A skin improvement agent made primarily from fermented extract

Abstract

Provided is a composition for the skin, the composition having, as an active ingredient, an extract obtained by fermenting and aging Aspergillus oryzae and two types of lactic acid bacteria. As a specific example, the present invention is characterized by having, as an active ingredient, an extract obtained by adding water to Aspergillus oryzae to induce saccharification and primary fermentation, subsequently adding Lactococcus cremoris SW01 to induce secondary fermentation, and then adding Lactobacillus plantarum.

Description

[Technical Field]

[0001] The present invention relates to a skin composition, a skin improving agent, and a skin improving agent having moisturizing properties, each of which contains as its main ingredient a fermentation extract produced by two types of microorganisms, koji mold and lactic acid bacteria.The skin improving composition may contain as its main ingredient Lactococcus cremoris itself or a fermentation extract produced by fermenting it. [Background technology]

[0002] Skin is affected by external factors such as infrared rays, ultraviolet rays, dryness, heat, and various pathogens, as well as internal factors such as natural aging, disease, and viruses. These factors are known to cause wrinkles, age spots, dryness, roughness, and rough skin. Much research has been conducted to improve and prevent these skin disorders and aging phenomena, and skin products have been developed and commercialized.

[0003] In recent years, the number of people using and the amount of consumption of fermented extracts obtained by fermentation and maturation using microorganisms has been increasing due to their effectiveness in maintaining and improving health. Foods and beverages containing fermented extracts have been commercialized for a long time, and recently, basic cosmetics using fermented extracts as the main ingredient have also been commercialized, rather than being consumed by humans as food.

[0004] One of the pharmacological effects of fermented plant extracts has been reported to be anti-aging (Patent Document 1). Other reported effects include tyrosinase inhibition (melanin production inhibition), AGE production inhibition (anti-glycation effect), and TNF-α and NO production inhibition (anti-inflammatory effect) (Patent Document 2). Hyaluronic acid synthesis promoters have been reported that contain as active ingredients a lactic acid bacteria fermentation product of pomegranate juice (Patent Document 3) and a lactic acid bacteria fermentation product of bean extract (Patent Document 4). As described above, various beneficial effects on the skin are expected. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6013670 [Patent Document 2] Patent Publication No. 2021-147361 [Patent Document 3] Patent Publication No. 2013-245170 [Patent Document 4] JP 2002-187838 A Summary of the Invention [Problem to be solved by the invention]

[0006] To date, there has been no knowledge about extracts that are produced by fermenting two types of microorganisms, koji mold and lactic acid bacteria, in stages. Furthermore, nano-type lactic acid bacteria (Lactbacillus plantarum) are derived from sunki pickles, which are eaten in the Kiso region of Nagano Prefecture, and there is little knowledge about their effects on the skin.

[0007] The present inventors have been conducting extensive research into the uses and effects of fermented extracts and fermented compositions of various microorganisms, and have developed the present invention based on the working hypothesis that beneficial fermented extracts can be produced by individually and stepwise fermenting multiple microorganisms with different properties. [Means for solving the problem]

[0008] The technical means used by the present invention to solve the technical problems are as follows.

[0009] The present invention provides a skin care composition containing, as an active ingredient, an extract obtained by fermentation and maturation using two types of bacteria: koji mold and lactic acid bacteria. The lactic acid bacteria may be of the genus Lactococcus or Lactobacillus.

[0010] Water is added to Aspergillus oryzae to cause primary fermentation, followed by the addition of lactic acid bacteria (Lactococcus lactis subsp. Cremoris H6 strain). SW01The present invention provides a composition for external use on the skin, which contains, as an active ingredient, an extract obtained by adding Lactobacillus plantarum to a mixture of lactic acid bacteria (Lactobacillus spp.) and subjecting it to secondary fermentation, followed by the addition of nano-type lactic acid bacteria (Lactbacillus plantarum). Alternatively, nano-type lactic acid bacteria (Lactbacillus plantarum) may be added, and a filter aid may be mixed and added, followed by tertiary fermentation to produce an extract.

[0011] After the primary fermentation (including fermentation and saccharification) The Aspergillus oryzae is sterilized, and after the secondary fermentation, the lactic acid bacteria is sterilized, and then filtered. Filter aids may be added together. The active ingredient may be an extract from which Aspergillus oryzae and lactic acid bacteria have been removed using a method known as Aspergillus oryzae. Aspergillus oryzae is sometimes called koji mold or rice koji.

[0012] The skin composition may be used as a skin improving agent having a moisturizing function and containing the above-mentioned skin composition as a main component.

[0013] The composition for skin may be used as an elastase activity inhibitor as a main component.

