KR20220093990A - Weissella cibaria strain and uses thereof - Google Patents

Abstract

The present invention relates to a strain of Weissella cibaria and uses thereof. The strain can be effectively used not only for preventing, alleviating, or treating skin-related conditions, but also for preventing, alleviating, or treating oral diseases by reducing sulfur compounds that cause bad breath and inhibiting oral microorganisms.

Description

Weissella cibaria strain and uses thereof

It relates to Weissella civaria strains and uses thereof.

The skin ecosystem provides various types of habitat for microorganisms, and a wide range of microorganisms live there. They form a symbiotic relationship with the human host, and are known to have many positive effects on the host. The skin constitutes various types of habitats, such as depressions and specialized crevices, and helps a wide range of microorganisms to grow. Basically, the skin forms a physical barrier and helps to defend against potential hazards and toxic substances from the outside. The skin becomes a connection point with the external environment and is also a gathering place for various microorganisms (fungi, bacteria, viruses and small larvae). In accordance with the selection of physical and chemical functions, microorganisms adapt to specialized niches to prepare habitats. In general, the skin is cold, acidic, and remains dry. Structurally, the epidermis forms a skin barrier, blocks the penetration of microorganisms and toxins, and plays an important role in maintaining moisture. The uppermost layer of the epidermis is composed of the stratum corneum. The epidermis has a so-called 'brick and mortar structure', and the skin tissue undergoes a continuous self-healing process, and the squames that have undergone the final differentiation process repeat the process of constantly being removed from the skin tissue.

On the other hand, some microorganisms that live on the skin inhabit the oral cavity and adjacent organs and cause bad breath. There are people who complain of discomfort in daily life due to such bad breath. In order to suppress bad breath, it is most effective to inhibit the growth of anaerobic bacteria that produce volatile sulfur compounds, which are the main components of bad breath. A disinfectant such as alcohol or a metal salt such as zinc chloride was used to remove or inhibit the growth of anaerobes or the generation of volatile sulfur compounds by anaerobes. However, these metal salts or disinfectants inhibit the occurrence of not only anaerobic bacteria that cause bad breath but also other microorganisms in the oral cavity. Gargling and spitting out of the mouth. In addition, these substances are diluted by saliva and swallowed together with saliva into the esophagus, so that the effect in the oral cavity is short, about 20 minutes to 2 hours, and then the microorganisms multiply again in the oral cavity to cause bad breath. In addition, there is a problem that it cannot be disinfected to the back of the tongue, which is the main growth site of anaerobic bacteria causing bad breath. Recently, there has been an attempt to suppress the growth of anaerobic bacteria by using lactic acid bacteria and fermented products of lactic acid bacteria. Lactic acid bacteria refer to bacteria that ferment carbohydrates to produce lactic acid as final metabolites, and lactic acid bacteria exist in the oral cavity and digestive tract of humans and animals. Typically, the lactic acid bacteria are used in the manufacturing process of fermented foods such as kimchi or yogurt, and the lactic acid bacteria used in food include Enterococcus genus, Lactobacillus genus, Lactococcus genus, Leukonostok ( Leuconostoc ) genus, Streptococcus genus, Weissella genus, and the like. However, these lactic acid bacteria have a problem in that when the lactic acid bacteria are administered to the oral cavity, they are swallowed directly into the esophagus while being diluted with saliva like metal salts or disinfectants, making it difficult to remain in the oral cavity. In addition, Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus casei ( Lactobacillus casei ), Lactobacillus salivarius ( Lactobacillus salivarius ), such as lactic acid bacteria can suppress the growth of anaerobic bacteria in vitro by making strong acids, but In the oral cavity, the strong acid produced by lactic acid bacteria is neutralized by the buffering action of saliva, making it difficult to suppress the growth of other bacteria. Moreover, since strong acids cause dental caries, there is a problem in that oral hygiene is not good if these bacteria are administered to the oral cavity for a long time. Therefore, there is a need for the development of microorganisms for reducing bad breath.

One aspect is to provide a CXO-1 strain belonging to the Weissella sp .

Another aspect is to provide a lysate or culture solution of the strain.

Another aspect is to provide a cosmetic composition comprising the strain, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof.

Another aspect is to provide a pharmaceutical composition for the treatment of wounds comprising the strain, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof.

Another aspect is to provide an oral composition comprising the strain, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof.

Another aspect is to provide a pharmaceutical composition for the prevention or treatment of oral diseases comprising the above strain, its lysate, culture medium, extract of the culture medium, or a mixture thereof.

One aspect provides a CXO-1 strain belonging to Weissella sp . In one embodiment, the Weissella cibaria strain may be a strain deposited with accession number KCCM12889P. In addition, the Weissellacibaria strain may be a strain comprising 16s rRNA of SEQ ID NO: 1.

In one embodiment, the strain increases the expression of anti-aging and anti-wrinkle factors, for example, COL1A1 or decreases the expression of MMP-1, and hyaluronan synthase 3 (Hyaluronan synthase), which is a factor related to skin moisturizing and skin barrier 3, HSA3) was confirmed to increase the expression of or filaggrin. In addition, it was confirmed that the wound healing effect of the strain is excellent. Therefore, the strain according to an aspect may have a skin beauty improvement effect, for example, skin aging inhibition, skin barrier strengthening, skin wrinkle inhibition, or skin moisturizing effect.

