KR102422611B1 - Antimicrobial composition comprising a novel Bacillus Velezensis strains or compounds isolated therefrom - Google Patents

Abstract

The present invention relates to a novel Bacillus velezensis , Bacillus velezensis NST6 strain, a culture solution thereof, a supernatant thereof, an extract thereof, a fraction thereof or a compound isolated therefrom, and a compound isolated therefrom, and uses thereof. The novel strain Bacillus belegensis NST6 strain according to the present invention, its culture medium, its supernatant, its extract, its fraction, or a basilomycin D analog, which is a compound isolated therefrom, is a pathogen, particularly Staphylococcus spp. As it exhibits excellent antibacterial activity against It can be usefully used for the purpose of pollution prevention, etc.

Description

TECHNICAL FIELD Antimicrobial composition comprising a novel Bacillus Velezensis strains or compounds isolated therefrom

The present invention relates to a novel Bacillus belegensis strain, a culture solution thereof, a supernatant thereof, an extract thereof, a fraction thereof or a compound isolated therefrom, and a use thereof.

Although CNS (Coagulase-negative staphylococci, CoNS) is in the normal flora, it occasionally causes infections in infants, the elderly and immunosuppressed patients as the use of insertion devices increases. Currently, 35 species and 17 subspecies of Staphylococcus belonging to the CNS are known. However, the main problem in clinical practice is Staphylococcus saprophyticus , Staphylococcus aureus , Staphylococcus aureus , and Staphylococcus epidermidis 3 species of Staphylococcus aureus.

Among them, Staphylococcus epidermidis in particular is a species of Staphylococcus aureus, a Gram-positive bacterium, also called Staphylococcus epidermis. It is present in the skin and mucous membranes and is not generally pathogenic, but there is a risk of infection in patients with compromised immune system. In particular, it is known that 75% of CNS infections are due to Staphylococcus epidermidis.

Meanwhile, until now, synthetic compounds or extracts of naturally occurring natural products have been mainly used for the development of new cosmetic materials. Recently, in addition to synthetic compounds and natural extracts, development of bio-cosmetic materials using enzymes or microorganisms has been gradually made, and among the ingredients naturally produced by living things, many studies have been conducted to use microorganism-derived ingredients as cosmetic materials. is losing As cosmetics using such microorganism-derived ingredients, products containing substances that prevent skin damage or have antifungal activity are known.

Many physiologically active substances produced by microorganisms are widely used commercially in fields such as food, pharmaceuticals, and fermented products as well as the aforementioned cosmetics, but the microorganisms themselves are very diverse depending on the classification such as species and strains. Accordingly, the types of microorganisms that can be an antibacterial target for each microorganism may also be very diverse, and sometimes only specific bacteria may exhibit an antibacterial function.

According to the Korea Centers for Disease Control and Prevention's report, about 70% of antibiotics are produced by microorganisms, and so far, more than 4,200 antibiotics have been classified in the microbial group. A certain strain produces a number of structurally and biosynthetically related antibiotics and can produce two or more different antibiotics. The production of these antibiotics is not absolutely species specific. That is, strains belonging to the same species may produce completely different antibiotics, and even if they belong to the same species, there may be many strains that do not produce antibiotics. In addition, strains of microorganisms belonging to different taxonomic classes may produce the same antibiotic. Therefore, in terms of antibiotic production, it is correct to say that it is strain specific rather than species specific.

In addition, in general, antibiotics that are simultaneously active against several types of microorganisms such as bacteria, fungi, and protozoa are highly toxic and cannot be used clinically, and only antibiotics that are active against a special class of microorganisms have selectivity (specificity). ), so there are many cases where it is developed as a drug.

On the other hand, with respect to Bacillus velezensis , the novel Bacillus velegensis (Patent Document 1), Bacillus velegensis, which does not exhibit antibacterial activity against enteric fermented bacteria, but exhibits antibacterial activity against fungi and food poisoning-inducing bacteria. Including a biological control agent for antifungal containing the strain culture solution and the supernatant, studies on many Bacillus belegensis strains and materials isolated therefrom have been made. However, it is known that the antibacterial activity against certain pathogens of the Bacillus belegensis strain, in particular, dermatophytes, is insignificant.

Under this background, the present inventors identified Bacillus belegensis NST6, a novel Bacillus genus bacterium expected to have antibacterial activity against Staphylococcus in the process of studying microorganisms living in the forest area of Gangwon-do, Korea, and the Bacillus belegen It was confirmed that cis NST6 exhibited strong antibacterial activity against Staphylococcus sp. strain. Accordingly, the Bacillus belegensis NST6 was developed as an antibiotic suitable for the killing and prevention of Staphylococcus epidermidis belonging to the CNS. Since the possibility of the effect of suppressing unnecessary increase in medical expenses can be attained, the present invention has been completed.

Korean Patent Publication No. 10-2015-0132723

It is an object of the present invention to provide a Bacillus velezensis NST6 strain of accession number KCTC14112BP.

Another object of the present invention is to use any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient. Including, Staphylococcus ( Staphylococcus ) It is to provide an antibacterial composition for the sp.

Another object of the present invention is the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or any one or more selected from the group consisting of a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient. It is to provide a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus sp.

Another object of the present invention is the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or any one or more selected from the group consisting of a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient. It is to provide a cosmetic composition for preventing or improving infectious diseases caused by Staphylococcus sp.

