Wound healing acceleration agent, and epidermal cell growth acceleration agent

Abstract

Provided is a new wound healing acceleration agent derived from a natural product having high safety.　A wound healing acceleration agent according to the present invention contains a koji mold fermentation product of tea leaves or an extract of the koji mold fermentation product of tea leaves.

Description

Wound healing promoter and epidermal cell proliferation promoter

[0001] The present invention relates to a wound healing promoter and an epidermal cell proliferation promoter, which contain a tea leaf fermentation product with koji mold or an extract thereof.

[0002] Acute skin wounds, such as incisions, lacerations, abrasions, and surgical wounds, often pose a significant health burden. Treatment of these acute skin wounds typically involves cleaning, disinfection, and suturing, but scarring can occur after treatment. Therefore, there is a need for agents that can induce rapid wound closure and promote wound healing without scarring.

[0003] For example, Patent Document 1 proposes a wound healing time shortening agent containing glycerin ester, and Patent Document 2 describes that a special lysophospholipid has the effect of promoting wound healing.

[0004] Meanwhile, in recent years, the value of traditional Japanese koji-fermented foods, such as sake, shochu, amazake, miso, and soy sauce, has been rediscovered, leading to increased popularity of koji. Koji is made by steaming rice, rice bran, barley, or beans to grow koji mold. For example, koji mold grown on rice is called rice koji. While koji mold is known to produce a variety of enzymes that enable the production of fermented foods, research into new functions of koji mold and the development of applications based on these functions are progressing beyond this field. For example, Patent Document 3 reports that koji mold fermentation products have the effect of improving male infertility, and Patent Document 4 reports that koji mold fermentation products have the effect of promoting hair growth and hair restoration.

[0005] JP 2020-050645 A JP 2023-164036 A International Publication No. 2020 / 008821 International Publication No. 2020 / 226022

[0006] Various wound healing promoters have been proposed, such as those in Patent Documents 1 and 2 mentioned above, and there is hope for wound healing promoters derived from highly safe natural products, such as those obtained from edible plants and microorganisms used in fermented foods.

[0007] Furthermore, Patent Documents 3 and 4 each describe koji mold fermentation products obtained by fermenting grains or tea leaves with koji mold, but the use of koji mold fermentation products to promote wound healing has not been investigated to date, and the effectiveness thereof has been unknown.

[0008] Therefore, the present invention has been made in view of the above points, and an object of the present invention is to provide a novel wound healing promoter derived from a natural product, which is highly safe.

[0009] To solve the above problems, the wound healing promoter of the present invention contains a koji mold fermentation product of tea leaves or an extract of the koji mold fermentation product of tea leaves. The koji mold fermentation product and extract thereof have the effect of promoting epidermal cell proliferation and inhibiting bacterial proliferation (wound protection effect), thereby inducing rapid wound closure and promoting wound healing. Furthermore, the koji mold fermentation product of tea leaves is highly safe because it is obtained from tea leaves, which are an edible plant, and koji mold, which is used in fermented foods.

[0010] Furthermore, the koji mold is preferably white koji mold or black koji mold, and more specifically, it is preferably Aspergillus luchuensis mut. kawachii or Aspergillus luchuensis. This allows a koji mold with excellent wound healing promoting effects to be selected as the koji mold that produces the koji mold fermentation product, which is the active ingredient of the wound healing promoter of the present invention. Furthermore, since white koji mold and black koji mold are koji molds used in the production of fermented foods, the koji mold fermentation products obtained from these are highly safe and can be used on a daily basis.

[0011] It is also preferred that the extract of the koji mold fermentation product of the present invention is a water extract or an extract containing an aqueous solvent of the koji mold fermentation product, thereby enabling a wound healing promoter having an excellent wound healing promoting effect to be obtained safely, easily, and at low cost.

[0012] Furthermore, the "wound" in the wound healing promoter of the present invention is preferably a skin wound. Furthermore, the wound healing promoter of the present invention is preferably one that promotes the proliferation of epidermal cells and also preferably one that inhibits bacterial proliferation on the skin. By applying the wound healing promoter of the present invention to a skin wound such as an incision, laceration, abrasion, or surgical wound, epidermalization is promoted, resulting in rapid wound closure and accelerated wound healing.

[0013] The method for producing a wound healing promoter of the present invention includes a step of seeding koji mold on tea leaves or a fermentation raw material derived from tea leaves, and a step of fermenting the fermentation raw material with the koji mold to obtain a koji mold fermentation product, thereby obtaining a wound healing promoter that has an effect of promoting epidermal cell proliferation and an effect of inhibiting bacterial proliferation (wound protection effect), and has an excellent wound healing promoting effect.

[0014] In the method for producing the wound healing promoter of the present invention, the koji mold is preferably a white koji mold or a black koji mold, thereby selecting a koji mold suitable for producing the koji mold fermentation product, which is the active ingredient of the wound healing promoter of the present invention.

[0015] The method for producing the wound healing promoter of the present invention preferably also includes a step of extracting the obtained koji mold fermentation product with water or an aqueous solvent to obtain an extract of the koji mold fermentation product, which allows a wound healing promoter having excellent wound healing promoting effects to be obtained safely, easily, and at low cost.

[0016] The epidermal cell proliferation promoter of the present invention contains a koji mold fermentation product of tea leaves or an extract of the koji mold fermentation product of tea leaves. The koji mold fermentation product and extract thereof have the effect of promoting the proliferation of epidermal cells in the skin, contributing to the promotion of skin turnover and the improvement of the skin's barrier function. Furthermore, the koji mold fermentation product of tea leaves is highly safe because it is obtained from tea leaves, which are an edible plant, and koji mold used in fermented foods.

[0017] According to the present invention, it is possible to provide a wound healing promoter and an epidermal cell proliferation promoter that have the following excellent effects: (1) Rapid wound closure occurs, accelerating wound healing. (2) Accelerated wound healing reduces the likelihood of scarring at the wound site. (3) Contributes to promoting skin turnover and improving the skin's barrier function. (4) High safety is achieved because the active ingredients are tea leaves, commonly known as "tea," and a product fermented by koji mold, a popular fermented food. (5) A liquid formulation can be obtained simply by extracting the koji mold fermentation product with water, allowing for safe, simple, and low-cost production.