Another aspect provides a lysate of the strain, a culture solution, or an extract of the culture solution. Specific details of the strain are the same as described above.

As used herein, the term "culture medium" may be used interchangeably with "culture supernatant", "conditioned culture medium" or "conditioned medium", and may be used to provide nutrients so that the B. sibira strain can grow and survive in vitro. It may mean the entire medium including the strain obtained by culturing the strain for a certain period of time, its metabolites, and extra nutrients in a medium that can. In addition, the culture solution may refer to a culture solution obtained by culturing the strain in which the cells are removed from the culture solution. On the other hand, the liquid from which the cells have been removed among the above 　 culture solution 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, 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" of the present invention refers to the strain itself of the present invention, and includes the strain isolated and selected from a skin sample, or the strain isolated from the culture solution by culturing the strain. The cells can be obtained by centrifuging the 　 culture medium to take the part that has sunk to the lower layer, or by gravity 　 sink to the lower layer of the culture solution, leaving it still for a certain period of time and then removing the upper liquid It can be obtained.

The culture medium may include the culture medium itself obtained by culturing the strain, its concentrate, or a freeze-dried product or a culture supernatant obtained by removing the strain from the culture medium, its concentrate or freeze-dried product.

초과 또는 40

미만 중 어느 온도에서 일정 시간, 예를 들면, 4 내지 50시간 동안 배양하여 수득된 것일 수 있다. The culture medium is a culture medium (eg, R2A medium or TSA medium) of 10

over or 40

It may be obtained by culturing for a certain time at any temperature of less than, for example, 4 to 50 hours.

In one embodiment, the culture supernatant of the strain can be obtained by centrifuging or filtering the strain culture solution to remove the strain.

In another embodiment, the concentrate may be obtained by concentrating the supernatant obtained after filtering the strain culture solution itself, or the culture solution using a centrifuge or filter.

The culture medium and culture conditions for culturing the Weissella sibaira strain may be appropriately selected or modified by those of ordinary skill in the art.

As used herein, the term “lysate” may refer to a product obtained by disrupting the cell wall of the strain itself by chemical or physical force.

As used herein, the term "culture solution extract" means extraction from the culture solution or its concentrate, and includes an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or these prepared or purified products, and fractions obtained by fractionation thereof. can do.

Another aspect provides the use of the strain, a lysate of the strain, a culture solution, or an extract of the culture solution. Specifically, it provides a composition comprising a Weissella sibaira strain, a lysate thereof, a culture solution or an extract of the culture solution thereof.

Uses of the strain may include improving skin condition, improving skin beauty, preventing, improving or treating skin diseases.

The skin condition improvement or skin beauty improvement may be skin aging inhibition or improvement, anti-wrinkle, skin elasticity, skin regeneration, skin barrier strengthening, skin moisturizing or wound improvement.

As used herein, the term "skin aging" refers to the tangible and intangible changes that appear on the skin with age, for example, a phenomenon in which the thickness of the epidermis becomes thin, the number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle , wound recovery, skin barrier function, epidermal moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage defense, chemical removal ability, immune response, sensory function, and decreased body temperature control.

The strain or its culture medium may be for improving skin aging caused by extrinsic factors or intrinsic factors. The extrinsic factor refers to various external factors, such as ultraviolet light (light), and the intrinsic factor is also referred to as a chronological factor and refers to a factor mainly caused by the passage of time. That is, the skin aging specifically refers to not only the premature aging symptoms induced by external stimuli such as ultraviolet rays, pollution, cigarette smoke, chemical substances, etc., but also a natural aging phenomenon that occurs as the proliferation of skin cells decreases with aging. It is a concept that includes all of wrinkles, elasticity reduction, skin sagging and dryness. In addition, wrinkles include those that cause wrinkles by changing the components constituting the skin tissue by stimulation by changes in internal and external factors.

The aging may be photoaging. As used herein, the term “photoaging” is a phenomenon induced by external environmental factors, and the most representative factor is ultraviolet rays. Ultraviolet rays cause damage to biological components such as activation of proteolytic enzymes, chain cleavage of matrix proteins, and abnormal cross-linking, and repetition of these mechanisms leads to apparent skin aging.

As used herein, the term “wrinkle” refers to a state in which the elasticity of the skin is lost and loosened, and for example, the skin may be folded. The "skin wrinkle prevention or improvement" may refer to any action of preventing or improving wrinkles by inhibiting the expression of factors related to wrinkles, or increasing the total amount of collagen.

The "skin barrier strengthening" may refer to any action that enhances the function of the skin barrier that is located at the outermost part of the skin and prevents loss of moisture and nutrients. In addition, "damage to the skin barrier function" may refer to any change that appears in the skin due to the deterioration or damage of the function of the skin barrier. For example, it may include increased skin wrinkles, dryness, dermatitis, atopic dermatitis, allergic dermatitis, acne, and the like.

The "skin moisturizing" may refer to any action of maintaining skin moisture or preventing moisture loss.

The "wound" is also called "wound" and means damage to the living body. The loss may be due to external factors such as, for example, physical damage, radiation, stimulation by chemical substances, etc., proliferation of microorganisms, or internal tolerance such as stress. The wounds may include all kinds of wounds, such as cleavage, puncture, cleavage, acne, fissure, bronchial, and school spear. "Wound improvement" may refer to any action that lightens or reduces the extent of a wound on the face or body, and "wound healing" may refer to a series of biological reactions to repair the wound.