Another object of the present invention is the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or any one or more selected from the group consisting of a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient. It is to provide a food composition for preventing or improving infectious diseases caused by Staphylococcus sp.

Another object of the present invention is to use any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient. It is to provide a quasi-drug composition for preventing or improving infectious diseases caused by Staphylococcus sp.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides a Bacillus velezensis NST6 strain of accession number KCTC14112BP.

In the present invention, the term "Bacillus ( Bacillus ) genus" refers to rod-shaped aerobic, or facultative anaerobic, Gram-positive bacteria widely distributed in nature, also called bacilli. Bacillus strains commonly found in nature include independent living types and pathogen types. It is known that under stressful environmental conditions, oval-shaped resistant spores can be produced and remain dormant for extended periods of time.

In the present invention, the term " Bacillus velezensis NST6" is a strain isolated and identified for the first time from a soil (loess) sample obtained from a region including rocks, pine rhizospheres, and mineral springs in Gangwon-do forest area, and a single colony thereof As a result of culturing by streaking in TSA medium to analyze the , a form generally considered to be of the genus Bacillus was observed ( FIG. 2 ). In addition, as a result of decomposing the entire protein sequence of the strain into fragments of 6 aa length and creating a phylogenetic tree according to the frequency of appearance of each oligopeptide (FIG. 1), it was confirmed that it was Bacillus belegensis, and was named Bacillus belegensis NST6, which was named Korea Life Insurance. It was deposited with the Korean Collection for TypeCulture (KCTC) of the Institute of Engineering on January 16, 2020 and was given an accession number KCTC 14112BP.

The Bacillus belegensis NST6 has a 16s rDNA nucleotide sequence represented by SEQ ID NO: 1.

Another aspect of the present invention is the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or any one or more selected from the group consisting of a basilomycin D analog, which is a compound isolated therefrom, is effective It provides an antibacterial composition for the Staphylococcus sp. strain, including as a component.

The terms used herein are the same as described above.

In the present invention, the term “antibacterial” refers to an activity of preventing or treating bacterial infection by inhibiting the growth or proliferation of microorganisms including microorganisms.

The antibacterial composition may exhibit antibacterial activity against Staphylococcus sp. The Staphylococcus sp. strain is, for example, Staphylococcus saprophyticus ( Staphylococcus saprophyticus ), Staphylococcus aureus ( Staphylococcus aureus ), Staphylococcus epidermidis ), multidrug-resistant bacteria Meti may include, but are not limited to, methicillin-resistant staphylococcus aureus (MRSA) 3089, MRSA 3090, MRSA 3095, and the like.

In the present invention, the term "culture medium" refers to the strain obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that Bacillus belegensis NST6 can grow and survive in vitro, its metabolites, and extra nutrients It refers to the entire medium including the like, but also includes a culture solution from which the strain is removed after culturing the strain. On the other hand, the liquid from which the cells are removed from the culture medium is also called "supernatant", and the culture solution is left for a certain period of time to take only the liquid from the upper layer except for the part that has sunk to the lower layer, or to remove the cells through filtration or centrifuge the culture solution to the lower part. It can be obtained by removing the precipitation of and taking only the liquid at the top. The "cell" refers to the Bacillus belegensis NST6 strain itself of the present invention, and includes the strain itself isolated and selected from the soil or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the culture solution and taking the part that has sunk to the lower layer, or it can be obtained by removing the upper liquid after leaving it for a certain period of time because it sinks to the lower layer of the culture solution by gravity.

In the present invention, the term "extract" refers to a result obtained by extracting a culture medium of Bacillus belegensis NST6 or a supernatant thereof. The extract is not limited thereto, as long as it can exhibit antibacterial activity, and may include an extract, a dilution thereof, a concentrate, a dried product obtained by drying the extract, a prepared product thereof, or a purified product thereof. As long as the extract can exhibit antibacterial activity, the method is not particularly limited, and it can be extracted by methods known in the art, for example, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction. Examples of the extraction solvent include, but are not limited to, lower alcohols having 1 (C1) to 4 (C4) carbon atoms, such as water, methanol, ethanol, butanol, and the like; polar solvents such as chloroform, ethyl acetate, and acetonitrile; and the like. The solvent may be used alone or in combination of two or more. When the solvent is mixed and used, for example, it may be mixed at a mixing ratio (v/v) of 1 to 10:10 to 1, but is not limited thereto. The extraction temperature may be 20 to 100° C., and the extraction period may be about 1 hour to 10 days, but is not limited thereto.

In one embodiment of the present invention, the supernatant of Bacillus belegensis NST6 is mixed with chloroform (FIG. 4), ethyl acetate, chloroform: methanol in 9:1 (v/v) or 8:2 (v/v) ratio of chloroform and It was extracted with a methanol mixed solvent, and the extract exhibited antibacterial activity against Staphylococcus epidermidis (FIG. 3).

The extract may be further filtered at any one or more time points selected from before or after extraction with respect to the supernatant of Bacillus belegensis NST6, but is not limited thereto.

