics that the water temperature and contained components are difficult to change and the water quality is stable.

[0276] In the hydrocharcoal according to this embodiment, the deep ocean water is reduced by hydrogen. By performing a reduction treatment using hydrogen, a state in which hydrogen is occluded is obtained, and an effect of rapidly dispersing coconut shell activated carbon when dissolved in water is exhibited. In this embodiment, an example of mixing with coconut shell activated carbon or the like after performing a reduction treatment with hydrogen on deep ocean water is shown, but the present invention is not limited to this, and it is also possible to perform a reduction treatment with hydrogen after the carbon-containing composition is completed. Note that the present invention does not necessarily have to perform a reduction treatment with hydrogen on deep ocean water. In addition, deep ocean water may be handled in a powder state for convenience, but the present invention is not limited to powder.

[0277] In addition, it is also possible to blend chlorogenic acid into hydrocharcoal that contains inulin and functional edible charcoal and also contains citric acid, Undaria pinnatifida, and hydrogen, and mix this with a lot of commercially available dark-roasted coffee for drinking. In this case, while functional edible charcoal adsorbs caffeine and acrylamide, it becomes possible to supplement the chlorogenic acid and citric acid that were lost during dark roasting. As a result, it becomes possible to expect effective utilization for hypertension and hyperglycemia. When blending chlorogenic acid, either chlorogenic acid alone may be used, or a method of blending an extract of raw coffee beans rich in chlorogenic acid may be adopted. In addition, "dried mango leaf extract" may be additionally blended into the hydrocharcoal. That is, the charcoal adsorbs caffeine, and when mixed with coffee, it becomes decaffeinated. On the other hand, by blending raw coffee bean extract, it becomes possible to supplement the valuable antioxidant "chlorogenic acid" that was lost from dark-roasted coffee. Since there is a group of people who dislike the loss of the stimulant effect of caffeine, by adding dried mango leaf extract, which is a caffeine alternative raw material and has a stimulant effect, it supports decaffeination overall and enables healthy coffee drinking. In addition, it has been confirmed that the "dried mango leaf extract" has an effect of improving mental energy and an effect of improving motor function.

[0278] (I) Regarding the synergistic effect by simultaneously ingesting reduced-fermentation lactic acid bacteria, functional edible charcoal, and hydrogen First, the adsorption of methyl mercaptan by functional edible charcoal will be explained. The inventors conducted an adsorption test for methyl mercaptan on (a) Ina red pine charcoal powder, (b) functional coconut shell activated carbon powder, (c) Kamakura siliceous bamboo charcoal powder, and (d) Wakayama Bichu activated carbon powder. The equipment used was a "gas chromatograph mass spectrometer QP5050A manufactured by Shimadzu Corporation", and the reagent used was a "methyl mercaptan standard solution 1 μg / μL manufactured by Wako Pure Chemical Industries, Ltd.". The test method is as follows. That is, 0.1 g of the reagent was placed in a vial for GCMS headspace, and 10 μL of the standard solution was injected while running along the inner wall surface of the vial. Immediately sealed, left standing for 20 minutes as it is, and then methyl mercaptan was analyzed by GCMS. The results are as shown in the following table.

Table 74

[0279] Next, the AGEs control effect by the collaboration of functional edible charcoal and reducing fermented lactic acid bacteria will be explained. It has been found that AGEs generated in food can be adsorbed by functional edible charcoal. Fig. 10 is a diagram explaining the mechanism of glycation. As shown in Fig. 10, when the blood glucose level in the human body rises, it is heated by body temperature and AGEs are generated in the body. The reducing fermented lactic acid bacteria suppress the generation of AGEs in the body, and the functional edible charcoal adsorbs AGEs in food. Thus, due to the combined function by the collaboration of reducing fermented lactic acid bacteria and functional edible charcoal, it becomes possible to take comprehensive measures against the two major glycation factors.