[0014] The skin composition may be used as a collagen production promoter as a main component.

[0015] The composition for skin may be a β-hexosaminidase activity inhibitor containing the composition for skin as a main ingredient. Also, the composition for skin improvement has multifaceted effects as an elastase activity inhibitor, a collagen production promoter, and a β-hexosaminidase activity inhibitor containing the composition for skin as a main ingredient.

[0016] The composition for skin may be used as a composition for external use on the skin, the composition for skin being the main component. [Effects of the Invention]

[0017] According to the present invention, it has become possible to carry out the fermentation process of koji mold (Aspergillus oryzae) and lactic acid bacteria individually and stepwise. In the fermentation process of the present invention, raw materials, additives, and other ingredients other than the target koji mold and lactic acid bacteria are used as little as possible, and a fermentation filtrate (fermentation extract, fermentation composition) has been developed that maximizes the extraction of only the components produced by the target koji mold and lactic acid bacteria through microbial fermentation.

[0018] This fermented extract and composition has been shown to improve skin function and have preventative effects against skin disorders. Furthermore, it is predicted that the above-mentioned preventative effects can be enhanced by adding nano-type lactic acid bacteria (Lactbacillus plantarum) after the fermentation process.

[0019] The present invention will be further described below in conjunction with the accompanying drawings and examples, but the present invention is not limited to the examples. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is an explanatory diagram showing the process for producing a rice koji lactic acid bacteria fermentation liquid of the present invention. [Figure 2] Graph showing elastase activity in rice koji lactic acid bacteria fermentation liquid (various concentrations) of the present invention [Figure 3] Graph showing collagen production in the rice koji lactic acid bacteria fermentation liquid of the present invention [Figure 4] Graph showing the inhibition of β-hexosaminidase activity in the rice koji lactic acid bacteria fermentation liquid of the present invention

[0021] The present inventors focused on microorganisms, namely, koji mold and two types of lactic acid bacteria, and obtained a fermentation extract (SW01, the test subject of this example) as described below.

[0022] FIG. 1 is an explanatory diagram showing the fermentation process. For the primary fermentation, approximately 80 to 99% water and approximately 1 to 20% koji mold (including culture medium) are added and mixed by volume, and then fermentation is carried out. The koji mold used was Aspergillus oryzae (NBRC 100959). The fermentation temperature was 50 to 55°C, and the fermentation time was 17 to 22 hours.

[0023] After the fermentation time has elapsed, the temperature is raised to about 90 to 120°C and maintained at that temperature for 10 to 20 minutes, killing the koji mold with the high temperature and stopping the fermentation process.

[0024] The koji mold culture medium used in this invention is rice starch (powder). During the primary fermentation, starch, a medium component (rice starch component) present outside the koji mold cells, is decomposed into monosaccharides such as glucose using the fungal enzymes and saccharified. When 1% rice koji was used, the Brix was 0.0 and the glucose concentration was 0.03% before fermentation, but after fermentation, this increased to Brix 0.8 and glucose concentration 0.38%.

[0025] In this first fermentation, the koji mold simply breaks down rice starch, so the number of bacteria itself remains unchanged, but the OD660 value increases from 0.50 to 1.50, which correlates with an increase in Brix value.

[0026] The koji mold converts the medium components (rice starch components) outside the koji mold cells into saccharified monosaccharides such as glucose. These monosaccharides are then used as nutrients for the lactic acid bacteria that are added, making it possible to produce a fermentation filtrate that does not use ingredients, additives, or additives that are not derived from koji mold or lactic acid bacteria.

[0027] Next, Lactococcus cremoris SW01 (NITE BP-03847) was added to the fermented broth after fermentation was stopped, and fermentation by the lactic acid bacteria was promoted. The dosage of Lactococcus cremoris SW01 was 0.01 to 1% by weight of the total amount. The glucose previously saccharified was used as a nutrient source for the lactic acid bacteria. This lactic acid bacteria is known to be found in plant-based cheese. Lactococcus cremoris SW01 (SW01) was deposited at the National Institute of Technology and Evaluation, International Patent Organism Depositary Center on March 3, 2023 (NITE BP-03847). The secondary fermentation using this lactic acid bacteria was carried out at a fermentation temperature of 25°C to 35°C for a fermentation time of 17 to 22 hours. After the fermentation time had elapsed, the OD660 was shown to be 1.00 to 2.00. The OD660 value indicated that the number of lactic acid bacteria administered also increased. The OD660 value depends on the number of bacteria, and an increase in the value indicates an increase in the number of bacteria.