In the present invention, the term "fraction" refers to a result obtained by a fractionation method of separating a specific component or a specific group from a mixture containing various components. As long as the fraction can exhibit antibacterial activity, the method is not particularly limited, and various fractionation methods such as methods known in the art, for example, reverse phase chromatography, adsorption chromatography, ion exchange chromatography, solvent fractionation, etc. fractions can be obtained. Specifically, a method of fractionating the extract by passing it through reverse-phase high-performance liquid chromatography (HPLC) under a flow of a solvent may be used, but is not limited thereto. The type of the solvent is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include polar solvents such as water, alcohol, and acetonitrile; non-polar solvents such as hexane, ethyl acetate, acetone, and chloroform; and the like. The solvent may be used alone or in combination of two or more.

In one embodiment of the present invention, the supernatant of NST6 or the extract obtained by extracting the supernatant with chloroform was eluted with 40 to 60% acetonitrile using a Sep-Pak C18 cartridge to obtain a fraction (FIG. 6), and the fraction was It was confirmed that the inhibitory activity of the phylococcus sp. strain was excellent (Table 2).

In the present invention, the term "basilomycin D" refers to a substance belonging to the iturin family, which is one of the cyclic peptide groups, and includes seven α-amino acids (2 Asn, Glu, Tyr, Pro, Ser, Thr). and one β-amino acid, and has antibiotic activity.

The present inventors isolated and identified the antibacterial material produced and secreted by the novel Bacillus belegensis NST6 of the present invention, and as a result, it was confirmed that the isolated material is an analog of basilomycin D, and named as basilomycin D analog C15. (Figs. 7 to 11).

The basilomycin D analog may be derived from at least one selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, and its fractions, but is not limited thereto.

The chemical formula of the basilomycin D analog C15 is as shown below and in FIG. 11 .

[Formula 1]

In one embodiment of the present invention, it was confirmed that the basilomycin D analogue C15 inhibited the Staphylococcus sp. strain even at a low concentration of 2 μM, and 99.9% bactericidal activity against the Staphylococcus sp. strain at a concentration of 64 μM or more. was confirmed to represent (Fig. 12).

Another aspect of the present invention is effective for any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or basilomycin D analog, which is a compound isolated therefrom. It provides a pharmaceutical composition for preventing or treating infectious diseases caused by Staphylococcus sp.

The terms used herein are the same as described above.

As used herein, the term "prevention" refers to any action that suppresses or delays the onset of the infectious disease by administration of the composition, and "treatment" means that the symptoms caused by the infectious disease are improved or It means any action that is changed for the better.

Since the active ingredient of the present invention exhibits excellent antibacterial activity against Staphylococcus sp. strains, the pharmaceutical composition according to the present invention can be applied to various infectious diseases caused by Staphylococcus sp. strains.

The Bacillus belegensis NST6 strain of the present invention, its culture medium, its supernatant, its extract, its fraction, or a pharmaceutical comprising any one or more selected from the group consisting of a basilomycin D analog, which is a compound isolated therefrom, as an active ingredient The composition may further comprise an appropriate carrier, excipient or diluent conventionally used in the preparation of pharmaceutical compositions. At this time, the content of the active ingredient included in the composition is not particularly limited thereto, but may include 0.0001 wt% to 10 wt%, preferably 0.001 wt% to 1 wt%, based on the total weight of the composition.

The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have one dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The composition of the present invention can be administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. It may be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but for a desirable effect, the culture medium of Bacillus belegensis NST6 of the present invention, its supernatant, and its The extract or its fraction is preferably administered at 0.0001 to 100 mg/kg per day, preferably at 0.001 to 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The composition may be administered to various mammals such as rats, livestock, and humans by various routes, and the method of administration includes without limitation as long as it is a conventional method in the art, for example, oral, rectal or intravenous, muscle, It may be administered by subcutaneous, intrauterine dural or intracerebrovascular injection.

In addition, the pharmaceutical composition of the present invention can be used in the form of veterinary drugs as well as pharmaceuticals applied to humans.

Another aspect of the present invention is effective for any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or basilomycin D analog, which is a compound isolated therefrom. It provides a cosmetic composition for preventing or improving infectious diseases caused by Staphylococcus sp.

The terms used herein are the same as described above.

The "improvement" refers to suppressing or inhibiting the symptoms of bacterial infection occurring on the skin, or alleviating the symptoms of infection that have already appeared.

In the present invention, the cosmetic composition comprises a solution, an external ointment, a cream, a foam, a nourishing lotion, a softening lotion, a pack, a softening water, an emulsion, a makeup base, an essence, a soap, a liquid detergent, a bath agent, a sunscreen cream, a sun oil, Suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations, patches and sprays can be prepared in a formulation selected from the group consisting of, but not limited

In the present invention, the cosmetic composition may further include one or more cosmetically acceptable carriers to be formulated in general skin cosmetics, and common ingredients include, for example, oil, water, surfactant, humectant, and lower alcohol. , a thickener, a chelating agent, a pigment, a preservative, a fragrance, etc. may be appropriately mixed, but is not limited thereto.

The cosmetically acceptable carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

When the formulation of the present invention is an ointment, paste, cream or gel, as a carrier component, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohard propellants such as, but not limited to, locarbon, propane/butane or dimethyl ether. These may be used alone or in combination of two or more.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier may be used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butylglycol oil, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used, but are limited thereto doesn't happen These may be used alone or in combination of two or more.

When the formulation of the present invention is a suspension, as a carrier component a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters; Crystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used, but is not limited thereto. These may be used alone or in combination of two or more.

