Selective antibacterial application of micrococcus luteus
By using Micrococcus luteus and its derivatives, selective antibacterial activity against harmful Staphylococcus aureus was demonstrated, which solved the problem of skin microbiome imbalance, improved skin health, and reduced the occurrence of skin diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CUTISBIO CO LTD
- Filing Date
- 2024-07-18
- Publication Date
- 2026-04-21
AI Technical Summary
Existing technologies are insufficient to effectively maintain the balance of the skin microbiome, leading to impaired skin barrier function and weakened skin immune response, which in turn triggers various skin diseases such as atopic dermatitis, seborrheic dermatitis, and acne.
Micrococcus luteus, its lysates, culture media, or extracts exhibit selective antibacterial activity, primarily targeting harmful staphylococci such as Staphylococcus aureus and methicillin-resistant Staphylococcus aureus, while having minimal impact on beneficial bacteria such as Staphylococcus epidermidis.
By selectively inhibiting the growth of harmful bacteria, the skin microbiome is maintained in balance, improving skin health and reducing the occurrence of skin diseases.
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Abstract
Description
Technical Field
[0001] This invention relates to the selective antimicrobial use of Micrococcus luteus against harmful Staphylococcus sp. More specifically, this invention relates to the selective antimicrobial use of a composition comprising Micrococcus luteus, its lysate, culture medium, or extract against harmful Staphylococcus sp. Background Technology
[0002] Because the surface of human skin contains a large amount of metabolic waste such as keratin, sweat, and sebum, various microorganisms inhabit and form the skin microbiome, also known as the resident flora of the skin. Beneficial and harmful bacteria coexist within this resident flora; the healthier the skin, the more beneficial bacteria are present, while in atopic or acne-prone skin, harmful bacteria are more prevalent. Therefore, it is well known that maintaining the balance of the resident flora in the skin helps maintain a healthy skin condition.
[0003] Normally, the resident flora on the skin form a skin barrier to prevent the invasion of other, more harmful microorganisms (pathogens) and to aid in the skin's immune function. An imbalance between beneficial and harmful bacteria on the skin can lead to impaired skin barrier function and a weakened skin immune response, and can be a contributing factor to various skin diseases such as atopic dermatitis, seborrheic dermatitis, acne, and psoriasis. Therefore, selectively inhibiting the activity of harmful bacteria without affecting beneficial bacteria could be helpful in treating a variety of skin diseases.
[0004] In this invention, studies were conducted on resident skin flora, and the results confirmed that Micrococcus luteus exhibits selective antibacterial activity against harmful Staphylococcus sp., thereby completing this invention. Summary of the Invention
[0005] The problem the invention aims to solve Therefore, the present invention provides an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus sp.
[0006] Specifically, the present invention provides an antibacterial cosmetic composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0007] The present invention provides an antibacterial pharmaceutical composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0008] The present invention provides an antibacterial topical skin composition comprising Micrococcus luteus, or its lysates, culture or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0009] The present invention provides a quasi-pharmaceutical composition for antibacterial use, comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0010] The present invention provides an antibacterial food or health functional food composition comprising Micrococcus luteus, or its lysates, culture medium or extract, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0011] In addition, the present invention provides a strain of Micrococcus luteus, which has the accession number KACC 81251BP.
[0012] In addition, the present invention provides a lysate, culture medium or extract of the aforementioned Micrococcus luteus strain.
[0013] means for solving problems One aspect of the present invention provides an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media or extracts as an active ingredient, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus.
[0014] In this invention, *Micrococcus luteus* can be a live bacterium (living bacteria) or a dead bacterium (dead bacteria). More specifically, dead bacteria can be bacteria that have been killed by heat treatment.
[0015] In one embodiment, *Micrococcus luteus* can be a strain of *Micrococcus luteus* deposited with accession number KACC 81251BP.
[0016] As used herein, the term "lysis products" refers to solutions or suspensions of microbial cells, such as lysed Micrococcus luteus, in aqueous culture media. Cell lysis products include macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, and lipids, and / or small molecules or fractions such as amino acids, sugars, and fatty acids. Additionally, lysis products also include cell debris, which can be smooth or granular.
[0017] As a method for achieving cell lysis of microorganisms, various known methods can be used, and any method capable of achieving cell lysis of microorganisms can be used. For example, cell opening / destruction can be carried out by enzymatic, chemical, or physical means. Non-limiting examples of enzymes and enzyme mixtures are proteases such as proteinase K, lipases, or glycosidases; non-limiting examples of chemicals are ion carriers, detergents such as sodium dodecyl sulfate, acids, or bases; non-limiting examples of physical means are high pressure such as French extrusion, osmotic pressure, or temperatures such as heat or cold. In addition, methods using enzymes, acids, bases, etc., other than proteolytic enzymes, can also be appropriately combined.
[0018] The term "culture medium" as used herein may be used interchangeably with "culture supernatant," "conditioned culture medium," or "conditioned medium," and may refer to the whole culture medium containing the microorganisms, their metabolites, and residual nutrients obtained by culturing *Micrococcus luteus* in a culture medium for a period of time, wherein the culture medium for culturing *Micrococcus luteus* is a culture medium that can provide nutrients so that *Micrococcus luteus* can grow and survive in vitro. Furthermore, the culture medium may also refer to the culture medium from which bacterial cells have been removed. On the other hand, the liquid from which bacterial cells have been removed from the culture medium is also called the "supernatant," which can be obtained by: taking only the supernatant liquid excluding the portion that settles to the lower layer after the culture medium has been left to stand for a period of time; removing bacterial cells by filtration; or removing the lower precipitate by centrifuging the culture medium and then taking only the supernatant liquid. The term "bacterial cells" refers to the microbial organism of the present invention, including microbial organisms screened by isolation from skin samples, etc., or microorganisms isolated from the culture medium after culturing the microorganisms. The bacteria can be obtained in the following ways: either by centrifuging and taking the portion that sinks to the bottom layer; or, since they will sink to the bottom layer of the culture medium due to gravity, they can be obtained by letting them stand for a period of time and then removing the upper layer of liquid.