[0018] 1 is a flowchart illustrating a schematic diagram of a method for producing a koji mold fermented product of tea leaves and an extract thereof according to an embodiment of the present invention.

[0023] In Example 2, (a) photographs showing the healing process of incision wounds in mice of the test group, comparison group, and control group, and (b) a graph showing the repair level of the incision wounds in mice of the test group, comparison group, and control group. In Example 3, (a) photographs showing the progress of a scratch assay of epidermal keratinocyte cells, and (b) a graph showing the remaining wound area at the scratch site of the epidermal keratinocyte cells. In Example 4, (a) photographs showing the progress of a scratch assay of fibroblasts, and (b) a graph showing the remaining wound area at the scratch site of the fibroblasts. In Example 5, (a) a graph showing the turbidity of an E. coli culture solution, and (b) a graph showing the results of an E. coli colony assay. In Example 6, (a) a graph showing the cell proliferation of fibroblasts under endoplasmic reticulum stress induction, and (b) a graph showing the results of gene expression analysis of endoplasmic reticulum stress markers. 1 is a graph showing (a) a representative histogram of cell surface expression of CD86 by flow cytometry and (b) the relative fluorescence intensity (RFI) value of CD86 for each sample in Example 7.

[0023] FIG. 1 is a graph showing (a) the results of measuring cell viability of epidermal keratinocyte cells and (b) the results of measuring cell viability of the fibroblast cell line NIH3T3 in Example 7.

[0019] First, with reference to FIG. 1, a method for producing the koji mold fermented tea leaves P1 contained in the wound healing promoter of the present invention will be described.

[0020] As shown in FIG. 1 , the method for producing a koji mold fermented product P1 of tea leaves according to an embodiment of the present invention is broadly composed of step S0 of preparing tea leaves as the fermentation raw material, step S1 of adding water to the tea leaves to allow them to absorb it, step S2 of steaming the tea leaves, step S3 of seeding the steamed tea leaves with koji mold, and step S4 of carrying out fermentation.

[0021] (Preparation of Tea Leaves) First, step S0 of preparing tea leaves as the fermentation raw material shown in FIG. 1 will be described. Tea leaves from the tea plant (Camellia sinensis) can be used as the fermentation raw material in the present invention. The tea leaves may include not only tea leaves but also stems and branches harvested from the tea plant. The tea leaves used as the raw material for the koji mold-fermented product of the present invention are preferably fresh tea leaves as they are harvested. However, they may also be tea leaves refrigerated after harvesting, tea leaves heat-treated after harvesting, tea leaves oxidized and fermented with the oxidizing enzymes contained in the tea leaves after harvesting, or tea obtained through a tea-making process. Tea residues remaining after brewing green tea, oolong tea, black tea, or the like can also be used. Furthermore, to improve the efficiency of the subsequent steaming process and koji mold cultivation, the tea leaves or tea stems may be crushed to a certain size so that they are not too large.

[0022] (Water absorption treatment) Next, the water absorption treatment step S1 will be described. In this step, water is added to the tea leaves or the tea leaves are soaked in water to absorb water. The amount of water absorption is not particularly limited, but specifically, the moisture content of the tea leaves is adjusted to 20 to 60%, preferably 30 to 50%.

[0023] (Steaming Treatment) Next, the steaming treatment step S2 will be described. The tea leaves absorbed in the above-mentioned step are placed in a steamer, the lid is closed, and the steamer is heated to expose the tea leaves to steam and heat them. The steaming treatment time is preferably approximately 30 to 120 minutes, more preferably approximately 45 to 90 minutes, and particularly preferably approximately 60 minutes. By performing this step, undesirable bacteria in the raw materials are sterilized, and in the subsequent fermentation step, fermentation is carried out with koji mold as the dominant species. Furthermore, when raw tea leaves are used as the fermentation raw material, this step sterilizes undesirable bacteria in the tea leaves and inactivates the oxidizing enzymes contained in the tea leaves, allowing koji mold to efficiently propagate on the tea leaves in the subsequent step. In addition to a steamer, steaming treatment can also be carried out using a steaming device capable of introducing steam into the device. After steaming, the tea leaves are removed from the steamer, spread evenly on a table, and cooled to approximately 30 to 40°C. This allows koji mold to be seeded on the tea leaves.

[0024] (Seeding of Koji Mold) Next, step S3 of seeding (inoculating) koji mold onto tea leaves will be described. In this step, koji mold is seeded onto tea leaves that have been cooled after the steaming step described above. In the present invention, koji mold refers to a microorganism that is mainly used in producing koji-fermented foods, and specific examples include white koji mold, black koji mold, and yellow koji mold. Of these, white koji mold refers to a group of molds of the genus Aspergillus that form white-ochre conidia (a type of asexual spore) that are widely used in shochu production, and is known as a white mutant of black koji mold, which will be described later. Specific examples include Aspergillus ryukyuensis mut. kawachii (Kawachi white koji mold; Aspergillus luchuensis mut. kawachii [also known as Aspergillus luchuensis var. kawachii kitahara]). Black koji mold refers to a group of Aspergillus molds that form black or dark brown conidia and are used in the production of distilled alcoholic beverages such as awamori in Okinawa and sweet potato shochu in Kagoshima. Specific examples include, but are not limited to, Aspergillus ryukyuensis (Aspergillus awamori), Aspergillus ryukyuensis val. kawachi (Kawachi black koji mold), Aspergillus saitoi, Aspergillus inui, Aspergillus usami, and Aspergillus aureus. Yellow koji mold refers to a group of Aspergillus molds that form yellow or yellow-green conidia and are primarily used in the production of sake, miso, soy sauce, and the like. Specific examples include, but are not limited to, Aspergillus oryzae, Aspergillus oryzae val. kawachi (Kawachi yellow koji mold), and Aspergillus sojae. In the present invention, from the viewpoint of excellent wound healing promoting effect, it is preferable to use white koji mold or black koji mold, and it is more preferable to use white koji mold.Among these, the white koji mold Aspergillus luchuensis mut. kawachii, the black koji mold Aspergillus luchuensis var. kawachii, Aspergillus luchuensis, or a combination thereof is preferably used, with Aspergillus ryukyuensis mut. kawachi being particularly preferred. Seeding of these koji molds can be carried out, for example, by mixing the koji mold with tea leaves placed in a sterilized bag and stirring. It is preferred to sow koji mold spores, and to add and sow koji mold spores so that there are 500,000 or more, preferably 1,000,000 or more, per 1 g (dry weight) of fermentation raw material. For example, if 1 g of seed koji contains 2 billion spores, then about 0.5 g (0.05 w / w%) of seed koji should be added to 1 kg of tea leaves. After seeding the koji mold on the tea leaves, it is preferable to thoroughly stir the mixture to disperse the koji mold throughout the tea leaves.