When the formulation of the present invention is a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolsate, isethionate, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugar, etc. may be used as carrier components. may, but is not limited thereto. These may be used alone or in combination of two or more.

Another aspect of the present invention is effective for any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or basilomycin D analog, which is a compound isolated therefrom. It provides a food composition for preventing or improving infectious diseases caused by Staphylococcus sp.

The terms used herein are the same as described above.

The "improvement" refers to any action in which the symptoms of the subject suspected of the infectious disease and the invention are improved or beneficial by using the composition.

As a food-acceptable salt that can be included in the food composition of the present invention, an acid addition salt formed by a food-acceptable free acid or a metal salt formed by a base is useful. As an example, an inorganic acid and an organic acid may be used as the free acid. Hydrochloric acid, sulfuric acid, hydrobromic acid, sulfurous acid or phosphoric acid may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaic acid, gluconic acid, methanesulfonic acid, or the like may be used as the organic acid. In addition, as the metal salt, an alkali metal salt or an alkaline earth metal salt, sodium, potassium or calcium salt can be used. However, it is not limited thereto.

The food composition of the present invention includes the form of pills, powders, granules, needles, tablets, capsules or liquids, and the food to which the composition can be added includes, for example, various foods, for example, beverages; There are chewing gum, tea, vitamin complexes, and health supplements.

As a component that can be included in the food composition of the present invention, there is no particular limitation on other components other than containing an active ingredient as an essential component. may contain. The content of the active ingredient in the food composition may be suitably determined according to the purpose of use (prevention, improvement or therapeutic treatment). At this time, the content of the active ingredient included in the composition is not particularly limited thereto, but may include 0.0001 wt% to 10 wt%, preferably 0.001 wt% to 1 wt%, based on the total weight of the composition.

In addition, the food supplement additives may include food supplement additives conventional in the art, for example, flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like.

Examples of the natural carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavoring agents (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used as flavoring agents other than those described above.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, it may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages. These components may be used independently or in combination.

In the present invention, the health supplement may include health functional food and health food.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, "function (sex)" means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.

The food containing the food composition of the present invention can be prepared by a method commonly used in the art, and during the preparation, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of the drug using food as a raw material, and can be excellent in portability.

Another aspect of the present invention is effective for any one or more selected from the group consisting of the Bacillus belegensis NST6 strain, its culture medium, its supernatant, its extract, its fraction, or basilomycin D analog, which is a compound isolated therefrom. It provides a quasi-drug composition for preventing or improving infectious diseases caused by Staphylococcus sp.

The terms used herein are the same as described above.

As used herein, the term "quasi-drug" refers to items that are not instruments, machines, or devices among articles used for the purpose of diagnosing, treating, alleviating, treating, preventing or ameliorating diseases of humans or animals, and to the structure and function of humans or animals. It refers to items that are not used for the purpose of pharmacological influence, not instruments, machines, or devices, and includes external preparations for skin and personal care products.

When any one or more selected from the group consisting of a culture medium of Bacillus belegensis NST6 according to the present invention, a supernatant thereof, an extract thereof, and a fraction thereof according to the present invention is added to the quasi-drug composition as an active ingredient, the active ingredient is added as it is or other quasi-drug ingredients can be used together, and can be used appropriately according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

The external preparation for skin is not particularly limited thereto, but is preferably prepared and used in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels or gels. The personal care products are not particularly limited thereto, but are preferably soap, cosmetics, wet tissues, toilet paper, shampoo, skin cream, face cream, toothpaste, lipstick, perfume, makeup, foundation, cheek touch, mascara, eye shadow, sunscreen. It may be a lotion, a hair care product, an air freshener gel or a cleansing gel. In addition, another example of the quasi-drug composition of the present invention may include, but is not limited to, disinfectant cleaner, shower foam, gargrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

The new strain according to the present invention, Bacillus velezensis NST6 culture medium, supernatant, extracts, fractions, or basilomycin D analogues isolated therefrom are pathogens, particularly Staphylococcus spp. It is useful for preventing, improving, or treating microbial infections in the fields of antibacterial agents for plants/crops, pharmaceuticals, health functional foods and cosmetics that require antibacterial efficacy against Staphylococcus spp. It can be used, and it can be usefully used for the purpose of preventing contamination in the field of quasi-drugs.

1 is a phylogenetic tree of 22 Bacillus species including the NST6 strain.
2 is a diagram showing the morphology of Bacillus belegensis NST6.
3 is a diagram showing the antibacterial activity of the chloroform and methanol mixed solvent extract of Bacillus belegensis NST6 supernatant against Staphylococcus epidermidis.
4 is a schematic diagram showing a method for preparing an extract of Bacillus belegensis NST6 supernatant.
Figure 5 is a diagram showing the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the supernatant (supernatant) and chloroform extract (crude) of Bacillus belegensis NST6 for various pathogens.
6 is a schematic diagram showing a method for preparing a fraction of a Bacillus belegensis NST6 supernatant.
7 is a result of RP-HPLC separation of the chloroform extract of the Bacillus belegensis NST6 supernatant.
FIG. 8 is a result (part C14) analyzed in positive mode in MS-infusion of peak 1 of FIG. 7 .
9 is a result of analysis (C15 part) in positive mode in MS-infusion of the 2 peaks of FIG. 7 .
FIG. 10 is an HR-MS for more accurate analysis of the 2 peaks of FIG. 7, and shows the results of analyzing both positive and negative modes
11 is a chemical formula of basilomycin analog D(C15) showing antibacterial activity.
12 is a diagram showing the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the basilomycin analogs D (C14 and C15) against Staphylococcus sp. strains.

