KR102627009B1 - Bacterial strain which has antibacterial activity against skin harmful bacteria, and pharmaceutical compositions comprising the same - Google Patents

Abstract

The present disclosure relates to bacterial strains having antibacterial activity against skin harmful bacteria, and compositions containing the same, and more specifically, to skin containing Staphylococcus epidermidis microorganisms, or cultures thereof. It relates to a composition for inhibiting the proliferation or growth of harmful bacteria.

Description

Bacterial strain which has antibacterial activity against skin harmful bacteria, and pharmaceutical compositions comprising the same}

The present disclosure relates to bacterial strains having antibacterial activity against skin harmful bacteria, and compositions containing the same, and more specifically, to skin containing Staphylococcus epidermidis microorganisms, or cultures thereof. It relates to a composition for inhibiting the proliferation or growth of harmful bacteria.

The skin has a complex flora called the skin microbiome, which is home to a wide range of different types of microorganisms (e.g., various bacteria, fungi, viruses, etc.). Within these microbiomes, microorganisms interact with each other and have various effects on the health and disease of human hosts. Many studies have shown that microbiome imbalances can contribute to several types of human diseases, and in the case of the skin microbiome, there are differences between the microbiome on healthy skin and abnormal skin. are being reported. Among the skin microbiome, Staphylococcus epidermidis and Staphylococcus aureus are particularly noteworthy. Staphylococcus epidermidis is found in large numbers in healthy skin and is known to participate in maintaining the balance of the skin microbiome. In contrast, Staphylococcus aureus causes an imbalance in the skin microbiome and induces inflammation accompanied by symptoms of discomfort, itchiness and dryness, which in severe cases can cause skin diseases such as atopic dermatitis and psoriasis. It is known.

There is a need to develop strains that can be useful in maintaining the balance of the skin microbiome for the purpose of improving skin health.

Korean Patent Publication No. 10-2017-0056143 (May 23, 2017)

The first object of the present disclosure is to provide a Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP) microorganism that has the activity of inhibiting the proliferation or growth of skin harmful bacteria.

A second object of the present disclosure is to provide a culture of the above microorganisms.

The third object of the present disclosure is to provide a pharmaceutical composition for inhibiting the proliferation or growth of harmful bacteria on the skin, including the microorganism or its culture.

The first aspect provides a Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP) microorganism that has the activity of inhibiting the proliferation or growth of harmful bacteria on the skin.

The skin harmful bacteria may be one or more selected from the group consisting of Pseudomonas and Staphylococcus genus. More specifically, the skin harmful bacteria may be one or more selected from the group consisting of Pseudomonas aeruginosa and Staphylococcus aureus .

A second aspect provides a culture of the above-described microorganism.

The third aspect provides a pharmaceutical composition for inhibiting the proliferation or growth of harmful skin bacteria, including the above-described microorganisms or cultures thereof.

According to one aspect, Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP) or its culture, which has the activity of inhibiting the growth or growth of harmful skin bacteria, effectively inhibits the growth or proliferation of skin harmful bacteria. It can be suppressed.

Figure 1 shows an optical micrograph of Staphylococcus epidermidis LMT17-69 strain.
Figure 2 shows a comparison of the growth inhibition ability of Staphylococcus epidermidis standard strain ATCC 12228 and Staphylococcus epidermidis LMT17-69 against harmful bacteria ( P. aeruginosa and S. aureus ).

Hereinafter, the present invention will be described in detail.

One aspect of the present disclosure provides a Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP) microorganism that has the activity of inhibiting the proliferation or growth of skin harmful bacteria.

According to one aspect of the present disclosure, Staphylococcus epidermidis LMT17-69 significantly inhibits the growth or proliferation of harmful skin bacteria (e.g., Pseudomonas aeruginosa, Staphylococcus aureus, etc.) It has been confirmed that it can be suppressed.