[0025] (Fermentation) Next, we will explain step S4, which involves culturing koji mold seeded on tea leaves and fermenting them. In this step, the koji mold is propagated on the seeded tea leaves. First, the tea leaves to which the koji mold has been added are placed in a culture chamber maintained at around 30°C. As fermentation progresses and the temperature of the tea leaves rises over time, koji mold generally has difficulty growing above 40°C. Therefore, the temperature is lowered by ventilation, and the ventilation volume is adjusted so that the temperature of the tea leaves remains between 30°C and 42°C, preferably between 30°C and 40°C. The fermentation temperature is adjusted appropriately depending on the type of koji mold used for fermentation. Specifically, when yellow koji mold is selected as the koji mold, for example, the temperature of the tea leaves is adjusted to 30°C to 35°C for 12 to 30 hours after the start of cultivation by adjusting the room temperature of the fermentation chamber, for example. Thereafter, the room temperature and other conditions are similarly adjusted to a slightly higher temperature of 35°C to 40°C, and the koji mold fermentation product P1 is considered complete when the koji mold has been cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably 40 to 60 hours from the start of culture. On the other hand, when white koji mold or black koji mold is selected as the koji mold, for example, the temperature of the tea leaves is adjusted to 35°C to 40°C by adjusting the room temperature of the fermentation chamber, for 12 to 30 hours from the start of culture. Thereafter, the room temperature and other conditions are similarly adjusted to a slightly lower temperature of 30°C to 35°C, and the koji mold fermentation product P1 is considered complete when the koji mold has been cultured for a total of 1 to 4 days, preferably 36 to 72 hours, and more preferably 40 to 60 hours from the start of culture.

[0026] The above-described steps S1 to S4 of producing the koji mold fermented product P1 can also be performed using a machine (for example, a drum-type automatic koji-making machine) that can perform the washing of tea leaves, water absorption, steaming, seeding with koji mold, and fermentation (koji production) within the same device.

[0027] (Koji Mold Fermentation Product) The obtained koji mold fermentation product P1 is so-called "koji" of tea leaves, in which koji mold has proliferated on tea leaves. The koji mold is contained in the koji mold fermentation product P1 as viable bacteria (including spores). This koji mold fermentation product P1 has an excellent effect of promoting wound healing and is useful for treating or improving acute skin wounds such as incisions, lacerations, abrasions, and surgical wounds. The koji mold fermentation product P1 obtained by the above-described process contains a certain amount of moisture, but the moisture can be removed by natural drying or low-temperature dehumidification drying so as not to kill the koji mold. The koji mold fermentation product P1 from which the moisture has been removed can also be pulverized into powder or granules. By reducing the moisture content of the koji mold fermentation product P1, the activity and growth of the koji mold in the koji mold fermentation product P1 can be suppressed during storage. Furthermore, refrigerated storage enables the koji mold fermentation product P1 to be stored for a long period of time while the koji mold remains viable. Even after the water has been removed, the wound healing promoting effect of the koji mold fermentation product P1 can be effectively maintained. Furthermore, when the koji mold fermentation product P1 obtained as described above is used as a wound healing promoter, it is also possible to sterilize the koji mold fermentation product P1 by a known method. Even after the sterilization, the wound healing promoting effect of the koji mold fermentation product P1 can be effectively maintained.

[0028] Next, a method for producing the extract P2 of the koji mold fermentation product contained in the wound healing promoter of the present invention will be described. As shown in Figure 1, the method for producing the extract P2 of the koji mold fermentation product according to an embodiment of the present invention generally comprises step S5 of adding an extraction solvent to the koji mold fermentation product P1 obtained by steps S1 to S4 described above to extract it.

[0029] (Extraction) The extraction step S5 will be described in detail below. In this extraction step S5, an extraction solvent is added to the koji mold fermentation product P1, and an extraction treatment is performed to obtain an extract P2 of the koji mold fermentation product. The extraction solvent is not particularly limited as long as it can extract components having a wound healing-promoting effect from the koji mold fermentation product P1, and examples thereof include water or a water-containing solvent, polar protic solvents such as alcohols, polar aprotic solvents such as dimethyl sulfoxide and acetone, and non-polar solvents such as hexane. Of these, water or a water-containing solvent is preferably used as the extraction solvent from the viewpoints of extraction efficiency, safety for the human body, and ease of handling. Furthermore, the water-containing solvent in the present invention refers to a solvent containing at least water, and includes solvents in which other components such as alcohols and inorganic salts are dissolved or mixed in water. Examples of alcohols include ethanol, 1,3-butylene glycol, isopropanol, propylene glycol, and glycerin, and it is also possible to use one or more of these in combination. In addition, the extraction solvent may contain other components such as pH adjusters, preservatives, extraction aids, etc. in addition to the alcohols and inorganic salts mentioned above, as long as the components do not interfere with the extraction of useful components from the koji mold fermentation product P1.

[0030] The method for extracting the koji mold fermentation product P1 is not particularly limited, as long as it can extract components having a wound healing-promoting effect from the koji mold fermentation product P1. Specifically, for example, the koji mold fermentation product P1 is preferably added to an extraction solvent and immersed in the extractant for extraction. When the koji mold fermentation product P1 is a dried product having a moisture content of less than 10%, 1 g to 300 g of this is preferably added to 1 L of extraction solvent, more preferably 5 g to 150 g, and particularly preferably 10 g to 100 g. The extraction conditions are not particularly limited, as long as they can extract components having a wound healing-promoting effect from the koji mold fermentation product P1. For example, the extraction temperature is preferably about 0 to 30°C, more preferably about 2 to 20°C, and particularly preferably about 4 to 10°C. In this case, the extraction time is preferably about 2 hours to 5 days, more preferably about 6 hours to 3 days, and particularly preferably about 10 hours to 1.5 days.