[0019] In one embodiment, the *Micrococcus luteus* culture of the present invention can be prepared using a culture medium readily selected by those skilled in the art from those used for culturing microorganisms, depending on the purpose. Specifically, a culture medium for culturing *Sphingomonas*, such as MH (Mueller Hinton broth) medium or Tryptic Soy Broth (TSB) medium, can be used, but the invention is not limited thereto. According to one embodiment, the *Micrococcus luteus* culture of the present invention is prepared by inoculating *Micrococcus luteus* into a microbial culture medium and following microbial culture methods known in the art (e.g., static culture, etc.).
[0020] The culture medium may include a culture medium obtained by culturing microorganisms, its concentrate or lyophilized form, or a culture supernatant obtained by removing microorganisms from a culture medium, its concentrate or lyophilized form.
[0021] The above-mentioned culture medium can be obtained by culturing Micrococcus luteus in a suitable culture medium (e.g., MH medium) at any temperature from 10°C to 40°C for a certain period of time (e.g., 4 to 50 hours).
[0022] In one embodiment, the culture supernatant of microorganisms can be obtained by centrifuging or filtering the microbial culture to remove microorganisms.
[0023] In another embodiment, the concentrate can be obtained by concentrating the microbial culture medium, or the supernatant obtained by centrifugation or filtration of the culture medium.
[0024] The culture medium and culture conditions used to cultivate this Micrococcus luteus can be appropriately selected or modified by those skilled in the art.
[0025] As used herein, the term "extract" means a substance extracted from a dissolved product, culture medium or its concentrate, and it may be an extract, a dilution or concentrate of an extract, a dried product obtained by drying an extract, or a crude extract or purified product of these, or a fraction thereof.
[0026] Relative to the total weight, it may contain 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt% of microorganisms, or their lysates, cultures, or extracts.
[0027] As used herein, the term "containing...as an active ingredient" means adding microorganisms or their lysates, cultures, or extracts to a degree that can produce the desired effect, and also includes various formulations in which various ingredients are added as byproducts for purposes such as drug delivery and stabilization.
[0028] In this specification, the term "antibacterial" refers to having the following activities: (i) Inhibit, reduce or prevent the growth of bacteria; (ii) Inhibit or reduce the ability of bacteria to cause infection in an object; or (iii) To inhibit or reduce the ability of bacteria to multiply or maintain infectivity in the environment.
[0029] In this invention, *Micrococcus luteus*, or its lysates, culture media, or extracts, can exhibit selective antibacterial activity against harmful staphylococci. Specifically, *Micrococcus luteus* exhibits antibacterial activity against harmful staphylococci, but shows weak antibacterial activity or no effect on harmless beneficial skin bacteria.
[0030] The harmful staphylococci refer to those that invade the surface or tissues of the host's skin and cause skin problems such as rashes and infections (abscesses, pus, erythema, etc.). The *Micrococcus luteus*, or its lysates, cultures, or extracts, may exhibit antibacterial activity against harmful staphylococci (e.g., *Staphylococcus aureus*, methicillin-resistant *Staphylococcus aureus* (MRSA), and *Staphylococcus capitis*, which are bacteria that cause skin diseases, but are not limited thereto. However, it has been confirmed that the *Micrococcus luteus*, or its lysates, cultures, or extracts, have weak antibacterial activity against *Staphylococcus epidermidis* (a beneficial bacterium of the skin) and almost no antibacterial activity against *Staphylococcus hominis* (a beneficial bacterium of the skin). In addition, it was confirmed that the aforementioned Micrococcus luteus, or its lysates, culture media, or extracts, showed almost no antibacterial activity against Micrococcus luteus, Candida albicans SC5314, Corynebacterium propinguum, Pseudomonas aeruginosa ATCC10145, and Escherichia coli DH5α, other than Staphylococcus aureus.
[0031] In one specific instance, the Micrococcus luteus, or its lysates, culture media, or extracts, may exhibit selective antibacterial activity against one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus capillus.
[0032] In one specific instance, the Micrococcus luteus, or its lysates, culture media, or extracts, may exhibit selective antibacterial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus capillus.
[0033] The antibacterial compositions of the present invention can be used for a variety of purposes and uses requiring antibacterial activity. Specifically, they can be used as cosmetics, pharmaceuticals (e.g., topical skin preparations), quasi-pharmaceuticals, food, health functional foods, detergents, cleaning agents, preservatives, food additives, or feed additives, but are not limited thereto.
[0034] In one specific instance, an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media, or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus, may be a cosmetic composition.