Hereinafter, the present invention will be described in more detail by way of Examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these examples, and it will be apparent to those of ordinary skill in the art to which the present invention pertains.

Example 1. Isolation of novel microorganisms of the present invention

Soil (loess) samples were obtained from rocks, pine roots, and mineral springs among forest areas in Gangwon-do. The collected loess samples (1 g each) were suspended in 9 ml of sterile water, diluted 10 times, evenly distributed in TSA (Tryptic soy agar) medium, and cultured at 37° C. for one day, and the strain was isolated therefrom. As a result, a total of 20 strains were isolated, and the optimum medium, optimum temperature, and pH were tested for these strains. Each strain was stored in TS broth (Tryptic soy broth) at -80 ℃ in a medium to which 80% glycerol was added. Among about 20 strains isolated from soil, various harmful pathogens (MRSA, Staphylococcus aureus ) and about 10 candidate groups screened by the replacement culture method were selected. In order to analyze a single colony of the selected strain, as a result of culturing by streaking on a TSA medium, a form generally determined to be of the genus Bacillus was observed (FIG. 2).

Example 2. Molecular biological identification of selected strains

The entire protein sequence of the strain selected in Example 1 was decomposed into 6 aaa length fragments to create a phylogenetic tree according to the frequency of occurrence of each oligopeptide (Qi et al. 2015, Ha, Zuo 2004; 2015, Zuo 2004) .

As a result, it was confirmed that the selected strain was Bacillus velezensis (951 bp). The identified microorganism was named Bacillus belegensis NST6 and deposited at the Korea Research Institute of Bioscience and Biotechnology Genetic Resources Center (KCTC), an international depository under the Budapest Treaty (accession number: KCTC 14112BP).

Example 3. Antibacterial activity assay of the supernatant extract of the selected strain extracted with an organic solvent

3-1. Selection of extraction organic solvent ratio showing maximum antibacterial activity

In order to select the ratio of the extracted organic solvent optimized for the antibacterial activity of the Bacillus belegensis NST6 strain extract, which is a novel microorganism, a disk diffusion method or an agar diffusion assay was performed. Bacillus belegensis NST6 strain was cultured in TSB medium (BD Difco, USA) at 180rpm, 37°C for 20 hours. After culturing each Bacillus to 2x10 ⁶ CFU/ml, centrifugation was performed at 7,000 rpm and 4° C. for 20 minutes to obtain a supernatant of each Bacillus from which the cells were removed.

The supernatant of NST6 was extracted using silica gel column chromatography using a chloroform:methanol (9:1 ~ 1:9 v/v) mixed solvent mixed with 0.5 ml of methanol with a concentration gradient.

Each extract was concentrated under a reduced pressure by rotary evaporation (evaporator, EYELA, USA), and then eluted (elution). Each eluted extract was redissolved in a minimum amount of HPLC (High Performance Liquid Chromatography) grade methanol, and then filtered with a 0.45 µm 13 mm non-sterile syringe filter (0.45 µm 13 mm non-sterile syringe filter, FUTECS).

Staphylococcus epidermidis, which is a Staphylococcus epidermis , was filtered in a Muller Hilton medium in which 2x10 ⁶ CFU/ml was cultured, and 20 ul of each filtered extract was loaded and dried. A paper disk of 6 mm in diameter (Thick, ADVANTEC) was used, and the same amounts of HPLC grade methanol and ethyl acetate (EtOAC) were used as controls. The paper disks were cultured for 24 hours at 37° C. for 24 hours to measure the inhibition of the growth of 13 pathogens through the size of the clear ring (claer zone).

As a result, the extract showing a clear ring of 0.6 mm or more for Staphylococcus epidermidis was chloroform: methanol 9:1 v / v mixed solvent extract (15 mm) and chloroform: methanol 8: 2 v / v mixed solvent extract ( 20 mm) (Fig. 3).

3-2. Selection of extracted organic solvents showing maximum antibacterial activity

In order to select an extraction organic solvent optimized for the antibacterial activity of a novel microorganism, Bacillus belegensis NST6 strain extract, Bacillus belegensis NST6 strain and NSL4 and NSN9, which are other strains as controls, were cultured, and the supernatant of each Bacillus was cultured. obtained.

The supernatant of NST6, NSL4 or NSN9 was extracted with chloroform (CHCl ₃ ) mixed with 0.5 to 1 ml of methanol (NST6 CHCl ₃ , L4 CHCl ₃ , N9 CHCl ₃ ) ( FIG. 4 ).

In addition, the supernatant of NST6 was extracted with ethyl acetate (EtOAC) or extracted with chloroform:methanol (v/v) mixed solvent (9:1, 8:2 v/v) using silica gel column chromatography. (NST6 EtOAC, NST6 9:1, NST6 8:2).

The obtained extract was subjected to the disk diffusion method of Example 3-1 for 8 pathogens, and the results are shown in Table 1 below. The same amount of HPLC grade methanol was used as a control.