[0028] After the fermentation time has elapsed, the temperature is raised to approximately 90 to 120°C and maintained at that temperature for 10 to 20 minutes, thereby killing the lactic acid bacteria with high temperature and stopping the fermentation process.

[0029] The pH of the fermented liquid was 3.5 to 4.5, and insoluble matter such as rice residue was filtered to increase the transparency of the fermented extract.

[0030] The degree of filtration can be varied: for stronger filtration, filter aids such as diatomaceous earth derived from fossil minerals or perlite derived from volcanic rock are used, while for weaker filtration, mesh filtration is used.

[0031] In this example, the OD value after filtration changed from 1.00 to 2.00 to 0.00 to 0.05. That is, this filtration treatment removes insoluble matter such as residue, and a fermentation filtrate (fermentation extract, fermentation composition) can be obtained.

[0032] As a final step, nano-type lactic acid bacteria (Lactbacillus plantarum) is added to the filtered fermented liquid at a concentration of 0.01-0.05%. This nano-type lactic acid bacteria (Lactbacillus plantarum) is a sterilized lactic acid bacteria (20-25% lactic acid bacteria, the rest is dextrin powder). Lactbacillus plantarum is known to be derived from sunki pickles.

[0033] Generally, lactic acid bacteria have the disadvantage of clumping when sterilized. For this reason, nano-sized lactic acid bacteria are processed by nano-processing (high-pressure homogenization) to prevent clumping and make them easier to handle as raw materials. By adding nano-sized lactic acid bacteria after filtration, it has become possible to stabilize the components of the fermented extract (fermented composition).

[0034] Substances derived from nano-sized lactic acid bacteria (cell walls, nucleic acids, proteins, sugars, etc.) were adopted in light of the possibility that they may have beneficial effects such as strengthening the immune system of the epithelium (epidermis) and normalizing homeostatic functions.

[0035] By adding nano-type lactic acid bacteria (Lactbacillus plantarum), the OD660 changed from 0.00-0.05 to 0.25-1.00. The fermented extract (fermented composition) was then maintained at 85-100°C for 10-30 minutes to sterilize the fermented liquid. This was done to prevent spoilage and prevent contamination due to secondary contamination.

[0036] Next, tests were carried out on the functions and effects of the skin composition according to the present invention. First, an elastase activity inhibition test (anti-wrinkle test) was carried out.

[0037] The dermis, located inside the epidermis, is mainly composed of collagen, elastin, and hyaluronic acid. Elastin is produced by dermal fibroblasts and plays an important role in maintaining the flexibility and elasticity of the skin. Elastin is a "elastic fiber" that is flexible and stretchable (elastic), and has the property of returning to its original shape when force is released. Elastin and collagen coexist in a symbiotic relationship, bundling them together.

[0038] Elastase, an elastin-degrading enzyme, is known to be induced by stimuli such as inflammation, aging, ultraviolet rays, active oxygen, and stress. When elastin is broken down by elastase, the cross-linked structure between collagen and elastin becomes brittle, causing skin elasticity to be lost and wrinkles to form. Therefore, inhibiting elastase activity is expected to have the effect of restoring or maintaining dermal elasticity and preventing aging.

[0039] In this example, elastase derived from human dermal fibroblasts (NHDF-Ad) was used to evaluate elastase activity by measuring the amount of nitroaniline produced using N-succinyl-Ala-Ala-Ala-p-nitroanilide as a substrate. Based on the elastase activity, the skin dermal elasticity maintenance and anti-aging functions (activity inhibition rate) of the test sample, rice koji lactic acid bacteria fermentation liquid SW01 (hereinafter referred to as SW01), were investigated.

[0040] A control reaction using only elastase derived from human dermal fibroblasts (NHDF-Ad) and the substrate N-Succinyl-Ala-Ala-Ala-p-nitroanilide was used, and its elastase activity was set at 100%. The reaction conditions for this experiment were 37°C and 2 hours. Furthermore, administration of 50 nM of phosphoramidon, a standard elastase inhibitor, demonstrated elastase inhibition, confirming the effectiveness of this experimental system.

[0041] As a result, when the evaluation sample SW01 was added at a concentration of 4 mg / mL, the elastase inhibitory activity was found to be 14.9% (p<0.01) compared to the control, and the inhibitory activity was also found to be concentration-dependent (Figure 2). Therefore, it was suggested that SW01 has elastase inhibitory activity and has the effect of contributing to the recovery of dermal elasticity and the maintenance of anti-aging functions in the skin.