[0280] Next, the control action of the two major aging factors, oxidation and glycation, through the collaboration of hydrogen, functional edible charcoal, and reducing fermented lactic acid bacteria will be explained. It is said that about 90% of the active oxygen in the human body is generated in the intestines. The factors are bad substances such as food additives and heavy metals, and bad bacteria that increase with age. Hydrogen reacts with active oxygen itself and is converted into safe and harmless water. And it is the smallest antioxidant in the universe with an atomic weight of 1, and it can be expected to have the function of spreading throughout the body and preventing oxidation. However, since it is not possible to continuously drink hydrogen, as a fundamental approach, it is important to simultaneously ingest functional edible charcoal, oligosaccharides, water-soluble dietary fiber, and lactic acid bacteria. Functional edible charcoal adsorbs bad substances, and oligosaccharides, water-soluble dietary fiber, and lactic acid bacteria promote bowel movements. In addition, by adding care for bad bacteria by reducing fermented lactic acid bacteria, a triple action of eliminating active oxygen by hydrogen, adsorbing and removing bad substances by functional edible charcoal, and eliminating bad bacteria by reducing fermented lactic acid bacteria enables a total approach to oxidation factors. As a result, it becomes possible to approach all of "oxidation" and "glycation", which are the two major factors of aging.

[0281] (J) A combination of multiple types of oligosaccharides, KT-11 lactic acid bacteria, which "has a high immunostimulatory effect and can be expected to have a synergistic effect with complex lactic acid bacteria", green papaya extract, which "decomposes proteins, which are factors that deteriorate the intestinal environment due to putrefaction, into amino acids", deep ocean water, and hydrogen, expecting a synergistic effect These are expected to significantly promote intestinal care. In commercialization, the product power may be enhanced by "coloring" using natural pigments. Here, the multiple oligosaccharides include galacto-oligosaccharide, xylo-oligosaccharide, fructo-oligosaccharide, lactulose such as lacto-oligosaccharide, and milk oligosaccharide. By using a combination of any of these, each oligosaccharide can serve as food for beneficial bacteria, and it can be expected that their respective advantages will produce a synergistic effect. In addition, among these oligosaccharides, there are reports of functions that react with intestinal bacteria to increase hydrogen gas generation in the intestine. It is expected that hydrogen gas is taken up from the intestinal tract into the blood, and it has a beneficial effect on the body's antioxidant mechanism such as an increase in the exhaled concentration of hydrogen gas. By collaborating with deep ocean water that has undergone a reduction treatment to generate hydrogen gas and hydrogen ions with electrons, it becomes possible to multiply the oligosaccharides and the hydrogen gas generated from intestinal bacteria, and maximizing hydrogen power can also be expected.

[0282] In addition, sake lees fermented product, re-fermented sake lees, sake lees decomposed product, sake lees processed product, or sake lees fermented extract powder may be added. It is known that these materials related to sake lees have functions of reducing visceral fat, normalizing the intestinal environment, and adsorbing oil and excreting lipids. Furthermore, it is also known that they have an anti-stress effect, an effect of improving endurance, and a beauty effect. Therefore, by blending these into the reduced fermented lactic acid bacteria powder according to this embodiment, a further synergistic effect is expected.

[0283] As described above, according to the reducing fermented lactic acid bacteria powder according to the present embodiment, it contains lactic acid bacteria cells which are good bacteria and lactic acid bacteria fermentation extracts, acts in the direction of increasing good bacteria in the intestine, and it is possible to obtain a bactericidal effect, antioxidant and anti-glycation effect against so-called "bad bacteria" in the living body. Thereby, it becomes possible to realize effective intestinal care and oral care, realize an anti-aging effect, and widely utilize it for beauty purposes. Furthermore, through collaboration with ionized apatite, silk hydrogen pearl powder, soy isoflavone, and hydrochacole, in addition to the antioxidant and anti-glycation effects, high synergistic effects can be expected, such as effects on beautiful skin, hair, nails, hair growth, and utilization for osteoporosis countermeasures and locomotive syndrome countermeasures by strengthening bones.

Claims

1. A method for producing equol using a lactic acid bacteria fermentation extract comprising a lactic acid bacteria production substance, comprising: Brown sugar, soybean pulp, or soybean-derived lactic acid bacteria fermentation extract, 22% or more by weight of plum extract from the Rosaceae family; mixing soy isoflavones including daidzin or daidzein to form a mixture; fermenting the mixture using hydrogen and deep sea water.

2. Cherry blossom extract, 2. The method for producing equol according to claim 1, further comprising adding at least one of deep sea water minerals, Great Salt Lake minerals, and lactic acid bacteria, and reduced fermented fulvic acid containing fulvic acid.

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