[0035] In this invention, the above-mentioned cosmetic material composition can be prepared into dosage forms including lotion, emollient, skin, skin softner, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nourishing lotion, massage cream, nourishing cream, moisturizing cream, hand cream, hand cleanser, foundation, serum, nourishing serum, mask (pack), soap, facial foam, facial cleanser, facial cream, body lotion, shower gel, suspension, gel, powder, paste, sheet mask (mask pack), and tablet. Such dosage form compositions can be prepared according to conventional methods in the art. Those skilled in the art can readily select the mixing amounts of the added ingredients, such as moisturizers, as described above, without impairing the purpose and effects of this invention.
[0036] In addition to the effective ingredients disclosed in this invention, the above-mentioned cosmetic material compositions may further include functional additives and ingredients contained in ordinary cosmetic material compositions, and may also include commonly used purified water, thickeners, preservatives, stabilizers, solubilizers, surfactants, carriers, fragrances, or combinations thereof. The above-mentioned functional additives may include ingredients selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular weight peptides, high molecular weight polysaccharides, sphingolipids, and seaweed extracts. Examples of the above-mentioned carriers include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, color developers, fragrances, etc. Compounds / compositions used with the above-mentioned alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, colorants, fragrances, etc., are well known in the art, and those skilled in the art can select and use appropriate corresponding substances / compositions. In addition, as needed, the cosmetic material composition may also contain UV blockers, antioxidants (butylated hydroxyanisole, propyl gallate, isoascorbic acid, tocopheryl acetate, butylated hydroxytoluene, etc.), preservatives (methylparaben, butylparaben, propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters (triethanolamine, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, disodium hydrogen phosphate, etc.), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexanediol, diglyceride, betaine, glyceryl polyether-26, methyl gluceth-20, etc.), lubricants, etc.
[0037] Furthermore, in cosmetic material compositions of various dosage forms, appropriate ingredients can be selected and mixed according to the dosage form or intended use of the cosmetic material. Since the mixing ingredients and methods can follow conventional techniques, their detailed description will be omitted in this invention.
[0038] In another specific embodiment of the invention, an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus, may be an antibacterial pharmaceutical composition.
[0039] As used herein, the term "pharmaceutical composition" can refer to a molecule or compound that, when administered to a subject, imparts certain beneficial effects. Beneficial effects may include: enabling diagnostic decisions; improvement of a disease, symptom, disorder, or pathology; reduction or prevention of the onset of a disease, symptom, disorder, or pathology; and normal responses to a disease, symptom, disorder, or pathology.
[0040] As used herein, the term "prevention" means the partial or complete delay or prevention of the onset or recurrence of a condition, disorder, or its associated symptoms; prevention of the acquisition or re-acquisition of a condition or disorder; or reduction of the risk of acquiring a condition or disorder. For example, the aforementioned prevention refers to all actions taken to inhibit or delay the occurrence of skin lesions or symptoms by applying the compositions according to the invention.
[0041] The term “treatment” as used in this article includes suppressing, reducing or eliminating the development of disease.
[0042] As used in this article, the term “improvement” can refer to any behavior that at least reduces parameters (e.g., the severity of symptoms) associated with the relief or treatment of a condition.
[0043] The pharmaceutical compositions of the present invention may contain *Micrococcus luteus*, or its lysates, culture media, or extracts, as an active ingredient in the pharmaceutical composition having antibacterial activity, or as an adjuvant to an active ingredient having pharmaceutical uses. The pharmaceutical compositions may be prepared according to methods well known to those skilled in the art.
[0044] The above-described pharmaceutical compositions can be administered orally or non-orally as desired, and can be used in the form of conventional pharmaceutical preparations. Non-oral administration refers to administration via routes other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous administration. When the above-described pharmaceutical compositions of the present invention are used as pharmaceutical products, they may further contain one or more active ingredients having the same or similar functions.
[0045] The above-described pharmaceutical compositions may be further prepared by comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other conventional additives (such as antioxidants, buffers, antibacterial agents, etc.) may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the drug into dosage forms for injection (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules, or tablets. Moreover, the drug may be preferably formulated according to each disease or ingredient using appropriate methods in the art.
[0046] When formulating the above-mentioned pharmaceutical compositions, commonly used diluents or excipients such as fillers, expanders, binders, wetting agents, disintegrants, and surfactants can be used. Formulations for non-oral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable esters (such as ethyl oleate) can be used as non-aqueous solvents and suspensions. As a base for suppositories, semi-synthetic fatty acid esters (Witepsol), polyethylene glycol (Macrogol), Tween 61, cocoa butter, laurin oil, and glycerin gelatin can be used.
[0047] To improve the stability and absorption of the above-mentioned pharmaceutical composition, carbohydrates (such as glucose, sucrose or dextrose), antioxidants (such as ascorbic acid or glutathione), chelating agents, and low molecular weight proteins or other stabilizers may be used as pharmaceutical agents.
[0048] In another specific embodiment of the present invention, the pharmaceutical composition may be an antibacterial topical skin composition.
[0049] Topical skin agents can be creams, gels, ointments, skin emulsifiers, skin suspensions, transdermal patches, lotions, or combinations thereof. These topical skin agents may be appropriately mixed, as needed, with ingredients used in common cosmetics or pharmaceuticals, such as aqueous, oily, powdered, alcoholic, moisturizing, thickening, UV absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients, or combinations thereof. This topical skin preparation can also be appropriately mixed with metal occlusive agents (disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), pharmaceuticals (caffeine, tannins, verapamil, licorice extract, glycyrrhizin, hot water extract of *Lysimachia christinae* fruit, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or salts, etc.), sugars (vitamin C, magnesium ascorbate phosphate, ascorbate glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.).