In another embodiment, the strain may be used together with other strains belonging to the genus Weissella having a skin improvement effect to show a synergistic effect. The strain belonging to the genus Weissella is, for example, Weissella confuse ( Weissella confuse ), Weissella ubarum ) and the like.

The composition comprises 0.001 wt% to 80 wt%, for example, 0.01 wt% to 60 wt%, 0.01 wt% to 40 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, based on the total weight of the composition %, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20% by weight, 0.05% to 10% by weight, 0.05% to 5% by weight, 0.1% to 60% by weight, 0.1% to 40% by weight, 0.1% to 30% by weight, 0.1% to 20% by weight, 0.1% by weight % to 10% by weight, or 0.1% to 5% by weight of the strain, a lysate thereof, a culture medium, or an extract of the culture medium thereof.

In one embodiment, the composition may be a cosmetic composition.

The cosmetic composition may have a cosmetic formulation of, for example, a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, an essence, a pack, a gel, an ampoule, or a skin adhesion type.

Components included in the cosmetic composition may include components commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances. may include

In another embodiment, the composition may be a composition for external application to the skin.

In the present specification, the external preparation for skin may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermally delivered patch, drug-containing bandage, lotion, or a combination thereof. The external preparation for skin is a component usually used in external preparations for skin such as cosmetics or pharmaceuticals, for example, an aqueous component, an oily component, a powder component, alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, a preservative, an antioxidant, a surfactant, a fragrance , colorant, various skin nutrients, or a combination thereof may be appropriately formulated as needed. The external preparation for skin includes metal-blocking agents such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belapamil, licorice extract, glablidine, and kaline. Fruit hot water extracts, various herbal medicines, tocopherol acetate, glitylittic acid, tranexamic acid and derivatives or salts thereof, vitamin C, magnesium ascorbate phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can be mix|blended suitably.

In another embodiment, the composition may be a pharmaceutical composition.

The pharmaceutical composition may further include a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, and the lubricant may be magnesium stearate, talc, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be carboxymethyl cellulose calcium, sodium starch glycolate, anhydrous calcium monohydrogen phosphate, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or a combination thereof. The lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

The pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may be granules, powders, solutions, tablets, capsules, dry syrups, or a combination thereof. The parenteral dosage form may be an injection.

In another embodiment, the composition may be a health functional food composition.

The health functional food composition may be used alone or in combination with other foods or food ingredients of the strain or its culture, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prophylactic, health or therapeutic treatment). In general, in the production of food or beverage, the composition of the present specification may be added in an amount of 15 parts by weight or less based on the raw material. There is no particular limitation on the type of the health functional food. Among the types of health functional food, the beverage composition may contain various flavoring agents or natural carbohydrates as an additional component like a conventional beverage. The natural carbohydrates include 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 the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The health food composition can also be added to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent used, or a combination thereof. The health functional food composition may also contain natural fruit juice, fruit juice beverage, fruit flesh for the production of vegetable beverage, or a combination thereof.

Another aspect provides a method of preventing, ameliorating or treating a skin condition in a subject comprising treating or administering to the subject in need thereof an effective amount of the composition. Specific details of the skin condition are the same as described above.

Another aspect provides a pharmaceutical composition for wound treatment comprising the strain, its lysate, culture medium, extract of the culture medium, or a mixture thereof as an active ingredient. Another aspect provides an external composition for wound treatment comprising the strain, its lysate, culture medium, extract of the culture medium, or a mixture thereof as an active ingredient. Another aspect provides a method for treating a wound comprising administering to a subject in need thereof an effective amount of the strain, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof. The specific details of the wound are the same as described above.

As used herein, the terms "administering", "introducing", and "implanting" are used interchangeably and into a subject by a method or route that results in at least partial localization of a composition according to one embodiment to a desired site. It may mean the arrangement of the composition according to one embodiment of the.

Administration may be administered by methods known in the art. Administration may be administered directly to a subject by any means, for example, intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. can The administration may be systemically or locally.

The subject may be a mammal, such as a human, cow, horse, pig, dog, sheep, goat, or cat. The subject may be an individual in need of skin beauty improvement, for example, skin moisturizing, skin barrier strengthening, skin wrinkle improvement effect.

The administration is 0.1 mg to 1,000 mg, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg , 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 10 mg to 50 mg, or 10 mg to 25 mg may be administered. However, the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art The dosage may be appropriately adjusted in consideration of these factors. The number of administration may be once a day or twice or more within the range of clinically acceptable side effects, and may be administered to one or two or more places for the site of administration, and total daily or at intervals of 2 to 5 days The number of days of administration may range from 1 to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For animals other than humans, the dose is the same as that of a human per kg, or the above dose is converted, for example, by the volume ratio (for example, average value) of the target animal and the organ (heart, etc.) of the human One dose can be administered.

As described above, the strain according to one aspect can increase the expression of skin moisturizing and skin barrier-related hyaluronic acid synthase 3 (Hyaluronan synthase 3, HAS3, filagrin) and regenerate damaged cells. In addition, the strain can increase the expression of COL1A1 reduced by ultraviolet light, and decrease the increased expression of MMP-1. That is, the increase in markers such as HAS3 and filaggrin means that filaggrin, a precursor of NMF, and intercellular lipids are normalized in keratinocytes through the formation of a normal stratum corneum. Therefore, to prevent skin aging and maintain skin health from external environmental changes such as atmospheric drying, ultraviolet rays and various pollutants, to strengthen the skin barrier to prevent moisture loss in the skin tissue while promoting the maintenance of moisture in the skin tissue It can form a stratum corneum.