“Skin harmful bacteria” referred to in the present disclosure are common bacteria that also exist on healthy skin and mucous membrane surfaces when the balance of the skin microbiome is disrupted by changes in the external environment, such as weakened immunity or exposure to a polluted environment. Microorganisms that can cause skin diseases such as mild skin infections, inflammation with symptoms of discomfort, itching, and dryness, atopic dermatitis, and eczema, as well as life-threatening diseases such as pneumonia, endocarditis, and sepsis. refers to The skin harmful bacteria may be, but are not limited to, one or more selected from the group consisting of Pseudomonas and Staphylococcus genus. More specifically, the skin harmful bacteria may be one or more selected from the group consisting of Pseudomonas aeruginosa and Staphylococcus aureus .

In the present disclosure, “microbiome” is a compound word of microbial community (microbiota) and genome, and refers to the entire microbial community and genome that coexist with humans, animals and plants, soil, sea, lakes, rock walls, and the atmosphere. In the present disclosure, the skin microbiome refers to a group of microorganisms that form a complex interrelationship between microorganisms, microorganisms, and hosts and microorganisms existing in the skin, and refers to, for example, a part of the human microbiome.

In the present disclosure, “inhibition” of the proliferation or growth of harmful bacteria on the skin means that the growth or proliferation of harmful bacteria is reduced by 10% compared to the control group that was not treated with Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP). means the ability to cause a reduction of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 100%. For example, the inhibition is 10% to 90%, 20% to 80%, 20% to 70%, 20% to 50%, 30% to 90%, 30% to 60%, 30% to 70% compared to the control group. , may cause a reduction of 30% to 50%, 40% to 60%, or 40% to 50%.

In one embodiment, the Staphylococcus epidermidis LMT17-69 (Accession Number KCTC 14632BP) microorganism may be live or dead.

In the present disclosure, “dead cells” is the opposite concept of live cells and refers to a form in which live cells and metabolites obtained through fermentation are sterilized by heating, pressurizing, drug treatment, etc. to prevent bacterial growth. “Dead cells” can also be used interchangeably with terms such as dead lactic acid bacteria cells, cultures of lactic acid bacteria, extracts of lactic acid bacteria, lysates of lactic acid bacteria, and components of lactic acid bacteria cells.

Another aspect of the disclosure provides a culture of the microorganism.

In the present disclosure, “culture” may be used interchangeably with “culture” or “fermentation” to allow Staphylococcus epidermidis LMT17-69 strain to grow and survive in vitro. It may refer to the entire medium containing the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients. The culture may refer to the culture solution itself obtained by cultivating the strain, the culture solution obtained by removing the bacterial cells from the bacterial culture solution obtained by removing the strain, a concentrate thereof, or a freeze-dried product. In one embodiment, the culture medium of the present disclosure is inoculated with a strain into a medium (e.g., TSB medium, R2A medium, TSA medium, etc.) and then incubated at room temperature, e.g., at a temperature between 10° C. and 40° C., for a certain period of time, e.g. For example, it may be obtained by culturing for 1 hour to 48 hours, 10 hours to 48 hours, 10 hours to 24 hours, 12 hours to 24 hours, or 6 hours to 12 hours.

Another aspect of the present disclosure provides a pharmaceutical composition for inhibiting the growth or proliferation of harmful skin bacteria containing the above-described microorganisms or cultures thereof as an active ingredient. The pharmaceutical composition may be used to treat one or more of skin diseases, including but not limited to inflammatory diseases, wounds, atopic dermatitis, psoriasis, or acne, severe eczema, ulcers, wounds, herpes, allergies, medicinal rash, pruritus, urticaria, and erythema. It can be used for prevention or treatment.

“Prevention” in this disclosure refers to delaying the onset of a disease, disorder or condition.

In this disclosure, “treatment” refers to the desired therapeutic effect by healing injured or damaged tissue or altering, altering, strengthening, improving, ameliorating and/or beautifying a pre-existing or recognized disease, disorder or condition. Refers to the alleviation or reduction (including partial reduction, significant reduction, almost complete reduction and complete reduction), resolution or prevention (whether temporary or permanent) of a disease, disorder or condition, so as to achieve a result.