[0031] As an extraction method other than those described above, the koji mold fermented product P1 can also be immersed in an extraction solvent and extracted under heating, pressure, or heating and pressure. Specifically, for example, it is preferred to perform heating and pressure extraction by autoclaving (for example, at 121°C and 2 atmospheres for 15 to 20 minutes, or at 115°C and 1.7 atmospheres for 30 to 40 minutes) which also serves as a sterilization treatment.

[0032] Furthermore, in this extraction step S5, it is also possible to combine extraction treatment under room temperature conditions (0 to 30°C) with extraction treatment under heat, pressure, or heated and pressurized conditions. For example, extraction treatment can be performed overnight at 4°C, followed by autoclaving at 121°C for 15 to 20 minutes, followed by extraction under heat and pressure. This allows components having wound healing-promoting activity to be extracted from the koji mold fermentation product P1 and also sterilizes the extract P2, thereby efficiently obtaining a wound healing promoter.

[0033] After the above-mentioned extraction process, the residue is removed by centrifugation, filtration, decantation, or the like to obtain extract P2 of the koji mold fermentation product. Examples of extract P2 of the koji mold fermentation product of the present invention include the extract itself, which is extract P2 of the koji mold fermentation product obtained in extraction step S5, as well as extracts obtained by sterilizing the extract by autoclaving, sterilizing by filtration, or the like; concentrates of extract P2 obtained by concentrating extract P2 under reduced pressure, and extract P2 of the koji mold fermentation product obtained by drying extract P2 by freeze-drying or the like to form a solid or powder. In all cases, the wound healing-promoting effect of extract P2 of the koji mold fermentation product can be effectively maintained.

[0034] In the present invention, promotion of wound healing means promoting the healing of skin wounds, and means that wound healing is promoted by, for example, shortening the time required for wound closure compared to a control that is not treated with the wound healing promoter of the present invention.

[0035] In the present invention, promotion of epidermal cell proliferation refers to promotion of the migration and proliferation of epidermal keratinocytes, and means that the migration and proliferation of epidermal keratinocytes is promoted compared to a control that is not treated with the epidermal cell proliferation promoter or wound healing promoter of the present invention. Promotion of epidermal cell proliferation contributes to the promotion of skin turnover and the improvement of the skin barrier function.

[0036] The wound healing promoter of the present invention contains the above-mentioned koji mold fermented tea leaves P1 or extract P2 of the koji mold fermented tea leaves as an active ingredient, and has the effect of promoting skin wound healing through the migration and proliferation of epidermal keratinocyte cells. Furthermore, the koji mold fermented tea leaves P1 or extract P2 of the koji mold fermented tea leaves also have a bacterial growth inhibitory effect. Therefore, the wound healing promoter containing the koji mold fermented tea leaves or extract P2 of the koji mold fermented tea leaves of the present invention can be used as a pharmaceutical, quasi-drug, topical agent, or cosmetic for treating or improving acute skin wounds such as incisions, lacerations, abrasions, and surgical wounds. Cosmetics include not only conventional cosmetics but also cosmetics classified as quasi-drugs, such as functional cosmetics and medicated cosmetics. The wound healing promoter of the present invention is made from tea leaves, commonly known as tea, as a fermentation raw material, and contains as an active ingredient a product fermented by koji mold, which is used in the production of fermented foods, and is therefore highly safe and can be used easily and with confidence regardless of age, gender, or underlying medical conditions.

[0037] The dosage of the wound healing promoter of the present invention is not particularly limited, as it varies depending on the target improvement or therapeutic effect, the administration method, the condition and type of wound, etc. For example, the wound healing promoter of the present invention preferably contains 0.001% to 10%, and more preferably 0.01 to 5%, of the koji mold fermented tea leaves P1. Furthermore, when extract P2 of the koji mold fermented tea leaves is incorporated into the wound healing promoter, the amount of the koji mold fermented tea leaves P1, when converted into the amount of the koji mold fermented tea leaves P1 used as the extraction raw material, is preferably incorporated in an amount equivalent to 0.001% to 10%, and more preferably 0.01 to 5%.

[0038] The wound healing promoter of the present invention can be prepared in various forms by conventional methods. In this case, it can be formulated using additives accepted as pharmaceutical additives, such as carriers and excipients for general formulations. Furthermore, in order to improve the bioavailability and stability of the koji mold fermentation product and extract thereof according to the present invention, a drug delivery system including formulation techniques such as microencapsulation, micronization, and inclusion with cyclodextrin, etc., can also be used.

[0039] The wound healing promoter of the present invention is preferably used in the form of an external preparation such as an ointment, cream, or lotion. Examples of external preparations include low-viscosity liquids, liquid preparations such as lotions, emulsions, gels, ointments, pastes, creams, adhesive bandages, wound dressings, patches, foams, packs, powders, and aerosols. It can also be used in the form of powders, granules, tablets, capsules, or oral liquid preparations, infusions, injections, and the like. When the wound healing promoter of the present invention is used as a cosmetic, it can be used as a lotion, cosmetic cream, cosmetic emulsion, beauty serum, cosmetic pack, cosmetic cleanser, soap, hair care agent, bath additive, and the like.

[0040] In addition, the wound healing promoter of the present invention can be appropriately blended with other medicinal ingredients and ingredients commonly used in topical skin preparations and cosmetics, such as water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, water-soluble polymers, surfactants, metal soaps, alcohols, polyhydric alcohols, pH adjusters, preservatives, fragrances, powders, thickeners, pigments or chelating agents, within the range that does not impair the effects of the present invention.

[0041] Furthermore, the epidermal cell proliferation promoter of the present invention contains the above-mentioned koji mold fermented tea leaf P1 or the extract P2 of the koji mold fermented tea leaf as an active ingredient, and has the effect of promoting epidermal cell proliferation through the migration and proliferation of epidermal keratinocyte cells. Therefore, the epidermal cell proliferation promoter containing the koji mold fermented tea leaf or the extract of the koji mold fermented tea leaf of the present invention can be used as a pharmaceutical, quasi-drug, topical agent, or cosmetic for promoting skin turnover and improving skin barrier function. Among these, cosmetics include not only conventional cosmetics but also cosmetics classified as quasi-drugs, such as functional cosmetics and medicated cosmetics. The epidermal cell proliferation promoter of the present invention uses tea leaves, commonly known as tea, as a fermentation raw material and contains as an active ingredient a product fermented by koji mold, which is used in the production of fermented foods, and is therefore highly safe and can be used easily and with confidence regardless of age, gender, chronic illness, etc.