In addition, the strain promotes the proliferation of damaged fibroblasts and extracellular matrix to regenerate the skin and restore the epithelial layer, so it is useful for skin regeneration or wound healing.

Another aspect provides an oral composition comprising the strain, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof as an active ingredient. Specific details of the strain are the same as described above.

In one embodiment, the strain reduces the concentrations of volatile sulfur compounds (VSC), methyl mercaptan and dimethyl sulfide, which are volatile sulfur compounds (VSC) generated by bad breath-inducing microorganisms, and it was confirmed that the amount of oral microorganisms was reduced. Specifically, the hydrogen sulfide (Hydrogen sulfide, H ₂ S) may cause bad breath due to oral contamination and diseases related to mental and physiological causes. In addition, the methyl mercaptan (Methyl mercaptan, CH ₃ SH) may cause bad breath due to oral diseases (periodontal disease). In addition, dimethyl sulfide (Dimethyl sulfide, (CH ₃ ) ₂ S) may cause transient bad breath due to food metabolism.

As used herein, the term "halitosis" is a problem experienced in daily life, and 90% of it is derived from periodontitis, tongue discoloration, poor oral hygiene, and inadequate prosthetics in the oral cavity, and the remaining 10% is gastrointestinal disease, carcinoma, diabetes, liver failure, renal failure and from the same systemic disease. More than 600 types of bacteria live in the oral cavity, including disease-causing bacteria.

Therefore, the strain according to an aspect may have an effect of improving oral contamination and various diseases, or bad breath caused by food metabolism, reducing plaque, reducing tartar, reducing dry mouth, or inhibiting oral bacteria.

In one embodiment, the oral composition may be formulated as a mouthwash, mouthwash, toothpaste, oral spray, denture cleaner, toothpaste whitening agent, and the like.

Another aspect provides a pharmaceutical composition for preventing or treating oral diseases comprising the strain, its lysate, culture medium, extract of the culture medium, or a mixture thereof as an active ingredient. Another aspect provides a method of treating an oral disease comprising administering to a subject in need thereof an effective amount of the composition. Another aspect provides a health functional food composition for preventing or improving oral diseases comprising the strain, its lysate, culture medium, extract of the culture medium, or a mixture thereof as an active ingredient. Specific details of the strain are the same as described above.

The oral disease is, for example, invasive periodontitis, juvenile periodontitis, acute or chronic periodontitis, alveolar bone destruction, chronic or intractable periodontitis, gingivitis, ulcerative gingivitis, acute paronychia, acute lumpy gingivitis, hormonal periodontitis, complex infection, apical It may be a disease, periodontal abscess, dental caries, apical abscess, pulpitis, and the like.

In one embodiment, after 2 weeks of use of the composition containing the strain, representative oral microorganisms Tannerella forsythia , Fusobacterium nucleatum , Prevotella nigrescens ( Prevotella ) nigrescens ), Streptococcus mitis and Streptococcus mutans were significantly reduced compared to before use, and it was confirmed that no adverse reactions due to long-term use were observed.

Therefore, the strain according to one aspect has excellent oral microbial inhibitory effect as well as excellent stability for the human oral cavity, so even if used for a long period of time, it does not affect the resistance of bacteria in the oral cavity. effective in treatment.

According to the Weissella sibaira strain according to an aspect, it can be usefully used for preventing, improving or treating skin-related conditions. In addition, it can be usefully used for the prevention, improvement or treatment of oral diseases by reducing the sulfur compounds causing bad breath and inhibiting oral microorganisms.

1 is a graph showing the effect of a strain according to an embodiment on the expression of HAS3.
2 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin.
3 is a graph showing the effect of a strain according to an embodiment on skin regeneration or wound recovery.
4 is a photograph showing the effect of a strain according to an embodiment on skin regeneration or wound recovery.
Figure 5a is a graph showing the effect of the strain according to one embodiment on the expression of COL1A1.
Figure 5b is a graph showing the effect of the strain according to an embodiment on the expression of MMP-1.
6A is a graph comparing changes in the concentration of hydrogen sulfide before, immediately after, and after 2 weeks of use of the composition according to an aspect.
6B is a graph comparing changes in the concentration of methylmercaptan before, immediately after, and after 2 weeks of use of the composition according to an aspect.
6c is a graph comparing changes in the concentration of dimethyl sulfide before, immediately after, and after 2 weeks of use of the composition according to an aspect.
Figure 7a is a graph comparing the amounts of Actinomycetem comitans ( Aa ) and gingivalis ( Pg ) before and 2 weeks after use of the composition according to an aspect.
Figure 7b is a graph comparing the amounts of bacteria forsythia ( Tf ) and bacteria denticola ( Td ) before and 2 weeks after use of the composition according to an aspect.
Figure 7c is a graph comparing the amounts of nucleatum bacteria ( Fn ) and intermediary bacteria ( Pi ) before and 2 weeks after use of the composition according to an aspect.
Figure 7d is a graph comparing the amount of bacteria nigrescens ( Pn ) and Mytisbacterium ( Smi ) before and 2 weeks after use of the composition according to an aspect.
7e is a graph comparing the amounts of bacteria mutans ( Smu ) and casei bacteria ( Lc ) before and 2 weeks after use of the composition according to an aspect.
7f is a graph comparing the total amount of microorganisms before and 2 weeks after use of the composition according to an aspect.
8 is a graph analyzing the results of a questionnaire evaluation regarding the efficacy and usability of a composition according to an aspect.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Isolation and identification of strains