Gram Stain Type Claer zone - Escherichia coli - - - Salmonella typhimurium - - + Bacillus subtils - - + Staphylococcus aureus NST6 CHCl ₃ 1.5 cm + Staphylococcus epidermidis NST6 CHCl ₃
NST6 EtOAc
NST6 9:1
NST6 8:2 1.8 cm
1.1 cm
1.3 cm
1.3 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3089 NST6 CHCl ₃ 1.2 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3090 NST6 CHCl ₃ 1.7 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3095 NST6 CHCl ₃ 1.5 cm

As a result, the NST6 CHCl ₃ extract exhibited excellent inhibitory activity against Staphylococcus sp. strains, specifically, Staphylococcus epidermidis, Staphylococcus aureus , and three MRSA strains, which are multidrug-resistant bacteria. . On the other hand, E. coli, Salmonella typhimurium ( Salmonella typhimurium ), Bacillus subtilis ( Bacillus subtills ) did not show inhibitory activity.

In particular, in Staphylococcus epidermidis, in addition to NST6 CHCl ₃ , it was confirmed that antibacterial activity was also shown in the extracts of NST6 EtOAC, NST6 9:1, and NST6 8:2, but in other strains, NST6 EtOAC, NST6 9:1, NST6 8:2 No antibacterial activity was found in the extract.

Therefore, from the above results, chloroform (CHCl ₃ ) was selected as an optimal solvent for the antibacterial activity of the Bacillus belegensis NST6 strain.

3-3. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assay

The supernatant and chloroform extract from the Bacillus belegensis NST6 strain culture medium were sequentially diluted twofold using DMSO (dimethyl sulfoxide). The diluted supernatant and extract samples and the control group were inoculated at 100 μL in a sterile 96-well plate. McFarland No. After culturing 10 pathogens diluted to a turbidity of 0.5, the cells were diluted to about 2Х10 ⁶ /mL. 100 μL of the diluted pathogen was inoculated into wells containing sequentially diluted chloroform extracts. The growth degree of the pathogen was measured visually, and the concentration of the chloroform extract that completely caused the growth of the pathogen was determined as the MIC.

In addition, in the same way as above, 10 pathogens were inoculated into the wells containing the chloroform extract, and the culture solution of the wells in which the growth inhibition of the pathogen was caused was diluted with 1% peptone and spread on an LBA (Laked Blood Agar) plate. After incubation at 37° C. for 24 hours, the minimum concentration of the chloroform extract in which pathogens did not grow at all was determined as MBC.

The above experiment was repeated 3 to 6 times to increase reliability.

As a result, the supernatant of the Bacillus belegensis NST6 strain itself had no antibacterial activity, but when the supernatant was extracted with chloroform, it specifically killed Staphylococcus epidermidis among 10 pathogens at a concentration of about 250 μg/mL. It was confirmed that it can be done (FIG. 5).

Example 4. Antimicrobial activity assay of the supernatant extract of the selected strain extracted by the sample pretreatment method

In order to prepare an extract showing antibacterial activity by using the supernatant of Bacillus belegensis NST6 as it is, an extraction method using a sample pretreatment method was sought. Bacillus belegensis NST6 was cultured in TSB (Tryptic soy broth) medium at 220rpm, 37°C for 20 hours, and then centrifuged at 7,000rpm, 4°C for 20 minutes to obtain a supernatant. After taking the supernatant and passing it through a 0.45 um pressure reduction filter, using Sep-Pak C18 cartridges, Waters, 20%, 40%, 60%, 80%, 90 containing 0.05% TFA (Trifluoroacetic acid) It was eluted with % or 100% acetonitrile (ACN), respectively (FIG. 6).

In addition, in Example 3, the Bacillus belegensis NST6 strain significantly increased the antibacterial effect on the Staphylococcus sp. strain when extracted using an organic solvent of chloroform, and separation and purification were attempted using a Sep-Pak C18 cartridge. . After dissolving the chloroform extract of NST6 by re-extraction with 30% ACN, it was eluted with 30%, 60% or 90% ACN containing 0.05% TFA, respectively (FIG. 6).

The disk diffusion method of Example 3-1 was performed for 8 pathogens using the eluted extract, and the results are shown in Table 2 below. The same amount of HPLC grade methanol was used as a control.

Gram Stain Type Claer zone - Escherichia coli - - - Salmonella typhimurium - - + Bacillus subtils - - + Staphylococcus aureus ACN 40% 1.1 cm + Staphylococcus epidermidis ACN 40% 1.2 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3089 ACN 40% 1.1 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3090 ACN 40% 1.2 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3095 ACN 40% 1.1 cm

As a result, when the NST6 supernatant was eluted with 40% ACN using the Sep-Pak C18 cartridge, the size of the clear ring for the Staphylococcus sp. strain was 1.1 to 1.2 cm, and the Staphylococcus sp. strain inhibitory activity was excellent. confirmed that. On the other hand, no inhibitory activity was observed in E. coli, Salmonella typhimurium, and Bacillus subtilis.

In addition, the disk diffusion method of Example 3-1 was performed for 8 pathogens using an extract eluted with 60% ACN using a Sep-Pak C18 cartridge with the chloroform extract of the NST6 supernatant, and the results are shown in the table below. 3 is shown.