[0050] The topical skin composition according to the present invention may contain 0.00001% to 80% by weight of microorganisms, their lysates, cultures or extracts thereof, relative to the total weight of the composition.
[0051] The aforementioned skin includes all parts of the body, including the face, hands, arms, legs, feet, chest, abdomen, back, buttocks, and scalp.
[0052] Another aspect of the present invention provides a method for preventing, improving or treating a subject's condition, the method comprising the steps of administering or applying Micrococcus luteus, or its lysate, culture medium or extract, to the subject.
[0053] The subject's condition may be one requiring antibacterial treatment, specifically, antibacterial treatment against harmful Staphylococcus aureus.
[0054] In this specification, the terms “application,” “introduction,” and “transplantation” are used interchangeably and can refer to a method or approach by at least partially localizing a composition of a particular example to a desired site, thereby distributing the composition of a particular example into a subject.
[0055] Administration can be performed using methods known in the art. It can be administered directly to an individual in any manner, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal, or subcutaneous administration. The administration can be systemic or local.
[0056] The individuals mentioned above can be mammals, such as humans, cattle, horses, pigs, dogs, sheep, goats, or cats. These individuals may be those requiring antimicrobial treatment.
[0057] The above-mentioned administration can be to administer 0.00001 mg to 1000 mg of the composition according to the invention to each individual daily, for example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg of the composition according to the invention. However, the dosage can be prescribed in various ways based on factors such as formulation method, route of administration, patient age, weight, sex, pathological state, diet, administration time, route of administration, excretion rate, and response sensitivity. Those skilled in the art can consider these factors to appropriately adjust the dosage. Dosage can be administered once daily, or more than twice daily within the clinically acceptable range of side effects. Regarding the administration site, administration can be performed at one or more sites. Regarding the duration of administration, it can be administered daily or at intervals of 2 to 5 days, ranging from 1 to 30 days. If necessary, the same treatment can be repeated after an appropriate period. For animals other than humans, the dosage per kg can be the same as for humans, or a converted dosage can be used, such as a dosage converted based on the volume ratio of the target animal's organs (heart, etc.) to those of humans (e.g., average values).
[0058] Furthermore, the present invention provides a composition comprising Micrococcus luteus, or its lysate, culture medium or extract, for antimicrobial treatment against harmful Staphylococcus aureus.
[0059] Another aspect of the present invention provides a selective antimicrobial method for harmful Staphylococcus aureus, the method comprising the step of treating the bacteria with Micrococcus luteus, or its lysates, culture media or extracts.
[0060] At this point, the description of the selective antimicrobial activity against Micrococcus luteus, or its lysates, culture media and extracts, and against harmful Staphylococcus aureus is as described above.
[0061] In various fields requiring antimicrobial treatment, the methods of treating bacteria with Micrococcus luteus, or its lysates, cultures, or extracts, can be appropriately applied according to conventional methods well known to those skilled in the art. The treatment volume of Micrococcus luteus, or its lysates, cultures, or extracts, can be appropriately determined based on the intended use.
[0062] In another specific embodiment of the invention, an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus, may be a quasi-pharmaceutical composition.
[0063] As used herein, the term "quasi-drug" refers to articles used for the diagnosis, treatment, improvement, relief, treatment, or prevention of human or animal diseases whose effects are milder than those of pharmaceuticals. For example, under the Pharmaceutical Affairs Law, quasi-drugs refer to articles other than those used as pharmaceuticals, including fiber / rubber products used for the treatment or prevention of human and animal diseases, articles and similar items that have a minor or non-direct effect on the human body and are not instruments or machines, disinfectants and insecticides used to prevent infectious diseases, etc.
[0064] The type or dosage form of the quasi-pharmaceutical composition of the present invention is not particularly limited, but it can be bandage, gauze, absorbent cotton, adhesive bandage, disinfectant cleaner, bath foam, mouthwash, wet wipes, laundry soap, hand sanitizer, humidifier filler, face mask or filter filler, etc.
[0065] When the compositions of the present invention are included in quasi-pharmaceuticals for selective antimicrobial use, the compositions may be used directly or together with other quasi-pharmaceutical ingredients, and may be used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined according to the purpose of use, and the quasi-pharmaceutical compositions based on the present invention may contain 0.01% to 20% by weight of microorganisms, their lysates, cultures, or extracts relative to the total weight of the composition.
[0066] In another specific embodiment of the present invention, an antibacterial composition comprising Micrococcus luteus, or its lysates, culture media or extracts, and exhibiting selective antibacterial activity against harmful Staphylococcus aureus, may be an antibacterial food composition or an antibacterial health food composition.
[0067] The food or health food composition may be used alone with Micrococcus luteus, or its lysates, culture media, or extracts, or may be used in combination with other food or food ingredients, and used appropriately according to conventional methods. The amount of active ingredients mixed may be appropriately determined according to the purpose of use (preventive, health care, or therapeutic treatment). Generally, when manufacturing food or beverages, up to 15 parts by weight of the composition described in this application may be added relative to the raw materials.
[0068] There are no particular restrictions on the types of health functional foods mentioned above. Examples of foods in which the above substances may be added include dosage forms selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, and health beverages, and include all health foods in the conventional sense. For example, beverage compositions in the category of health functional foods may contain various flavorings or natural carbohydrates as additional ingredients.