In the present disclosure, prevention or treatment is performed by reducing the growth or proliferation of harmful bacteria on the skin by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, compared to the case where the composition of the present disclosure is not administered. More than 30%, more than 35%, more than 45%, more than 50%, more than 55%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100 %, or may be inhibited by 90% to 100%.

In the present disclosure, the composition may comprise at least 1×10 ⁶ CFU/ml, at least 1×10 ⁷ CFU/ml, at least 1×10 ⁸ CFU/ml, and at least 1×10 ⁹ CFU/ml. You can. For example, the composition comprises no more than 1×10 ¹⁵ CFU/ml, no more than 1×10 ¹⁴ CFU/ml, no more than 1×10 ¹³ CFU/ml, no more than 1×10 ¹² CFU/ml. can do. For example, the composition can kill the microorganism of the present disclosure at 1×10 ⁶ to 1×10 ¹⁵ CFU/ml, 1×10 ⁷ to 1×10 ¹⁴ CFU/ml, 1×10 ⁸ to 1×10 ¹³ CFU/ml. ml, 1×10 ⁹ to 1×10 ¹² CFU/ml, 1×10 ¹⁰ to 1×10 ¹² CFU/ml, and 1×10 ¹¹ to 1×10 ¹² CFU/ml. Additionally, it may contain 0.01 to 50% by weight, or 0.1 to 20% by weight, of the microorganism or its culture based on the weight of the composition.

In one embodiment, the composition may be formulated to further include a pharmaceutically acceptable carrier or additive in addition to the microorganism of the present disclosure or its culture. The carrier or additive may include excipients, stabilizers, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, lubricants, flavoring agents, coloring agents, diluents, dispersants, surfactants, antioxidants, buffers, bacteriostatic agents, etc. . The carrier may be, for example, any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, such as phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, for example, suitable for pharmaceutical administration. These can be oil/water emulsions, various types of wetting agents, liposomes, dispersions and coatings. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known and conventional methods.

In the present disclosure, the pharmaceutical compositions can be administered topically (e.g., but not limited to, epidermal or transdermal application, etc.) and parenterally (e.g., but not limited to, subcutaneously, intravenously, intramuscularly, intradermally, or intraperitoneally). Can be applied to a subject by any topical or systemic route known in the art, including intramucosal, buccal, sublingual, transmucosal, intranasal or intrabronchial application, etc.).

In the present disclosure, the subject can be a mammal. The mammal may be, but is not limited to, a human, dog, cat, horse, or pig.

Another aspect of the disclosure provides a method of inhibiting the growth or proliferation of skin harmful bacteria in a subject comprising applying to the subject a microorganism of the disclosure, or a composition of the disclosure.

In this disclosure, “application” means providing a microorganism of this disclosure, or a composition of this disclosure, to a subject. Microorganisms of the disclosure, or compositions of the disclosure, can be applied by a variety of suitable routes known in the art.

In the present disclosure, the microorganism of the present disclosure, or the composition of the present disclosure can be applied in an effective amount. In the present disclosure, an “effective amount” refers to an amount sufficient to achieve a beneficial or desired result as described herein when applied to a subject. In the context of this disclosure, an “effective amount” refers to an amount sufficient to inhibit the proliferation or growth of harmful skin bacteria when applied to a subject. The “effective amount” may vary depending on the administration method, formulation, age and weight of the subject, and the severity of the disease and/or condition to which it is applied, and may vary according to the judgment of a person skilled in the art of medicine and veterinary medicine. For example, the “effective amount” may be 0.01 mg to 200 mg of the above-mentioned microorganism, or culture thereof, per kg of body weight, or 0.1 mg to 400 mg of the microorganism, or culture thereof, per kg of body weight. Additionally, the “effective amount” may be applied as more than 1x10 ⁶ CFU, more than 1x10 ⁷ CFU, more than 1x10 ⁸ CFU, and more than 1x10 ⁹ CFU per subject. For example, the strain may be applied at 1x10 ¹⁵ CFU or less, 1x10 ¹⁴ CFU or less, 1x10 ¹³ CFU or less, and 1x10 ¹² CFU or less per subject. For example, the strain may be applied at 1x10 ⁶ CFU to 1x10 ¹⁵ CFU, 1x10 ⁷ CFU to 1x10 ¹⁴ CFU, 1x10 ⁸ CFU to 1x10 ¹³ CFU, 1x10 ⁹ CFU to 1x10 ¹² CFU, 1x10 ¹⁰ CFU to 1x10 ¹² CFU per subject. You can. In the present disclosure, the microorganism or its culture, which is an active ingredient, may be applied once or more times a day, for example, two or more times or divided into several times.