Samples collected from human oral saliva and kimchi were inoculated into a liquid or solid medium (Becton Dickinson, Cockeysville, MD) containing MRS. After inoculation, 100 colonies formed after culturing for 48 hours in an incubator at 28°C were purely separated and cultured, and cultured in an incubator at 28°C for 48 hours. The cultured colonies were subjected to 16s rRNA gene sequence identification. The primers used were designed to amplify in response to bacteria only (27F, 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R, 5'-GGTTACCTTGTTACGACTT-3). PCR amplification was carried out in 30 cycles of 95°C for 1 minute, 55°C for 1 minute, 75°C for 1 minute and 30 seconds each, and finally, after treatment at 72°C for 8 minutes, it was stored at 4°C. After the PCR reaction was completed, the DNA sequences of the isolated and cultured species were determined using ABI-3730XL (ABI, USA). The nucleotide sequence of the 16S rRNA region determined from the isolated and cultured microbial colonies was compared and analyzed with other strains registered in the BLAST program provided on the website of the National Center for Biotechnology Information (NCBI) in the United States. Only novel species were selected and used, and among them, a novel microorganism Weissella cibaria with less than 97% homology (hereinafter referred to as "CXO-1") was selected. The selected CXO-1 strain was deposited with the Korea Microbial Conservation Center on December 3, 2020 and was given an accession number KCCM12889P, and the CXO-1 strain has a 16s rRNA sequence of SEQ ID NO: 1 (complementary DNA).

[Experimental example]

Skin barrier strengthening and skin moisturizing activity

The effect of the culture medium of the CXO-1 strain isolated in Example 1 on skin barrier strengthening and skin moisturizing activity was analyzed. Specifically, HaCaT cells, a human keratinocyte cell line, were cultured in DMEM medium (Dulbecco'smodified Eagle's Medium, Gibco 1210-0038) containing 10% fetal bovine serum, and all cultures were cultured at 37°C 5°C. % CO ₂ in an incubator. The cultured cell line was treated with the culture solution of the CXO-1 strain (1%, 10% (w/w)) and further cultured for 24 hours.

-actin 유전자에 대한 보정을 통해 최종적으로 분석하였고, 그 결과를 도 1 및 도 2에 나타내었다. As a positive control, 1 μM of retionic acid was used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and RNA was quantified at 260 nm with nanodrops, and cDNA was synthesized in an amplifier using 2 μg of RNA each. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, the target gene, HAS3 (hyaluronic acid synthase), a factor related to skin barrier strengthening and moisturizing, and primers and cDNA for filaggrin were added together with a real-time PCR machine (Step One Plus, Real-time polymerase chain reaction was performed in Applied Biosystems, USA). The expression level of the gene is

It was finally analyzed through correction for the -actin gene, and the results are shown in FIGS. 1 and 2 .

1 is a graph showing the effect of a strain according to an embodiment on the expression of HAS3.

2 is a graph showing the effect of a strain according to an embodiment on the expression of filaggrin.

As a result, as shown in FIGS. 1 and 2, the strain according to one embodiment restores the damaged skin barrier by significantly increasing the expression of HAS3 and filaggrin in a concentration-dependent manner, and strengthens the skin barrier to increase skin defense. can give In addition, it is excellent in moisturizing the skin and is effective in preventing or treating moisture-related diseases.

Skin regeneration and wound healing effect

In order to evaluate the skin regeneration and wound healing efficacy of the CXO-1 strain isolated in Example 1, a cell migration assay was performed. Specifically, 96-well ImageLock?? Hs68 human fibroblasts were seeded on a plate (No. 4379; Essen BioScience) at 2Х10 ⁴ cells/well. Experiments were conducted at 90% cell confluent. On the second day, WoundMaker® (Essen BioScience, USA) was washed with 45 ml of 70% ethanol solution for 5 minutes, and then washed with 45 ml of distilled water for 5 minutes. All cell layers in each well of the plate were scratched using the washed WoundMaker®. Then, the medium was removed and washed with PBS. After filling the serum-free medium, each test substance was treated. On the third day, using the IncuCyte ZOOM (Essen BioScience, USA) program, the degree of wound recovery was measured and graphed at 2-hour intervals for 8 hours. In addition, the degree of wound recovery was visually confirmed by checking the photograph of the cell measured with a microscope in the device.

3 is a graph showing the effect of a strain according to an embodiment on skin regeneration or wound recovery.

4 is a photograph showing the effect of a strain according to an embodiment on skin regeneration or wound recovery.

As a result, as shown in FIGS. 3 and 4, it was confirmed that the wound healing effect was excellent in the group treated with the serum-free medium and the culture solution of the CXO-1 strain isolated in Example 1 compared to the untreated control group. . In particular, it was confirmed that about 60% recovery after 8 hours in the group treated with the culture solution of the CXO-1 strain at a concentration of 1%.