[0069] The aforementioned natural carbohydrates can be monosaccharides (such as glucose and fructose), disaccharides (such as maltose and sucrose), polysaccharides (such as dextrin and cyclodextrin), and sugar alcohols (such as xylitol, sorbitol, and erythritol). As sweeteners, natural sweeteners (such as sematrandine and stevia extract) and synthetic sweeteners (such as saccharin and aspartame) can be used. This health food composition may also contain: nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts; alginic acid and its salts; organic acids; protective colloid thickeners; pH adjusters; stabilizers; preservatives; glycerin; alcohols; and carbonating agents or combinations thereof for use in carbonated beverages. This health functional food composition may also contain fruit pulp or combinations thereof for use in the manufacture of natural fruit juices, fruit juice beverages, and vegetable beverages.
[0070] Furthermore, when the antibacterial composition of the present invention is used as a detergent material, there are no particular limitations on the articles to which the antibacterial composition can be applied. Specifically, examples include hand sanitizers, shower gels, shampoos, conditioners, facial cleansing foams, facial cleansing gels, soaps, mouthwashes, toothpastes, bathroom cleaners, kitchen cleaners, etc.
[0071] Furthermore, when the antibacterial composition of the present invention is used as a preservative or antiseptic material, there are no particular limitations on the articles to which the antibacterial composition can be applied; specifically, examples include cosmetics, food, and pharmaceuticals.
[0072] Furthermore, when the antibacterial composition of the present invention is used as an additive material, there are no particular limitations on the articles to which the antibacterial composition can be applied; specifically, food and animal feed are examples.
[0073] In one embodiment, the present invention provides a *Micrococcus luteus* strain deposited with accession number KACC 81251BP. The aforementioned *Micrococcus luteus* strain may be a strain comprising the 16S rRNA of SEQ ID NO: 3.
[0074] The strain exhibits selective antimicrobial activity against harmful Staphylococcus. Specifically, the *Micrococcus luteus* strain exhibits antimicrobial activity against harmful Staphylococcus, but shows weak antimicrobial activity or no effect on harmless beneficial skin bacteria.
[0075] The stated *Micrococcus luteus* strain exhibits antibacterial activity against harmful staphylococci, such as *Staphylococcus aureus*, methicillin-resistant *Staphylococcus aureus* (MRSA), and *Staphylococcus capillus*, which cause skin diseases, but is not limited thereto. However, it has been confirmed that the stated *Micrococcus luteus* strain exhibits weak antibacterial activity against *Staphylococcus epidermidis*, which is a beneficial bacterium on the skin, and almost no antibacterial activity against *Staphylococcus hominis*, which is also a beneficial bacterium on the skin. Furthermore, it has been confirmed that the stated *Micrococcus luteus* strain exhibits almost no antibacterial activity against *Micrococcus luteus*, *Candida albicans* SC5314, *Corynebacterium spp.*, *Pseudomonas aeruginosa* ATCC10145, and *Escherichia coli* DH5α, which are not staphylococci.
[0076] In one specific example, the *Micrococcus luteus* strain can exhibit selective antibacterial activity against one or more of *Staphylococcus aureus*, methicillin-resistant *Staphylococcus aureus* (MRSA), and *Staphylococcus capitella*.
[0077] In one specific example, the *Micrococcus luteus* strain exhibits selective antibacterial activity against *Staphylococcus aureus*, methicillin-resistant *Staphylococcus aureus* (MRSA), and *Staphylococcus capillus*.
[0078] Another aspect of the present invention provides a lysate, culture medium, or extract of the aforementioned Micrococcus luteus strain.
[0079] The description of the *Micrococcus luteus* strain, its lysates, and culture medium is as described above.
[0080] Lysates, cultures, or extracts of the aforementioned *Micrococcus luteus* strains exhibit selective antibacterial activity against harmful staphylococci. Specifically, the selective antibacterial activity against harmful staphylococci is as described above for the *Micrococcus luteus* strains.
[0081] Invention Effects A composition comprising Micrococcus luteus, or its lysates, culture medium or extract, according to one aspect of the present invention, can exhibit selective antibacterial activity against harmful staphylococci, particularly Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus capillus, and thus can be effectively used as an antibacterial composition in cosmetics, quasi-pharmaceuticals, topical skin agents or pharmaceuticals, etc. Attached Figure Description
[0082] Figure 1This is the result of determining the growth inhibition ability of Staphylococcus aureus ATCC25923, methicillin-resistant Staphylococcus aureus (MRSA) USA 300, Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis, Staphylococcus capitulata, Micrococcus luteus, Candida albicans SC5314, Corynebacterium spp., Pseudomonas aeruginosa ATCC10145, and Escherichia coli DH5α when homogenized using a strain of a specific example of the present invention (N-CNT: negative control group; cell lysate: strain homogenate).
[0083] Figure 2 This is a graph showing the results of absorbance determination by WST8 analysis to detect the degree of Staphylococcus aureus biofilm formation when homogenized with a strain of a specific example of the present invention (control group: PBS, 0% cell lysate; experimental group: lysate, 20% cell lysate).
[0084] Figure 3 This is a graph showing the results of determining the cell viability of human keratinocytes when homogenized using a strain of a specific example of the present invention (control group: 10% PBS; experimental group: 10% cell lysate).