Gram Stain Type Claer zone - Escherichia coli - - - Salmonella typhimurium - - + Bacillus subtils - - + Staphylococcus aureus ACN 60% 1.0 cm + Staphylococcus epidermidis ACN 60% 1.3 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3089 ACN 60% 0.8 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3090 ACN 60% 0.9 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3095 ACN 60% 0.8 cm

As a result, when the chloroform extract of the NST6 supernatant was eluted with 60% ACN using the Sep-Pak C18 cartridge, the size of the clear ring for the Staphylococcus sp. strain was 0.8 to 1.3 cm, thereby inhibiting the Staphylococcus sp. It was confirmed that the activity was excellent. On the other hand, no inhibitory activity was observed in E. coli, Salmonella typhimurium, and Bacillus subtilis.

Example 5. Separation and purification of antibacterial substances derived from selected strains

After confirming the antibacterial activity against the Staphylococcus sp. strain from the extract of Bacillus belegensis NST6, separation and purification of antibacterial substances exhibiting antibacterial activity was attempted. The chloroform extract of Bacillus belegensis NST6 was separated and purified using reversed-phase HPLC (Shimadzu) using a C18 column (Waters μBondapak C18 3.9 Х 300 mm). As a mobile phase, 5 to 95% acetonitrile containing 0.05% TFA was used for separation and purification by applying a concentration gradient of 1 ml per minute for 50 minutes. (Fig. 7).

The disk diffusion method of Example 3-1 was performed on eight pathogens using samples corresponding to each peak including peaks 1 and 2 of FIG. 7, and the results are shown in Table 4 below. The same amount of HPLC grade methanol was used as a control.

Gram Stain Type Claer zone - Escherichia coli 1 peak
2 peak -
- - Salmonella typhimurium 1 peak
2 peak -
- + Bacillus subtils 1 peak
2 peak -
- + Staphylococcus aureus 1 peak
2 peak 0.8 cm
0.9 cm + Staphylococcus epidermidis 1 peak
2 peak 0.9 cm
1.4 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3089 1 peak
2 peak 0.8 cm
0.9 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3090 1 peak
2 peak 0.9 cm
1.0 cm multidrug resistant bacteria methicillin-resistant staphylococcus aureus (MRSA) 3095 1 peak
2 peak 0.8 cm
0.9 cm

As a result, a clear ring of 0.8 to 1.4 cm was confirmed in the Staphylococcus sp. strain, confirming that the material in the 1st and 2nd peaks had antibacterial activity against the Staphylococcus sp. strain.

As a result of analyzing the 1 peak of FIG. 7 using liquid chromatography-mass spectrometry (LC/MS) (Shimadzu, Japan/API 2000, USA), the lipopeptide inturin family basilomycin belonging to the eluate It was confirmed that the D analog C14 (hereinafter, C14) and the basilomycin D analog C15 (hereinafter, C15) were included, and as a result of LC/MS analysis of the 2 peaks in FIG. 7, it was found that the eluate contained C15. Confirmed. The results of MS analysis for C14 and C15 are shown in FIGS. 8 and 9, respectively.

For more accurate analysis, the results of analyzing C15 in positive and negative modes using high-sensitivity High Resolution Mass Spectrometry (HR-MS) (Q Exactive Mass Spectrometer, Thermo) are shown in FIG. 10 . The structural formula of C15 is shown in FIG. 11 .

Example 6. Antimicrobial activity assay of antimicrobial substances derived from selected strains

6-1. Antimicrobial activity assay of antibacterial substances

After dissolving 100 μM of each of the antibacterial substances C14 and C15, each of which is an antibacterial substance identified in Example 5, in a minimum amount of methanol, the disk diffusion method of Example 3-1 was performed for 5 types of pathogens using this, and the The results are shown in Table 5 below. The same amount of HPLC grade methanol was used as a control.

Gram Stain Type Claer zone - Escherichia coli C14
C15 -
- - Salmonella typhimurium C14
C15 -
- - Bacillus subtils C14
C15 -
- + Staphylococcus aureus C14
C15 -
1.0cm + Staphylococcus epidermidis C14
C15 -
1.0cm

As a result, a transparent ring of 1.0 cm was confirmed only in C15, confirming that C15 had antibacterial activity against two strains of Staphylococcus sp. C14 showed no antibacterial activity.

6-2. Antimicrobial activity assay by concentration of antimicrobial substances

The disk diffusion method of Example 3-1 was performed for Staphylococcus epidermidis using C15 and C14 for each concentration (70, 80, 90, 100, 110, 128, 1,028 μM), and the results are shown in Table 6 below shown in

Basilomycin D analogues density Claer zone C14 70 μM - 80 μM - 90 μM - 100 μM - 110 μM - 128 μM - 1,028 μM - C15 70 μM 0.8 cm 80 μM 0.8 cm 90 μM 0.8 cm 100 μM 0.8 cm 110 μM 0.9 cm 128 μM 1.0 cm 1,028 μM 1.2 cm

As a result, it was confirmed that C14 had no antibacterial activity against Staphylococcus epidermidis at all concentrations, but C15 had antibacterial activity at all concentrations.

6-3. MIC and MBC assay of antibacterial substances

As a result of confirming the MIC and MBC of C14 and C15 in the same manner as in Example 3-2 for three Staphylococcus sp. strains, C14 had no antibacterial activity, whereas C15 was a Staphylococcus sp. strain even at a low concentration of 2 μM. It was confirmed that it has a bactericidal activity of 99.9% against Staphylococcus sp. strains at a concentration of 64 μM or more (FIG. 12).