[0042] The epidermal cell proliferation promoter of the present invention preferably contains 0.001% to 10% of the koji mold fermented tea leaves P1, and more preferably 0.01 to 5%. Furthermore, when an extract P2 of the koji mold fermented tea leaves is added to the epidermal cell proliferation promoter, the amount of the koji mold fermented tea leaves P1, when converted into the amount of the koji mold fermented tea leaves P1 used as the extraction raw material, is preferably 0.001% to 10% and more preferably 0.01 to 5%.

[0043] The epidermal cell proliferation promoter of the present invention is preferably used in the form of an external preparation such as a lotion, emulsion, cream, ointment, etc. Examples of external preparations include low-viscosity liquids, liquid preparations such as lotions, emulsions, gels, ointments, pastes, creams, foams, packs, aerosols, patches, powders, etc. Furthermore, when the epidermal cell proliferation promoter of the present invention is used as a cosmetic, it can be used as a lotion, cosmetic cream, cosmetic emulsion, serum, cosmetic pack, cosmetic cleanser, soap, hair care agent, bath agent, makeup cosmetic, etc.

[0044] The epidermal cell proliferation promoter of the present invention can be used in combination with one or more of various commonly used medicinal ingredients, such as moisturizers, whitening agents, anti-inflammatory agents, cell activators, UV protection agents, blood circulation promoters, antioxidants, etc., within the scope that does not impair the effects of the present invention. Furthermore, ingredients commonly used in external skin preparations and cosmetics, such as water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, water-soluble polymers, surfactants, metal soaps, alcohols, polyhydric alcohols, pH adjusters, preservatives, fragrances, powders, thickeners, pigments, and chelating agents, can be appropriately blended.

[0045] Next, the present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to these examples and comparative examples.

[0046] Example 1 1. Preparation of Koji Mold Fermented Tea Leaves and Their Extracts In this example, fresh tea leaves harvested from tea plants were used as the fermentation raw material. Water was added to the tea leaves to achieve a moisture content of approximately 50%, and the tea leaves were then subjected to a water absorption treatment. They were then placed in a pressurized steam boiler and steamed. The steaming conditions were 115°C for 60 minutes, with two steaming treatments, each with a cooling interval. After the tea leaves were cooled to 30°C, seed koji (product name: Kawachi Genichiro Shoten Kawachikin White Koji, 2 billion spores per gram of seed koji) from white koji mold (Aspergillus luchuensis mut. Kawachii) was added to the tea leaves at a concentration of 0.1% by weight of the tea leaves, and the mixture was allowed to ferment for 3 days. The koji-making temperature was adjusted so that the temperature of the tea leaves was 35°C to 40°C for 24 hours after the addition of the seed koji, and then adjusted slightly lower for the next 48 hours to 30°C to 35°C. The koji-fermented tea leaves obtained after three days of koji-making were then subjected to a low-temperature drying process to a moisture content of 8%, yielding a koji-fermented tea leaf product (hereinafter also referred to as "tea koji"). This koji-fermented tea leaf product was pulverized in a mill to produce a powder.

[0047] Next, in this example, water (Milli-Q water) was used as the extraction solvent. 0.5 g of powdered koji mold fermented tea leaves was added to 10.0 mL of Milli-Q water, mixed, and suspended. This was left overnight in a thermostatic chamber at 4°C for 12 hours for extraction. After 12 hours, the suspension of koji mold fermented tea leaves and water was autoclaved at 121°C for 20 minutes. After autoclaving, the cooled suspension was divided into 1.0 mL aliquots in 1.5 mL microtubes and stored at -80°C. Each time the suspension was used in an experiment, it was thawed and centrifuged at 5000 rpm for 20 seconds to recover the supernatant, yielding an extract of koji mold fermented tea leaves (hereinafter also referred to as "tea koji extract").

[0048] Comparative Example 1 2. Preparation of Tea Leaf Extract In this comparative example, raw tea leaves were processed using the same materials and method as in Example 1, except that the addition (seeding) of koji mold was not performed, to obtain a powdered tea leaf sample. This powdered tea leaf sample was subjected to the same extraction process as in Example 1 to obtain a tea leaf extract.

[0049] Example 2 3. Investigation of the effect of promoting wound healing In this example, the tea koji extract prepared in Example 1 and the tea leaf extract prepared in Comparative Example 1 were administered to a mouse wound model to investigate the effect of promoting wound healing.

[0050] [Preparation of Mouse Wound Model] C57BL / 6 female mice (purchased from Sankyo Labo Service Co., Ltd.) were used. First, the hair on the back skin of the mice was shaved two days before creating a skin wound. Two days after shaving, the mice were anesthetized with isoflurane (induction 3%, maintenance 1-2%), and a 10.0 mm incision was made perpendicular to the midline of the dorsal skin using sterile scissors to create a skin incision.

[0051] [Incision Wound Treatment] Mice were divided into three groups: a test group, a comparison group, and a control group. The test group mice were treated with the tea koji extract prepared in Example 1, the comparison group mice with the tea leaf extract prepared in Comparative Example 1, and the control group mice with tap water. After creating an incision in the test group mice, sterile gauze was soaked in the tea koji extract prepared in Example 1 and the incision was covered with this gauze. A wound dressing (Tegaderm, 3M) was applied over the gauze, and the gauze containing the tea koji extract was fixed to the wound position. From the seventh day after incision creation, the sterilized gauze soaked in the tea koji extract was replaced with a new one once a day, and each time, the gauze was covered and fixed with the wound dressing. From the eighth day onwards, the gauze was not covered with gauze and only observation was performed. On the other hand, the mice in the comparison group and the control group were treated in the same way as the test group, except that the tea leaf extract prepared in Comparative Example 1 or tap water was used instead of the tea koji extract used in the test group.