That is, the strain according to one embodiment promotes skin regeneration by activating cells in the wound site, and thus can be used as a material for skin regeneration or wound healing.

Anti-aging activity assay

The effect of the CXO-1 strain culture medium isolated in Example 1 on factors related to aging skin aging and wrinkle generation by ultraviolet (UV) irradiation was analyzed.

-actin 유전자에 대한 보정을 통해 최종적으로 분석하였고, 그 결과를 도 5a 및 도 5b에 각각 나타내었다. Specifically, a human dermal fibroblast (Hs68) cell line was seeded in a 6-well plate at 3.5x10 ⁵ cells/well, and then cultured at 37° C., 5% CO ₂ condition in an incubator for 24 hours. After removing the medium and adding DPBS, 20 mJ/cm2 of UVB was irradiated or not. Immediately after UVB irradiation, after removing DPBS and replacing it with a medium without FBS, the culture solution of the CXO-1 strain was treated by concentration (1% (w/w), 10% (w/w)) and added for 24 hours cultured. As a negative control group, UV-treated and strain culture untreated groups were used. After that, RNA was isolated from the cells of each sample using trizol (RNA iso, DAKARA, Japan), and RNA was quantified at 260 nm using nanodrops, and cDNA was synthesized in an amplifier using 2 μg of each RNA. (C1000 Thermal Cycler, Bio-Rad, USA). Using the synthesized cDNA as a template, a real-time PCR machine (Step One Plus, Step One Plus, Real-time polymerase chain reaction was performed in Applied Biosystems, USA). The expression level of the gene is

It was finally analyzed through correction for the -actin gene, and the results are shown in FIGS. 5A and 5B, respectively.

Figure 5a is a graph showing the effect of the strain according to one embodiment on the expression of COL1A1, Figure 5b is a graph showing the effect of the strain according to one embodiment on the expression of MMP-1.

As a result, as shown in Figure 5a, compared to the untreated control group, the expression of COL1A1 reduced by UV light in the group treated with the serum-free medium and the culture solution of the CXO-1 strain isolated in Example 1 was concentration-dependently increase could be observed. In particular, it was confirmed that the group treated with the CXO-1 strain culture solution at a concentration of 10% had significantly higher COL1A expression compared to the serum-free medium at the same concentration. In addition, as shown in FIG. 5b, the expression of MMP-1 increased by UV in the group treated with the serum-free medium and the culture solution of the CXO-1 strain isolated in Example 1 was concentration-dependent as compared to the untreated control group. was found to decrease. In particular, it was confirmed that the group treated with the CXO-1 strain culture solution at a concentration of 10% significantly reduced the expression of MMP-1 compared to the serum-free medium at the same concentration.

That is, the strain according to one aspect has an excellent effect in preventing skin aging, such as reducing elasticity and inhibiting wrinkle production, by restoring collagen destroyed by ultraviolet rays by ultraviolet rays, and inhibiting the production of MMP-1 increased by ultraviolet rays .

bad breath analysis

He is aware of his own bad breath, and as a result of measuring with Oral Chroma ^TM (FIS Inc. Inc., J apan), which is a bad breath measuring device, 112 ppb of hydrogen sulfide, 26 ppb of methyl mercaptan and dimethyl sulfide ( For 20 volunteers aged 20 to 60 years with a concentration of dimethyl sulfide) 8 ppb or higher, take an appropriate amount (10 ml) of the composition containing the culture solution of Example 1 twice a day (morning and evening) in the mouth. Gargle for about 30 seconds and then spit out. Volatile Sulfur Compound (VSC), a parameter for detecting bad breath in the applicant's oral cavity, was collected before, immediately after, and 2 weeks after use using a 1 ㎖ syringe (TOP Corp., Taiwan), and the concentration of each component was measured and shown in Table 1 and FIGS. 5A to 5C.

Analysis items evaluation time Average (ppb) standard deviation (SD) Significance Probability
(p-value) rate of change (%) hydrogen sulfide
(H ₂ S) before use 461.85 268.04 - - immediately after use 48.90 82.65 0.000 ^* - 89.41 2 weeks after use 56.35 95.20 0.000 ^* - 87.80 methyl mercaptan
(CH ₃ SH) before use 179.15 140.34 - - immediately after use 56.50 39.39 0.000 ^*W - 68.46 2 weeks after use 32.15 32.56 0.000 ^*W - 82.05 dimethyl sulfide
((CH ₃ ) ₂ S) before use 85.20 92.48 - - immediately after use 1.6 2.64 0.000 ^*W - 98.12 2 weeks after use 8.15 30.84 0.000 ^*W - 90.43

^* Volatile Sulfur Compounds

If P<0.05, there is a significant difference compared to before use

^W Wilcoxon signed ranks test, Post-Hoc test (Bonferroni correction)

6A to 6C are graphs comparing changes in the concentration of volatile sulfur compounds (hydrogen sulfide, methyl mercaptan and dimethyl sulfide) before, immediately after, and after 2 weeks of use of the composition according to an aspect.

As a result, as shown in FIGS. 6a to 6c , it was confirmed that the concentration of volatile sulfur compounds, a parameter for bad breath, significantly decreased immediately after use and 2 weeks after use compared to before use (p<0.05). Specifically, as shown in Table 1, the rate of change of volatile sulfur compounds is 89.41% and 87.80% for hydrogen sulfide, 68.46%, and 82.05% for methyl mercaptan, and 98.12% and 90.43% for dimethyl sulfide. After 2 weeks, it was confirmed that each concentration decreased by 80% or more.