[0042] Next, we tested whether rice koji lactic acid bacteria fermentation liquid SW01 promotes collagen production. SW01 (sample) was added to human dermal fibroblasts (NHDF-Ad), and the amount of collagen in the culture supernatant after 72 hours was measured using ELISA. The results showed that collagen production per cell increased 1.5-fold in the test group where 500 μg / mL of SW01 was added compared to the control (Figure 3). This indicates that rice koji lactic acid bacteria fermentation liquid SW01 promotes collagen production in the dermal layer of the skin.

[0043] The above experiment demonstrated that the rice koji lactic acid bacteria fermentation liquor SW01 of the present invention simultaneously exerts the dual effects of elastase inhibition and collagen production promotion. This demonstrates that applying the rice koji lactic acid bacteria fermentation liquor SW01 of the present invention to the skin increases dermal fibroblasts and maintains firmness and dermal structure.

[0044] Next, we tested whether the rice koji lactic acid bacteria fermentation liquid SW01 (sample) contributes to anti-inflammatory and anti-allergic effects. It is known that when IgE bound to the cell surface of fat cells is cross-linked by an antigen, granulocytes containing histamine are released outside the cells, which causes allergic reactions.

[0045] The degree to which a test substance inhibits degranulation is assessed by measuring the activity of β-hexosaminidase, which is abundant in granules. This allows the effect of the test substance on allergic reactions to be evaluated. Rat basophilic leukemia cells RBL-2H3 are known to release granulocytes containing histamine and other substances extracellularly when IgE bound to the cell surface is crosslinked by antigen. This experimental system has been used in exploratory research to screen for substances with anti-allergic effects against type I allergies. In this example, RBL-2H3 cells are stimulated with an antigen, and the inhibition of degranulation is evaluated by measuring β-hexosaminidase activity (Figure 4).

[0046] The β-hexosaminidase activity observed when sterilized water was added dropwise as a control was defined as 100%, and the extent to which the β-hexosaminidase activity was inhibited by the addition of the rice koji lactic acid bacteria fermentation broth SW01 of the present invention, the test subject, was measured at various addition amounts. Quercetin was also used at 50 μg / ml as a positive control. The positive control reduced β-hexosaminidase activity to approximately 40%, demonstrating the suitability of this experimental system. The addition of 2.5 μg / ml of rice koji lactic acid bacteria fermentation broth SW01 reduced β-hexosaminidase activity to approximately 50%. This demonstrates that rice koji lactic acid bacteria fermentation broth SW01 inhibits β-hexosaminidase activity and exerts anti-allergic and anti-inflammatory effects. The p-value in Figure 4 is p<0.01.

[0047] From the above, it has been demonstrated that the rice koji lactic acid bacteria fermentation liquid SW01 of the present invention has various effects on skin improvement, such as inhibiting elastase activity, inhibiting β-hexosaminidase activity, and promoting collagen production. Skin improvement includes preventing minor skin diseases such as itching and inflammation from a health and hygiene perspective, thereby preventing pre-disease. [Accession number]

[0048] National Institute of Technology and Evaluation Postal code 292-0818 Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture Deposit date: March 3, 2023 Name of contracted microorganism: Lactococcus cremoris SW01 Accession number: BP-03847

[0049] TIFF0007743618000001.tif159151

Claims

1. A method for producing an extract that is an active ingredient in a skin composition, characterized by comprising the steps of using rice starch as a culture medium for Aspergillus oryzae, adding water to the Aspergillus oryzae containing the rice starch culture medium to perform primary fermentation to convert it into sugar, then adding lactic acid bacteria (Lactococcus cremoris) to perform secondary fermentation, and further adding nano-type lactic acid bacteria (Lactobacillus plantarum).

2. A method for producing the extract of claim 1, comprising: A method for producing an extract, comprising the steps of sterilizing Aspergillus oryzae after primary fermentation, sterilizing lactic acid bacteria after secondary fermentation, and then filtering to remove the Aspergillus oryzae and lactic acid bacteria.

3. A skin improvement agent having moisturizing properties, the main ingredient of which is an extract that is an active ingredient of a skin composition obtained by the manufacturing method described in claim 1 or 2.

4. An elastase activity inhibitor having as its main ingredient an extract that is an active ingredient of a skin composition obtained by the manufacturing method described in claim 1 or 2.

5. A collagen production promoter having as its main ingredient an extract that is an active ingredient of a skin composition obtained by the manufacturing method described in claim 1 or 2.

6. A β-hexosaminidase activity inhibitor, the main ingredient of which is an extract that is an active ingredient of a skin composition obtained by the manufacturing method described in claim 1 or 2.

7. A topical skin composition having as its main ingredient an extract that is an active ingredient of a skin composition, obtained by the manufacturing method described in claim 1 or 2.

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