[0052] Photographs of the incision wounds of mice in each group were taken daily until the wounds of the control group mice were completely healed. Wound length was evaluated using image processing software ImageJ (FIJI: an extension of ImageJ, version 1.53c, National Institutes of Health, USA), and the incision length of each mouse group at each time point was calculated. Data are presented as the mean ± standard error of the mean (SEM) for each group (n = 3). Comparisons between two groups were performed using unpaired two-tailed Student's t-tests, and comparisons between three or more groups were performed using one-way analysis of variance with Tukey or Dunnett's multiple comparison test using statistical analysis software (GraphPad Prism 9, GraphPad Software). * or † indicates p < 0.05, ** or †† indicates p < 0.01, and *** indicates p < 0.001. P<0.05 was considered statistically significant.

[0053] The results are shown in Figures 2(a) and 2(b). These results confirm that the test group (mice treated with the tea koji extract prepared in Example 1) required significantly less time for wound closure than the other groups, demonstrating rapid wound healing. Furthermore, the comparison group (mice treated with the tea leaf extract prepared in Comparative Example 1) and the control group required approximately the same number of days for wound closure.

[0054] Example 3 4. Investigation of the effect of promoting migration and proliferation of epidermal keratinocyte cells In this example, the tea koji extract prepared in Example 1 and the tea leaf extract prepared in Comparative Example 1 were each treated with mouse epidermal keratinocyte cell line PAM212 cells to investigate the effect of promoting migration and proliferation of epidermal keratinocyte cells.

[0055] Specifically, PAM212 cells were cultured in each well of a 24-well plate at 4 × 10 ５ Cells were seeded at 10.0% cells / well and cultured in DMEM medium containing 10.0% fetal bovine serum (FBS) until semi-confluent. A cross-shaped wound was made in each well by scraping the cells with a 10 μL pipette tip. The 10.0% FBS-containing DMEM medium was removed from each well and replaced with DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract prepared in Example 1, and culture was continued. The cross-shaped wound in the well was observed 0 hours and 12 hours after the replacement, and photographs were taken using an optical microscope.

[0056] The cells with a cross-shaped wound were cultured in the same manner as described above, except that DMEM medium (containing 1.0% FBS) containing 0.05% of the tea leaf extract prepared in Comparative Example 1 was used instead of the DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract prepared in Example 1. The cross-shaped wounds in the wells were observed 0 hours and 12 hours after the medium replacement, and photographs were taken with an optical microscope. As a control, a similar test was performed using DMEM medium (containing 1.0% FBS) containing 0.05% water (Milli-Q water) instead of the DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract.

[0057] Statistical analysis was performed in the same manner as in Example 2, and data were compared between the tea koji extract group, tea leaf extract group, and control group (Milli-Q water). Data are expressed as the mean ± standard error of the mean (SEM) for each group. ** indicates p<0.01, and *** indicates p<0.001.

[0058] The results are shown in Figures 3(a) and 3(b). These results demonstrate that tea koji extract promotes the migration and proliferation of epidermal keratinocytes, contributing to the promotion of epidermal cell proliferation and wound closure. Furthermore, tea leaf extract did not exhibit the same promoting effect on epidermal keratinocyte migration and proliferation as tea koji extract.

[0059] Example 4 5. Investigation of the effect of promoting fibroblast migration and proliferation In this example, the tea koji extract prepared in Example 1 and the tea leaf extract prepared in Comparative Example 1 were each treated with mouse fibroblast cell line NIH-3T3 cells to investigate the effect of promoting fibroblast migration and proliferation.

[0060] Specifically, NIH-3T3 cells were placed in each well of a 24-well plate at 3 × 10 ５ Cells were seeded at 10.0% cells / well and cultured in DMEM medium containing 10.0% fetal bovine serum (FBS) until semi-confluent. A cross-shaped wound was made in each well by scraping the cells with a 10 μL pipette tip. The 10.0% FBS-containing DMEM medium was removed from each well and replaced with DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract prepared in Example 1, and culture was continued. The cross-shaped wound in the well was observed 0 hours and 15 hours after the replacement, and photographs were taken using an optical microscope.

[0061] The cells with a cross-shaped wound were cultured in the same manner as described above, except that DMEM medium (containing 1.0% FBS) containing 0.05% of the tea leaf extract prepared in Comparative Example 1 was used instead of the DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract prepared in Example 1. The cross-shaped wounds in the wells were observed 0 hours and 15 hours after the medium replacement, and photographs were taken with an optical microscope. As a control, a similar test was performed using DMEM medium (containing 1.0% FBS) containing 0.05% water (Milli-Q water) instead of the DMEM medium (containing 1.0% FBS) containing 0.05% of the tea koji extract.

[0062] Statistical analysis was performed in the same manner as in Example 2, and data were compared between the tea koji extract group, the tea leaf extract group, and the control group (Milli-Q water). Data were expressed as the mean ± standard error of the mean (SEM) for each group.

[0063] The results are shown in Figures 4(a) and 4(b). These results demonstrate that neither the tea koji extract nor the tea leaf extract affects fibroblast proliferation. The results of Examples 3 and 4 demonstrate that the tea koji extract promotes the proliferation of epidermal cells, thereby facilitating wound closure and wound healing.

[0064] Comparative Example 2 6. Preparation of Tea Leaf Extract-r (without autoclaving) In this comparative example, fresh tea leaves were processed using the same materials and method as in Example 1, except that the addition (seeding) of koji mold was not performed, to obtain a powdered tea leaf sample. This powdered tea leaf sample was subjected to the same extraction process as in Example 1, except that autoclaving was not performed, to obtain Tea Leaf Extract-r (without autoclaving).

[0065] Example 5 7. Investigation of bacterial growth inhibitory effect In this example, the tea koji extract prepared in Example 1, the tea leaf extract prepared in Comparative Example 1, and the tea leaf extract-r (not autoclaved) prepared in Comparative Example 2 were each added to a culture medium for Escherichia coli, and the bacterial growth inhibitory effect was investigated.

[0066] [Measurement of Turbidity of Culture Solution] 2 mL of LB liquid medium containing 0% (control), 0.5%, 1.0%, 2.0%, 4.0%, and 5.0% of the tea koji extract prepared in Example 1 was prepared. Frozen stocks of Escherichia coli (DH5α) were thawed, and 2.0 μL of the thawed E. coli solution was added to the above-mentioned LB liquid medium, followed by incubation at 37°C for 8 hours. After incubation, 100 μL of each culture solution was dispensed into a 96-well plate, and the optical density at a wavelength of 600 nm was measured to determine the turbidity (OD ６００ ) was sought.