Through the results of the above examples, the novel strain Bacillus belegensis NST6 strain according to the present invention, an extract thereof, a fraction thereof, or a basilomycin D analog that is a compound isolated therefrom is a pathogen, particularly, Staphylococcus genus It was confirmed that the strain-specifically exhibited excellent antibacterial activity.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Korea Microorganism Conservation Center (Overseas) KCCM14112BP 20200116

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Antimicrobial composition comprising a novel Bacillus Velezensis strains or compounds isolated therefrom <130> KPA200060-KR <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1428 <212> DNA <213> Unknown <220> <223> 16srRNA of NST6 <400> 1 atgcaagttc gagcggacag atgggagctt gctccctgat gttagcggcg aacgggtgag 60 taacacgtgg gtaacctgcc tgtaagactg ggataattcc gggaaaccgg ggctaatacc 120 ggatggttgt ttgaaccgca tggttcagac ataaaaggtg gcttcggcta ccacttacag 180 atggacccgc ggcgcattag ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt 240 agccgacctg agagggtgat cggccacact gggactgaga cacggcccag actcctacgg 300 gaggcagcag tagggaatct tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga 360 gtgatgaagg ttttcggatc gtaaagctct gttgttaggg aagaacaagt gccgttcaaa 420 tagggcggca ccttgacggt acctaaccag aaagccacgg ctaactacgt gccagcagcc 480 gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc 540 ggtttcttaa gtctgatgtg aaagcccccg gctcaaccgg ggagggtcat tggaaactgg 600 ggaacttgag tgcagaagag gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga 660 tgtggaggaa caccagtggc gaaggcgact ctctggtctg taactgacgc tgaggagcga 720 aagcgtgggg agcgaacagg attagatacc ctggtagtcc acgccgtaaa cgatgagtgc 780 taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg 840 gggagtacgg tcgcaagact gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900 agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcctctgac 960 aatcctagag ataggacgtc cccttcgggg gcagagtgac aggtggtgca tggttgtcgt 1020 cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt 1080 tgccagcatt cagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg 1140 gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggacag 1200 aacaaagggc agcgaaaccg cgaggttaag ccaatcccac aaatctgttc tcagttcgga 1260 tcgcagtctg caactcgact gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc 1320 cgcggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccacga gagtttgtaa 1380 cacccgaagt cggtgaggta accttttagg agccagccgc cgaaggtg 1428

Claims (11)

Staphylococcus epidermidis ( Staphylococcus epidermidis ) Having an antibacterial activity, accession number KCTC14112BP Bacillus velezensis ( Bacillus velezensis ) NST6 strain.
The Bacillus belegensis NST6 strain of claim 1, its culture medium, its supernatant, its extract, its fraction, or a compound isolated therefrom, wherein any one or more selected from the group consisting of basilomycin D analogs represented by the following formula (1) is effective Including as a component, Staphylococcus ( Staphylococcus ) Antibacterial composition for spp.
[Formula 1]

.
delete According to claim 2, wherein the basilomycin D analog is the Bacillus belegensis NST6 strain of claim 1, its culture, its supernatant, its extract, and its fractions It is derived from any one or more selected from the group consisting of, antibacterial for composition.
The antibacterial composition according to claim 2, wherein the extract is a supernatant of Bacillus belegensis NST6 extracted with chloroform, ethyl acetate, or a mixed solvent of chloroform and methanol.
The antibacterial composition of claim 5, wherein the mixed solvent of chloroform and methanol is chloroform: methanol in a ratio of 9 to 8:1 to 2 (v/v).
The antibacterial composition according to claim 2, wherein the fraction is obtained by eluting an extract obtained by extracting a supernatant of Bacillus belegensis NST6 with acetonitrile.
The Bacillus belegensis NST6 strain of claim 1, its culture medium, its supernatant, its extract, its fraction, or a compound isolated therefrom, wherein any one or more selected from the group consisting of basilomycin D analogs represented by the following formula (1) is effective Staphylococcus ( Staphylococcus ) comprising as a component, a pharmaceutical composition for preventing or treating infectious diseases caused by strains:
[Formula 1]

. The Bacillus belegensis NST6 strain of claim 1, its culture medium, its supernatant, its extract, its fraction, or a compound isolated therefrom, wherein any one or more selected from the group consisting of basilomycin D analogs represented by the following formula (1) is effective A cosmetic composition for preventing or improving infectious diseases caused by Staphylococcus sp. strains, including as a component:
[Formula 1]

.
The Bacillus belegensis NST6 strain of claim 1, its culture medium, its supernatant, its extract, its fraction, or a compound isolated therefrom, wherein any one or more selected from the group consisting of basilomycin D analogs represented by the following formula (1) is effective Staphylococcus ( Staphylococcus ) Food composition for preventing or improving infectious diseases caused by strains, including as a component:
[Formula 1]

.
The Bacillus belegensis NST6 strain of claim 1, its culture medium, its supernatant, its extract, its fraction, or a compound isolated therefrom, wherein any one or more selected from the group consisting of basilomycin D analogs represented by the following formula (1) is effective Staphylococcus ( Staphylococcus ) quasi-drug composition for preventing or improving infectious diseases caused by strains, including as a component:
[Formula 1]

.

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