That is, the composition according to an aspect is effective in improving bad breath by significantly reducing the concentration of volatile sulfur compounds.

Oral Bacterial Analysis

After having the same subject as above use the composition containing the strain culture medium of Example 1 in the same way, before use and 2 weeks after use, the sample container was provided from the oral pathogenic microorganism test requesting institution (DENOMICS, Korea). After collecting a volunteer's saliva sample, changes in oral microorganisms and total amount of microorganisms in Table 2 below were analyzed using Oral Bacteria Dignosis (OBD) analysis service, and the results are shown in Table 3 below.

Analytical strains Characteristics of the strain One Aggregatibacter actinomycetemcomitans [ Aa ] Induction of invasive periodontitis and juvenile periodontitis 2 Porphyromonas gingivalis [ Pg ] Induction of acute/chronic periodontitis, destruction of alveolar bone 3 Tannerella forsythia [ Tf ] Chronic, intractable periodontitis, gingivitis induction 4 Treponema denticola [ Td ] Induction of ulcerative gingivitis, acute paronychia 5 Fusobacterium nucleatum [ Fn ] Acute massive gingivitis, induction of calculus formation 6 Prevotella intermedia [ Pi ] Hormonal periodontitis, combined infection induction 7 Prevotella nigrescens [ Pn ] Periodontal disease, periodontal abscess induction 8 Streptococcus mitis [ Smi ] Induction of dental caries, apical abscess 9 Streptococcus mutans [ Smu ] Induction of pulpitis, dental caries 10 Lactobacillus casei [ Lc ] Induction of dental caries

Analytical strains evaluation time Average
(copy/ml X 10 ³ ) Standard Deviation
(X 10 ³ ) Significance Probability
(P-value) rate of change (%) Aa before use 175.00 558.07 - - 2 weeks after use 62.50 119.68 0.465 ^W -64.29 Pg before use 1,139.16 1,954.58 - - 2 weeks after use 240.15 428.12 0.059 -78.92 Tf before use 5,563.70 12,098.04 - - 2 weeks after use 1,312.30 1,628.22 0.014 ^*W -76.41 Td before use 360.43 908.33 - - 2 weeks after use 65.46 102.87 0.421 ^W -81.84 Fn before use 519,635.00 1,781,383.15 - - 2 weeks after use 2,290.50 1,502.25 0.000 ^*W -99.56 Pi before use 2,138.45 4,308.09 - - 2 weeks after use 1,091.10 2,739.60 0.358 -49.06 Pn before use 132,565.00 510,358.53 - - 2 weeks after use 5,610.45 5,056.61 0.002 ^*W -95.77 Smi before use 184,240.90 144,154.23 - - 2 weeks after use 43,640.55 145,637.20 0.002 ^* -76.31 Smu before use 7,319.00 6,205.66 - - 2 weeks after use 891.55 1,333.29 0.000 ^* -87.82 Lc before use 0.06 0.18 - - 2 weeks after use 0.08 0.36 1.000 ^W +33.33

7A to 7E are graphs comparing the amount of microorganisms described in Table 2 before and 2 weeks after use of the composition according to an aspect.

7f is a graph comparing the total amount of microorganisms before and 2 weeks after use of the composition according to an aspect.

As a result, as shown in Table 3 and FIGS. 7a to 7e, a total of 5 bacterial species ( Tf, Fn, Pn, Smi and Smu ) were confirmed to be significantly reduced. Specifically, it was confirmed that the five strains significantly decreased to 76.41%, 99.56%, 95.77%, 76.31%, 87.82%, and 84.28%, respectively, after 2 weeks of use compared to before use.

In addition, as shown in FIG. 7f , it was confirmed that the total amount of microorganisms was significantly reduced by 84.28% after 2 weeks of use compared to before using the composition containing the culture medium of Example 1 .

That is, the composition according to one aspect can prevent or treat oral diseases such as periodontitis, gingivitis, apical disease, dental caries, pulpitis, and the like, as well as prevent tartar formation by significantly reducing oral microorganisms.

sensory evaluation

After allowing the 20 volunteers to use the composition containing the culture medium of Example 1 for 2 weeks, the efficacy of improving bad breath, reducing plaque, and reducing dry mouth was evaluated according to the evaluation criteria 1 of Table 4 below, and the results were obtained. It is shown in Table 5 below. In addition, according to the evaluation criteria 2 of Table 4 below, usability such as freshness, stimulation, flavor and taste were evaluated, and the results are shown in Table 6 below.

score Evaluation Criteria 1 Evaluation Criterion 2 One Not at all. Not very good. 2 Not like that. Not good. 3 I don't think so. It doesn't look good. 4 It seem to be like that. It's good. 5 Yes. good night. 6 it really is very good.

Item number (n) Response rate (%) bad breath improvement 19 95.00 plaque reduction 16 80.00 Reduce dry mouth 17 85.00

Item number (n) Response rate (%) refreshing 16 80.00 no irritation 19 95.00 incense 8 40.00 taste 9 45.00

Number (n): Number of applicants who selected 4, 5 and 6 points

Response rate (%):

8 is a graph analyzing the results of a questionnaire evaluation regarding the efficacy and usability of a composition according to an aspect.