[0067] In the same manner as above, except that LB liquid media containing 0.5%, 1.0%, 2.0%, 4.0%, and 5.0% of the tea leaf extract prepared in Comparative Example 1 were used instead of the LB liquid medium containing the tea koji extract prepared in Example 1, 2.0 μL of thawed E. coli liquid was added to each medium and cultured at 37°C. After culturing, 100 μL of each culture medium was dispensed into a 96-well plate, and the optical density at a measurement wavelength of 600 nm was measured to determine the turbidity (OD ６００ ) was sought.

[0068] Furthermore, 2.0 μL of thawed E. coli liquid was added to each of the LB liquid media and cultured at 37°C in the same manner as above, except that the LB liquid medium containing the tea koji extract prepared in Example 1 was replaced with LB liquid media containing 0.5%, 1.0%, 2.0%, 4.0%, and 5.0% of the tea leaf extract-r (not autoclaved) prepared in Comparative Example 2. After culturing, 100 μL of each culture solution was dispensed into a 96-well plate, and the optical density at a measurement wavelength of 600 nm was measured to determine the turbidity (OD ６００ The data were expressed as the mean ± standard error of the mean (SEM) for each group.

[0069] [Colony Assay] Of the cultures cultured as described above, the culture containing 5.0% tea koji extract was diluted 100-fold three times using LB liquid medium. Similarly, the culture containing 5.0% tea leaf extract and the culture containing 5.0% tea leaf extract-r were also diluted 100-fold three times using LB liquid medium. Additionally, as a control, a culture containing only LB liquid medium was also diluted 100-fold three times. 500 μL of each resulting dilution was plated on an LB plate, incubated overnight at 37°C, and the number of colonies was counted. Statistical analysis was performed using the same method as in Example 2 above, and data were compared between the control group and each test group. Data were expressed as the mean ± standard error of the mean (SEM) for each group.

[0070] The results of the turbidity measurement of the culture medium are shown in Figure 5(a), and the results of the colony assay are shown in Figure 5(b). Tea is known to have bacterial growth inhibitory effects, and these results demonstrate that the tea koji extract has the same bacterial growth inhibitory effect as the positive control, tea leaf extract-r. This indicates that the tea koji extract has the effect of inhibiting bacterial growth on the skin (wound protection effect) and promoting wound healing.

[0071] Furthermore, when comparing tea leaf extract-r (not autoclaved) with tea leaf extract (autoclaved), the tea leaf extract (autoclaved) had a slightly reduced bacterial growth inhibitory effect, whereas the tea koji extract had no reduction in bacterial growth inhibitory effect. Considering this, it was inferred that in addition to tea-derived components such as catechins, components derived from koji mold exerted bacterial growth inhibitory effects.

[0072] Example 6: 8. Study of ER Stress Inhibitory Effect ER stress has been reported to be involved in the differentiation of fibroblasts into myofibroblasts during skin wound healing (Matsuzaki, S. et al., "Physiological ER Stress Mediates the Differentiation of Fibroblasts," PLoS ONE 2015, 10, e0123578). While inducing myofibroblast-mediated skin contraction is important for wound closure, excessive wound contraction can lead to scarring and scar formation. Therefore, to minimize scarring and ensure clean wound repair, it is preferable to moderate (suppress) ER stress without excessive stress. Therefore, in this example, the tea koji extract prepared in Example 1 and the tea leaf extract prepared in Comparative Example 1 were each treated in NIH-3T3 cells, a mouse fibroblast cell line, in which ER stress had been induced, to examine the effects on ER stress.

[0073] [Cell proliferation assay] NIH-3T3 cells were plated at 4 × 10 in each well of a 96-well plate. ３Cells were seeded at 10.0% fetal bovine serum (FBS)-containing DMEM medium overnight. Tunicamycin was used as the ER stress inducer. For the ER stress induction test group, the liquid medium in each well was replaced with DMEM medium containing 0.25% tea koji extract prepared in Example 1 and 1.0 μg / mL tunicamycin. For the control group, the liquid medium in each well was replaced with DMEM medium containing 0.25% tea leaf extract prepared in Comparative Example 1 and 1.0 μg / mL tunicamycin. For the control group, water (Milli-Q water) was replaced with DMEM medium containing 0.25% water and 1.0 μg / mL tunicamycin. Then, the cells were cultured for 24 hours. On the other hand, as a control group in which ER stress was not induced, the liquid medium in each well was replaced with DMEM medium containing 0.25% tea koji extract prepared in Example 1, DMEM medium containing 0.25% tea leaf extract prepared in Comparative Example 1, and DMEM medium containing 0.25% water (Milli-Q water) as a control, and cultured for 24 hours. After 24 hours of culture, each well was photographed and analyzed using image processing software ImageJ to measure the remaining viable cell area in the other control and test groups relative to the cell area in the control group in which ER stress was not induced (untreated tunicamycin, containing 0.25% water). Statistical analysis was performed as described in Example 2 above, and data are expressed as the mean ± standard error of the mean (SEM) for each group. * indicates p<0.05.

[0074] [Gene expression analysis of endoplasmic reticulum stress markers] NIH-3T3 cells were placed in each well of a 24-well plate at 4 × 10 ５Cells were seeded at 10.0% fetal bovine serum (FBS)-containing DMEM medium overnight. The liquid medium in each well was replaced with DMEM medium containing 0.25% of the tea koji extract prepared in Example 1 and 1.0 μg / mL of tunicamycin, 0.25% of the tea leaf extract prepared in Comparative Example 1 and 1.0 μg / mL of tunicamycin, or, as controls, DMEM medium containing 0.25% water (Milli-Q water) and 1.0 μg / mL of tunicamycin, or DMEM medium containing 0.25% water (Milli-Q water) but no tunicamycin, and then cultured for 12 hours. After 12 hours, RNA was extracted from the cells, and the mRNA expression levels of endoplasmic reticulum stress markers HSPA5 (heat shock protein family A member 5), XBP-1 (X-box binding protein 1), and CHOP (C / EBP homologous protein) were measured by RT-qPCR analysis. HPRT was used as an internal standard, and its relative expression levels were calculated. The sequences of the PCR primers used are shown in Table 1 below. Statistical analysis was performed using the same method as in Example 2 above, and data are expressed as the mean value for each group ± standard error of the mean (SEM). * indicates p<0.05, and *** indicates p<0.001.