[0085] Figure 4 This is an experimental result used to confirm the properties of the active ingredient in the strain homogenate after heating or treating with proteinase K, specifically in the case of Staphylococcus aureus ATCC 25923, and measuring its antibacterial activity (heat treatment: 95°C, 10 minutes; proteinase K: 1 mg / mL). Figure 5 The results show the results of comparing the growth inhibition abilities of *Micrococcus luteus* strain CBN009 (a specific example of the present invention) and its standard strain (*Micrococcus luteus* ATCC 4698) on *Staphylococcus aureus* ATCC 25923, methicillin-resistant *Staphylococcus aureus* USA 300 (MRSA), *Staphylococcus epidermidis* ATCC 12228, *Staphylococcus hominis*, *Staphylococcus capitella*, *Micrococcus luteus*, *Candida albicans* SC5314, *Corynebacterium spp.*, *Pseudomonas aeruginosa* ATCC10145, and *Escherichia coli* DH5α when treated with homogenate of *Micrococcus luteus* strain CBN009 and its standard strain (*Micrococcus luteus* ATCC 4698). Detailed Implementation
[0086] The invention will be described in more detail below by way of examples. However, these examples are for illustrative purposes only, and the scope of the invention is not limited to these examples.
[0087] Example 1. Isolation and Identification of Strains Skin samples were collected from healthy Korean individuals and mixed in phosphate-buffered saline (PBS) solution. The mixture was then inoculated into LB (Luria-Bertani) medium using a serial dilution method. Bacterial colonies formed on plates after 63 hours of incubation at 34°C were screened and purified using the streaking method under the same medium and conditions. To confirm the molecular phylogenetic characteristics of the cultured strains, 16S rRNA gene sequence analysis was performed. PCR (Polymerase chain reaction) amplification was performed 32 times at 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute and 45 seconds, followed by a final treatment at 72°C for 5 minutes. Primers (SEQ ID NO: 1 and SEQ ID NO: 2) designed to achieve a specific reaction for common bacterial genes were used. The PCR amplification product was purified, and the base sequence (Macrogen, Korea) was confirmed. This sequence was then analyzed using the BLAST program from the National Center for Biotechnology Information (NCBI). Species exhibiting over 99% sequence homology with previously registered strains were identified as closely related. The strain isolated in this embodiment possessed the 16S rRNA sequence of SEQ ID NO: 3 (complementary DNA) and was identified as *Micrococcus luteus*. This strain was named *Micrococcus luteus* CBN009 and deposited at the Agricultural Genetic Resources Center on January 3, 2023, with accession number KACC81251BP.
[0088] Example 2. Cultivation and homogenization of the strain The strain isolated in Example 1 was placed in Mueller Hinton broth and cultured at 30°C. Cells were collected at the final time point of the exponential phase on the growth curve. The cells were centrifuged at 8000 rpm for 30 minutes and washed once with phosphate-buffered saline (PBS). The cells were then dissolved in PBS and homogenized by sonication. The homogenized strain homogenate was passed through a 0.2 µm filter to remove cells and quantified at 2 mg / mL using the Bradford assay for subsequent evaluation and activity analysis. For subsequent evaluation and activity analysis, these procedures were performed no more than one day after production.
[0089] Experimental Example 1. Antibacterial Activity The analysis examined whether the strain homogenate obtained in Example 2 exhibited antibacterial activity against harmful strains.
[0090] Specifically, antibacterial activity experiments were conducted against Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus USA 300 (MRSA), Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis, Staphylococcus capitulata, Micrococcus luteus, Candida albicans SC5314, Corynebacterium spp., Pseudomonas aeruginosa ATCC 10145, and Escherichia coli DH5α. First, to utilize the strains in the experiments, they were cultured in trypsin-soybean broth (TSB) or MH medium, with bacteria cultured in an aerobic chamber at 34°C and yeast in an aerobic chamber at 30°C. Cells were subcultured at the final time point of the exponential phase on the growth curve, achieving a passage ratio of 1 / 100 (seed / media). Cell lines reaching the initial exponential phase on the growth curve through subculturing were then diluted to OD. 600 A 0.01 dilution was used to evaluate its antibacterial ability.
[0091] The antibacterial activity of the strain homogenates was determined under the following conditions. First, on an MH agar plate, the dilutions of the 10 strains to be evaluated for antibacterial activity (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Micrococcus luteus, Candida albicans, Corynebacterium propinquum, Pseudomonas aeruginosa, and Escherichia coli) were spread twice with a sterile cotton swab, and then agar wells with a diameter of 0.8 cm were prepared. After inoculating 150 µL of the strain homogenate prepared in Example 2 into agar wells, the cultures were incubated at 30°C for 24 and 48 hours, and the resulting clear zones were observed. The results are shown below. Figure 1 middle.
[0092] Figure 1 The results show the inhibitory effects of the strain homogenized in Example 2 on the growth of Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus USA 300 (MRSA), Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis, Staphylococcus capitulata, Micrococcus luteus, Candida albicans SC5314, Corynebacterium spp., Pseudomonas aeruginosa ATCC10145, and Escherichia coli DH5α (N-CNT: negative control group; Cell lysate: strain homogenate).