Example

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Isolation and identification of Staphylococcus epidermidis LMT17-69 strain

1. Staphylococcus epidermidis ( Staphylococcus epidermidis ) Isolation of strains

A Staphylococcus epidermidis strain was isolated from a woman's breast milk.

First, the sample was spread on MRS medium (Difco, USA) and cultured anaerobically at 30°C. The sample pretreatment method was aseptically taken, diluted with 180 ml of 0.85% NaCl solution, and homogenized for 5 minutes with a stomacher. Afterwards, a tube containing 9 ml of 0.85% Nacl solution was prepared by sterilizing it, and samples were prepared by stepwise dilution into the prepared tube. Afterwards, it was plated on MRS plate medium and cultured at 37°C for 2 to 3 days, and the colonies that appeared were distinguished by shape and color and purified again. Afterwards, the 16s rRNA gene base sequences of the strains were deciphered through sequencing.

2. Identification of Staphylococcus epidermidis LMT17-69 strain

(1) Analysis of morphological characteristics

The selected Staphylococcus epidermidis LMT17-69 strain was cultured on TSA plate medium (Difco, USA) and the morphology of colonies was observed. The colony morphology of the selected Staphylococcus epidermidis LMT17-69 strain on TSA plate medium is shown in Table 1 below.

LMT17-69 form circle size 0.7-1.0mm color cream color opacity opacity bump convex surface lubricity aerobic growth + anaerobic growth +

Figure 1 shows an optical microscope photo of the selected Staphylococcus epidermidis LMT17-69 strain. As shown in Figure 1, the Staphylococcus epidermidis LMT17-69 strain was a protococcus and was similar to the typical characteristics of the genus Staphylococcus.

(2) 16S rDNA analysis

The 16S rRNA gene of the isolated Staphylococcus epidermidis LMT17-69 strain was amplified, and the nucleotide sequence of the amplified 16S rRNA gene was analyzed. The amplification was accomplished by performing PCR using Staphylococcus epidermidis LMT17-69 strain genomic DNA as a template and using the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2 (Bionics Co., Ltd.) as a primer set. . The 16S rDNA sequence of the isolated strain is shown in SEQ ID NO: 3. The nucleotide sequence of the identified 16S rDNA was compared with the nucleotide sequence of known 16S rDNA using EZBioCloud (https://www.ezbiocloud.net/). As a result, the 16S rDNA of Staphylococcus epidermidis LMT17-69 had 99.93% sequence identity with the Staphylococcus epidermidis species. Additionally, as a result of phylogenetic tree analysis, Staphylococcus epidermidis LMT17-69 was identical to the Staphylococcus epidermidis species. As a result, the Staphylococcus epidermidis LMT17-69 strain was confirmed as a new strain belonging to the Staphylococcus epidermidis species.

The present inventors named the LMT17-69 strain as Staphylococcus epidermidis LMT17-69 (accession number KCTC 14632BP) and transferred it to the Korea Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology in 2021. It was deposited on June 30, 2018.