[0075]

[0076] Figure 6(a) shows the results regarding cell proliferation under endoplasmic reticulum stress induction. According to these results, endoplasmic reticulum stress induction by tunicamycin treatment caused cell damage, thereby inhibiting fibroblast proliferation. However, by adding tea koji extract to the medium, the inhibition of fibroblast proliferation was significantly reduced compared to culture conditions in a medium without tea koji extract (control). Furthermore, Figure 6(b) shows the RT-qPCR quantification results for the endoplasmic reticulum stress markers HSPA5, XBP-1, and CHOP. According to these results, tea koji extract significantly reduced the mRNA expression of HSPA5 and CHOP, demonstrating its ability to reduce endoplasmic reticulum stress. Furthermore, no effect on XBP-1 was observed, while tea leaf extract significantly reduced only HSPA5. These results demonstrate that tea koji extract can suppress (alleviate) endoplasmic reticulum stress and inhibit cell damage caused by endoplasmic reticulum stress. Therefore, it is suggested that tea koji extract promotes wound healing with less scarring.

[0077] [Example 7] 9. Skin Sensitization Study Up to now, various studies have been conducted on the effect of tea koji extract on skin wound healing, but materials applied to the skin are generally required to be free of the risk of inducing skin allergies. Therefore, in this Example, the skin sensitization potential of tea koji extract was investigated using a sensitization test using the h-CLAT (human Cell Line Activation Test) method.

[0078] [h-CLAT test] Specifically, THP-1 cells (human monocytic leukemia cell line) were cultured and stimulated for 24 hours using RPMI 1640 medium containing 0.25%, 1.0%, and 4.0% of the tea koji extract prepared in Example 1, RPMI 1640 medium containing 0.25%, 1.0%, and 4.0% of the tea leaf extract prepared in Comparative Example 1, conditioned medium of rhododenol-treated human melanoma SK-MEL-37 cells as a positive control, and RPMI 1640 medium containing 0.25% water (Milli-Q water) as a control. After 24 hours, THP-1 cells were stained with an APC-labeled anti-human CD86 antibody (clone IT2.2), and CD86 expression in THP-1 cells was analyzed using a flow cytometer (product name: FACScanto II, BD Biosciences) and flow cytometry analysis software (FlowJo, version 10). Dead cells were identified using 7-aminoactinomycin D (7-AAD, Sigma-Aldrich). The relative fluorescence intensity (RFI) value of each sample was calculated using the following formula, and samples with an RFI greater than 150% (CD86 > 150%) were considered to be positive for skin sensitization.

[0079]

[0080] [Measurement of cell viability] Keratinocyte cell line PAM212 cells and fibroblast cell line NIH-3T3 cells were cultured for 48 hours in the presence (0.1%, 0.25%, 0.5%, 1.0%, 2.5%, 5.0%) or absence (0% (control)) of the tea koji extract prepared in Example 1. Similarly, keratinocyte cell line PAM212 cells and fibroblast cell line NIH-3T3 cells were cultured for 48 hours in the presence (0.1%, 0.25%, 0.5%, 1.0%, 2.5%, 5.0%) or absence (0% (control)) of the tea leaf extract prepared in Comparative Example 1. After 48 hours of culture, cell viability in each test group was measured using CellTiter-Glo® 2.0 Cell Viability Assay (Promega). The measurement results are shown in the graphs in Figures 8(a) and (b). Statistical analysis was performed in the same manner as in Example 2 above, and the data were expressed as the mean value for each group ± standard error of the mean (SEM). * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and NS indicates no significant difference.

[0081] A representative histogram of CD86 cell surface expression by flow cytometry is shown in Figure 7(a), and the RFI values ​​for each sample are graphed in Figure 7(b). The results show that the conditioned medium of rhododenol-treated human melanoma SK-MEL-37 cells, the positive control, significantly increased the relative fluorescence intensity (RFI) of CD86 (RFI = 2360.4). In contrast, the RFI values ​​of tea koji extract were less than 150% at all concentrations, ranging from low concentrations that did not affect the cell viability of various cells to high concentrations that reduced cell viability (see Figures 8(a) and 8(b)). This indicates that tea koji extract poses no potential risk of skin sensitization and is a safe material for use on the skin.

[0082] The present invention is not limited to the above-described embodiments or examples, and its technical scope also includes various modified designs within the scope that does not deviate from the gist of the invention described in the claims.

[0083] INDUSTRIAL APPLICABILITY The present invention provides a wound healing promoter that induces rapid wound closure and promotes wound healing, and is therefore widely useful in industries such as pharmaceuticals, quasi-drugs, and cosmetics.

Claims

1. A wound healing promoter comprising a product of fermentation of tea leaves with Aspergillus or an extract of said product of fermentation of tea leaves with Aspergillus.

2. The wound healing promoter according to claim 1, characterized in that the koji mold is white koji mold or black koji mold.

3. The wound healing promoter according to claim 1, wherein the koji mold is Aspergillus luchuensis mut. kawachii or Aspergillus luchuensis.

4. The wound healing promoter according to claim 1, characterized in that the extract of the koji mold fermentation product is a water extract or a water-containing solvent extract of the koji mold fermentation product.

5. A wound healing promoter according to any one of claims 1 to 4, wherein the wound is a skin wound.

6. A wound healing promoter according to any one of claims 1 to 4, which promotes the proliferation of epidermal cells.

7. A wound healing promoter according to any one of claims 1 to 4, which inhibits bacterial growth on the skin.

8. A method for producing a wound healing promoter, comprising the steps of seeding koji mold on tea leaves or a fermentation raw material derived from tea leaves, and fermenting the fermentation raw material with the koji mold to obtain a koji mold fermentation product.

9. A method for producing a wound healing promoter according to claim 8, characterized in that the koji mold is white koji mold or black koji mold.

10. A method for producing a wound healing promoter as described in claim 8 or 9, further comprising a step of extracting the koji mold fermentation product with water or a water-containing solvent to obtain an extract of the koji mold fermentation product.

11. An epidermal cell proliferation promoter comprising a product of fermentation of tea leaves with Aspergillus or an extract of the product of fermentation of tea leaves with Aspergillus.

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