[0093] like Figure 1As shown, clear zones were formed in *Staphylococcus aureus* ATCC 25923, *MRSA*, *Staphylococcus epidermidis*, and *Staphylococcus capitella* treated with homogenates of the strains from Example 2. Specifically, clear zones were formed in *Staphylococcus aureus* ATCC 25923, *MRSA*, and *Staphylococcus capitella*, which are harmful skin bacteria, thus confirming antibacterial activity. In *Staphylococcus epidermidis*, which is a beneficial skin bacterium, the clear zones were reduced and less distinct compared to *Staphylococcus aureus* ATCC 25923, *MRSA*, and *Staphylococcus capitella*, thus confirming weaker antibacterial activity. In *Staphylococcus hominis*, which is a beneficial skin bacterium, almost no clear zones were formed, thus confirming almost no antibacterial activity. Furthermore, in *Micrococcus luteus*, *Candida albicans* SC5314, *Corynebacterium spp.*, *Pseudomonas aeruginosa* ATCC 10145, and *Escherichia coli* DH5α, which are not Staphylococcus, almost no clear zones were formed even after homogenization with the strains from Example 2, thus indicating almost no antibacterial activity. These results indicate that the strain homogenate of Example 2 exhibits selective antibacterial activity against harmful Staphylococcus bacteria, particularly against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Staphylococcus capillus.
[0094] Experimental Example 2. Inhibition of Biofilm Formation This experiment was conducted to confirm whether the strain homogenate obtained in Example 2 could inhibit the biofilm formation of Staphylococcus aureus ATCC 25923.
[0095] Specifically, firstly, a frozen stock of Staphylococcus aureus ATCC 25923 strain was inoculated into MH medium and cultured at 34°C for 17 hours. Then, the strain was inoculated again into fresh MH medium and subcultured at 30°C to allow it to reach the early stage of the exponential phase on the growth curve.
[0096] Before the experiment, 160 µL of MH medium containing 1% glucose was added to all usable wells of a 96-well plate. Half of these usable wells were treated with 40 µL of PBS, and the remaining half were treated with 40 µL of cell homogenate with a protein concentration of 2 mg / mL. Then, 2 µL of Staphylococcus aureus subculture medium was added to each well, and the cells were grown at 30 °C and 200 rpm for 24 hours. The wells treated with PBS served as a negative control.
[0097] The wells were then washed twice with distilled water, and the biofilm was fixed by drying. The wells were then stained with 0.1% crystal violet solution. The wells were then washed three times with distilled water, and the biofilm was dissolved using 30% acetic acid solution. The absorbance at 560 nm was measured using a microplate reader, and the results are shown below. Figure 2 middle.
[0098] Figure 2 This is a graph showing the absorbance results determined by WST-8 analysis to detect the degree of Staphylococcus aureus biofilm formation when homogenized with the strain of Example 2 (control group: PBS, 0% cell lysate; experimental group: lysate, 20% cell lysate).
[0099] like Figure 2 As shown, in Staphylococcus aureus treated with strain homogenate, the biofilm formation was reduced to 27.4% compared to the PBS-treated group. This confirms that the strain homogenate significantly inhibits Staphylococcus aureus biofilm formation.
[0100] Experimental Example 3. Relative Cell Viability Evaluation To confirm whether the strain isolated in Example 1 was toxic to cells, cell viability was tested.
[0101] Specifically, firstly, the human epidermal keratinocyte cell line (HaCaT) was cultured in DMEM medium (Dubelcco's modified eagle medium) containing 10% fetal bovine serum (FBS) at a concentration of 2.5 × 10⁻⁶. 4 Cells were seeded at a density of 24-well plates, cultured overnight, and then cultured for 24 hours in FBS-free DMEM medium.
[0102] The cytotoxicity of the strain homogenate was evaluated using the WST assay. Cultured cell lines were treated with a 2 mg / ml homogenate of the strain in 10% of the culture medium or with 10% PBS, and cultured for 24 hours under the same conditions. A 10% PBS treatment group served as a negative control, with a total treatment volume of 500 µL per well. After removing the culture medium, the cells were washed once with 1×PBS, and DMEM medium containing WST-8 reagent was added. The cells were then cultured at 37°C for 0.5–4 hours. The absorbance at 450 / 625 nm was then measured using a microplate reader, and the results are shown below. Figure 3 middle.
[0103] Figure 3 This is a graph showing the results of WST analysis evaluating the viability of human keratinocytes treated with homogenates of the strains isolated in Example 1 (control group: 10% PBS; experimental group: 10% cell lysate).
[0104] like Figure 3 As shown, when treated with a 10% (v / v) homogenate of the strain, there was no significant difference in the viability of human keratinocytes compared to the negative control group treated with PBS, thus indicating that the strain homogenate is not cytotoxic. This result suggests that applying the strain homogenate to the skin is safe.
[0105] Experimental Example 4. Analysis of Antibiotics The antibacterial activity of Staphylococcus aureus ATCC 25923 was observed to change when the strain obtained in Example 2 was homogenized and heated or treated with a proteolytic enzyme (Protease K).
[0106] Specifically, as shown in Experiment 1 above, the strain homogenate of Example 2 exhibited antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, and Staphylococcus capillus. To investigate the source of this activity, this experiment was conducted. The same experimental method as in Example 1 was used, and Staphylococcus aureus ATCC25923 was used as the antibacterial target strain. After preparing the strain homogenate according to Example 2, it was divided into 1.5 mL portions and dispensed into three E-tubes. Each tube underwent the following treatments: one tube was directly stored in an ice bath, one tube was treated at 95°C for 10 minutes, and one tube was treated with proteinase K at a concentration of 1 mg / mL. 150 µL of the homogenate from each treatment was inoculated into agar wells and cultured at 30°C for 24 and 48 hours. The resulting clear areas were observed, and the results are presented. Figure 4 middle.
[0107] Figure 4 This is an experimental result obtained by treating Staphylococcus aureus ATCC 25923 with a homogenate of the strain after heating or treating it with proteinase K, and measuring its antibacterial activity. This is used to confirm the properties of the active substance in the strain homogenate (heat treatment: 95℃, 10 minutes, proteinase K: 1 mg / mL).