Example 2: Analysis of physiological activity characteristics of Staphylococcus epidermidis LMT17-69 strain

1. Sugar fermentation characteristics of Staphylococcus epidermidis LMT17-69 strain

The sugar metabolism characteristics of the selected Staphylococcus epidermidis LMT17-69 strain were confirmed using the API 50 CHL kit (BioMetrieux, France) according to the supplier's experimental method. Table 2 shows the sugar fermentation characteristics of the identified Staphylococcus epidermidis LMT17-69 strain.

number carbohydrate LMT17-69 24 hours 48 hours One glycerol + + 2 Erythritol - - 3 D-arabinose - - 4 L-arabinose - - 5 D-ribose - - 6 D-xylose - - 7 L-xylose - - 8 D-Adonitol - - 9 Methyl-βD-xylopyranoside - - 10 D-galactose + + 11 D-glucose + + 12 D-fructose + + 13 D-mannose - - 14 L-sorbose - - 15 L-rhamnose - - 16 Dulcitol - - 17 inositol - - 18 Mannitol - - 19 D-Sorbitol - - 20 Methyl αD-mannopyranoside - - 21 Methyl αD-glucopyranoside - - 22 N-Acetylglucosamine - - 23 Amygdalin - - 24 arbutin - - 25 Esculin - - 26 salicin - - 27 D-cellobiose - - 28 D-maltose + + 29 D-Lactose + + 30 D-Mellibiose - - 31 D-Saccharose + + 32 D-Trehalose - - 33 Inulin - - 34 D-Melegitos + + 35 D-raffinose - - 36 amidon - - 37 glycogen - - 38 xylitol - - 39 gentiobios - - 40 D-Turanos + + 41 D-lyxose - - 42 D-Tagatose - - 43 D-fucose - - 44 L-fucose - - 45 D-arabitol - - 46 L-arabitol - - 47 Potassium Glunate - - 48 Potassium 2-ketogluconate - - 49 Potassium 5-ketoglunate - -

Example 3: Evaluation of the activity of inhibiting growth or proliferation of harmful skin bacteria of Staphylococcus epidermidis LMT17-69 strain

To evaluate the activity of Staphylococcus epidermidis LMT17-69 strain to inhibit the growth or proliferation of harmful skin bacteria, an experiment was conducted using the following method. The harmful bacteria Staphylococcus aureus ATCC6538 and Pseudomonas aeruginosa ATCC9027 were inoculated at 1x10 ⁶ CFU/mL in 1 mL of sterilized Tryptic Soy Broth (TSB). A 0.4 μm cell culture insert was placed on top, and Staphylococcus epidermidis LMT17-69 was inoculated into the insert at 1x10 ⁶ CFU/mL in 1 mL of TSB. After culturing at 37°C for 6 hours, harmful bacterial samples were recovered and diluted in TSB. After plating on TSB plate medium and culturing at 37°C, the number of colonies on the plate medium was counted to estimate the number of bacteria.

Experimental results showed that the growth inhibition of Pseudomonas aeruginosa ATCC9027 and Staphylococcus aureus ATCC6538 was 25.2% and 57.2%, respectively, by a 10-fold concentrate of Staphylococcus epidermidis standard strain (ATCC 12228), whereas Staphylococcus epidermidis The growth inhibition by the 10-fold concentrate of Dis LMT17-69 was 50.2% and 64.3%, respectively, showing a significantly higher ability to inhibit the growth of harmful bacteria compared to the control group.

Taken together, as disclosed in the above examples and FIG. 2, the Staphylococcus epidermidis LMT17-69 strain of the present disclosure suggests that it can significantly inhibit the growth or proliferation of harmful skin bacteria.

Korea Research Institute of Bioscience and Biotechnology KCTC14632BP 20210630

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Claims (5)

Staphylococcus epidermidis ( Staphylococcus epidermidis), which has the activity of inhibiting the proliferation or growth of skin-harmful bacteria, wherein the skin-harmful bacteria are at least one selected from the group consisting of Pseudomonas aeruginosa and Staphylococcus aureus Staphylococcus epidermidis ) LMT17-69 (accession number KCTC 14632BP) microorganism. delete delete A culture of a microorganism according to claim 1, wherein the culture is a microorganism, a culture medium, a concentrate thereof, or a freeze-dried product of the microorganism. A pharmaceutical composition for inhibiting the growth or proliferation of harmful skin bacteria, comprising the microorganism of claim 1 or a culture thereof, wherein the harmful bacteria include Pseudomonas aeruginosa and Staphylococcus aureus. ) A composition that is one or more selected from the group consisting of.