As a result, as shown in FIG. 8 and Table 3, the composition containing the strain culture solution of Example 1 was evaluated to have excellent effects of improving bad breath, reducing plaque, and reducing dry mouth. In addition, as shown in Table 4, the composition was evaluated as being refreshing and having no irritation, so that the feeling of use was excellent overall.

Accordingly, the composition according to an aspect may provide an oral care product having excellent feeling of use as well as functionalities such as improving bad breath, reducing plaque, and reducing dry mouth.

Oral Stability Assessment

In order to evaluate oral stability, the 20 volunteers were allowed to use the composition containing the culture medium of Example 1 for 2 weeks, and then the test site of the volunteers was observed. Thereafter, the state of the test site was checked, recorded, and evaluated through Q&A. In the event of an adverse reaction, an adverse reaction report was prepared, and an expert judged the relationship between the adverse reaction and the composition.

As a result, no adverse events were observed in all volunteers during the trial period.

That is, the composition according to one aspect has an advantage in that it has excellent oral microbial inhibitory effect as well as excellent stability to the human oral cavity, so that even when used for a long period of time, it does not affect the resistance of bacteria in the oral cavity.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Korea Microorganism Conservation Center (Overseas) KCCM12889P 20201203

<110> Cosmax, INC. <120> Weissella cibaria strain and uses thereof <130> PN137159 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1431 <212> RNA <213> Artificial Sequence <220> <223> Weissella cibaria CXO-1 16s rRNA <400> 1 cagtcgaacg ctttgtggtt caactgattt gaagagcttg ctcagatatg acgatggaca 60 ttgcaaagag tggcgaacgg gtgagtaaca cgtgggaaac ctacctctta gcaggggata 120 acattggaa acagatgcta ataccgtata acaatagcaa ccgcatggtt gctacttaaa 180 agatggttct gctatcacta agagatggtc ccgcggtgca ttagttagtt ggtgaggtaa 240 tggctcacca agacgatgat gcatagccga gttgagagac tgatcggcca caatgggact 300 gagacacggc ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgaaa 360 gcctgatgga gcaacgccgc gtgtgtgatg aagggtttcg gctcgtaaaa cactgttgta 420 agagaagaat gacattgaga gtaactgttc aatgtgtgac ggtatcttac cagaaaggaa 480 cggctaaata cgtgccagca gccgcggtaa tacgtatgtt ccaagcgtta tccggattta 540 ttgggcgtaa agcgagcgca gacggttatt taagtctgaa gtgaaagccc tcagctcaac 600 tgaggaattg ctttggaaac tggatgactt gagtgcagta gaggaaagtg gaactccatg 660 tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctttctgga 720 ctgtaactga cgttgaggct cgaaagtgtg ggtagcaaac aggattagat accctggtag 780 tccacaccgt aaacgatgag tgctaggtgt ttgagggttt ccgcccttaa gtgccgcagc 840 taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg 900 acggggaccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc gaagaacctt 960 accaggtctt gacatccctt gacaactcca gagatggagc gttcccttcg gggacaaggt 1020 gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttattact agttgccagc atttagttgg gcactctagt gagactgccg 1140 gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta tgacctgggc 1200 tacacacgtg ctacaatggc gtatacaacg agttgccaac ccgcgagggt gagctaatct 1260 cttaaagtac gtctcagttc ggattgtagg ctgcaactcg cctacatgaa gtcggaatcg 1320 ctagtaatcg cggatcagca cgccgcggtg aatacgttcc cgggtcttgt acacaccgcc 1380 cgtcacacca tgagagtttg taacacccaa agccggtggg gtaaccttcg g 1431

Claims (10)

Deposited with accession number KCCM12889P, Weissella sp. belonging to Weissella cibaria ( Weissella cibaria ) CXO-1 strain. The lysate of the strain of claim 1, or a culture solution. A cosmetic composition comprising the strain of claim 1, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof. The cosmetic composition for improving skin condition or moisturizing according to claim 3 . The cosmetic composition of claim 4, wherein the skin condition is skin aging, skin barrier strengthening, skin wrinkles, skin elasticity, skin regeneration or wound improvement. A pharmaceutical composition for treating wounds comprising the strain of claim 1, a lysate thereof, a culture medium, an extract of the culture medium, or a mixture thereof as an active ingredient. An oral composition comprising the strain of claim 1, a lysate thereof, a culture solution, an extract of the culture solution, or a mixture thereof as an active ingredient. The oral composition according to claim 7, wherein the oral composition is for improving bad breath, reducing plaque, reducing tartar, reducing dry mouth, or inhibiting oral bacteria. A pharmaceutical composition for the prevention or treatment of oral diseases comprising the strain of claim 1, a lysate thereof, a culture medium, an extract of the culture medium, or a mixture thereof as an active ingredient. The method according to claim 9, wherein the oral disease is invasive periodontitis, juvenile periodontitis, acute or chronic periodontitis, alveolar bone destruction, chronic or intractable periodontitis, gingivitis, ulcerative gingivitis, acute paronychia, acute gingivitis gangrene, hormonal periodontitis, complex infection, A pharmaceutical composition for preventing or treating oral diseases that is at least one selected from the group consisting of apical disease, periodontal abscess, dental caries, apical abscess, and pulpitis.

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