[0108] like Figure 4 As shown, when the homogenate of this strain was heat-treated or treated with proteinase K, the clear regions became blurred or disappeared, indicating that the antibacterial activity was inhibited. In particular, when treated with proteinase K, the clear regions were confirmed to completely disappear. Therefore, it can be inferred that the antibacterial activity confirmed in the homogenate of this strain is attributable to any protein.
[0109] Experimental Example 5. Comparison of antibacterial activity with standard strains In this experiment, the antibacterial activity of the Micrococcus luteus CBN009 strain homogenate obtained in Example 2 was compared with that of its standard strain (Micrococcus luteus ATCC 4698).
[0110] Specifically, antibacterial activity was tested against Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus USA 300 (MRSA), Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis, Staphylococcus capitulata, Micrococcus luteus, Candida albicans SC5314, Corynebacterium spp., Pseudomonas aeruginosa ATCC10145, and Escherichia coli DH5α. First, to utilize the strains in the experiments, they were cultured in TSB or MH media, with bacteria cultured in an aerobic chamber at 34°C and yeast in an aerobic chamber at 30°C. At the final time point of the exponential phase on the growth curve, the cells were subcultured to a ratio of 1 / 100 (seed / media). Cell lines reaching the initial exponential phase on the growth curve through subculturing were then diluted to OD. 600 A 0.01 dilution was used to evaluate its antibacterial ability.
[0111] The antibacterial activity of the strain homogenate was determined under the following conditions. First, on an MH agar plate, dilutions of the 10 strains (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus capitis, Micrococcus luteus, Candida albicans, Corynebacterium propinquum, Pseudomonas aeruginosa, and Escherichia coli) to be evaluated were applied twice with a sterile cotton swab to prepare agar wells with a diameter of 0.8 cm. After inoculating 150 µL of the strain homogenate prepared in Example 2 into the agar wells, the plates were incubated at 30°C for 24 and 48 hours, and the resulting clear zones were observed. The results are shown below. Figure 5 middle.
[0112] Figure 5 The results show the inhibitory effects of the homogenate of Micrococcus luteus CBN009 obtained in Example 2 and its standard strain (Micrococcus luteus ATCC 4698) on the growth of Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus USA300 (MRSA), Staphylococcus epidermidis ATCC 12228, Staphylococcus hominis, Staphylococcus capitulata, Micrococcus luteus, Candida albicans SC5314, Corynebacterium spp., Pseudomonas aeruginosa ATCC10145, and Escherichia coli DH5α.
[0113] like Figure 5 As shown, clear zones were formed in Staphylococcus aureus ATCC25923, MRSA, Staphylococcus epidermidis, and Staphylococcus capitulata treated with homogenates of the Micrococcus luteus strain CBN009, and the same results were observed in homogenates of its standard strain (Micrococcus luteus ATCC 4698). These experimental results indicate that the selective antimicrobial activity of Micrococcus luteus against harmful Staphylococcus species is a unique characteristic of the Micrococcus genus, rather than a specific characteristic of the Micrococcus luteus strain CBN009.
[0114] Collection Number Name of the institution preserving the collection: Korea Agricultural Culture Preservation Center (Overseas) Collection Number: KACC81251BP Date of preservation: January 3, 2023.
[0115]
[0116] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings International Style
[0117]
[0118] 1 According to Article 6.4(d) of the Implementing Regulations of the Treaty of Budapest, this date is the date of obtaining international depositary status.
[0119] The Budapest Treaty on the International Recognition of Microbial Deposits for Use in Patent Proceedings International Style
[0120]
[0121] 1 According to Article 6.4(d) of the Implementing Regulations of the Treaty of Budapest, this date is the date of obtaining international depositary status.
Claims
1. An antibacterial composition, characterized in that, Containing Micrococcus luteus, or its lysates, culture media, or extracts, it exhibits selective antimicrobial activity against harmful Staphylococcus aureus.
2. The antibacterial composition according to claim 1, characterized in that, Micrococcus luteus, or its lysates, cultures, or extracts, exhibit antimicrobial activity against harmful Staphylococcus aureus, but show weak antimicrobial activity or have no effect on harmless beneficial bacteria on the skin.
3. The antibacterial composition according to claim 1 or 2, characterized in that, Micrococcus luteus, or its lysates, culture media, or extracts, exhibit selective antibacterial activity against one or more of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus capitella.
4. The antibacterial composition according to any one of claims 1 to 3, characterized in that, Micrococcus luteus is a strain of Micrococcus luteus with accession number KACC 81251BP.
5. The antibacterial composition according to any one of claims 1 to 4, characterized in that, The antibacterial composition is used in cosmetics, pharmaceuticals, topical skin agents, quasi-pharmaceuticals, food, health functional foods, or detergents.
6. A method for antibacterial treatment against harmful Staphylococcus aureus, characterized in that, include: The procedure of administering Micrococcus luteus, or its lysate, culture medium or extract, to a subject.
7. A composition comprising Micrococcus luteus, or its lysates, culture medium, or extract, characterized in that, The composition is used for antibacterial treatment against harmful Staphylococcus aureus.
8. A strain of Micrococcus luteus with accession number KACC 81251BP.
9. A lysate of the strain according to claim 8.
10. A culture medium for the strain according to claim 8.
11. An extract of the strain according to claim 8.