KR102354126B1 - Streptococcus thermophilus_Breezytail_AIR21 and Fermented Product Manufactured Using Thereof - Google Patents

Abstract

The present invention relates to a Streptococcus thermophilus_Breezytail_AIR21 strain, and a fermented product manufactured using the same. A culture solution of the novel Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) provided from the present invention is excellent in Staphylococcus aureus reduction, anti-inflammatory, anti-allergy, moisturizing, and skin barrier strengthening effects, thereby being excellent as a cosmetic composition when applied to a cosmetic base as an active ingredient.

Description

Streptococcus thermophilus_Breezytail_AIR21 strain and fermented product prepared using the same {Streptococcus thermophilus_Breezytail_AIR21 and Fermented Product Manufactured Using Thereof}

The present invention relates to a novel Streptococcus thermophilus_Breezytail_AIR21 strain having anti-allergic and companion animal skin improvement activity and a fermented product prepared using the same.

The skin ecosystem provides various types of habitat for microorganisms, and a wide range of microorganisms live there. Human beings are known to have a symbiotic relationship with them and 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 response to the selection of physical and chemical functions, microorganisms adapt to specialized niches to provide habitat. 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 and is separated from the keratinocytes. The epidermis has a so-called "brick and mortar structure". The skin tissue undergoes a continuous self-healing process, and the squames that have passed the final stage of differentiation repeat the process of constantly being removed from the skin tissue.

On the other hand, bacteria that ferment sugars to obtain energy and produce a large amount of lactic acid are collectively called lactic acid bacteria. While living in the intestine, lactic acid bacteria play a symbiotic relationship with the digestive system of the human body, and play a role in decomposing fiber and complex protein into important nutrients. In addition, by maintaining an acidic intestinal environment, it suppresses the growth of harmful bacteria, improves diarrhea and constipation, synthesizes vitamins, and inhibits blood cholesterol. Lactobacillus has the property of being able to bind strongly to intestinal mucosa and epithelial cells, so it helps a lot in intestinal function, and promotes the activity of macrophages and spleen to secrete and promote substances involved in immune response. In addition, it has an immunomodulatory function, so it is effective in atopy and allergy-related diseases.

In the cosmetic field, lactic acid bacteria cultures have been used since commercialization in 1955, mainly Streptococcus genus, Lactobacillus genus, Lactococcus genus, Leuconostoc genus, Bifidobacterium. ( Bifidobacterium ) Research is being actively carried out on the form of raw materials such as filtration or extraction by directly fermenting the culture medium using the genus, etc.

Among them, as a technology applied to various uses using the genus Streptococcus , Republic of Korea Patent Publication No. 10-2173646 "Skin-derived Streptococcus salivarius sbk4 new strain and functional cosmetic composition using the same", Korean Patent Publication No. 10-2154927 「Functional cosmetic composition for skin improvement containing chlorogenic acid, ferroic acid, rasveratrol and fermented Streptococcus thermophilus as active ingredients」, Korean Patent Publication No. 10-0926047 "Atopic dermatitis improving agent composition" etc. are disclosed.

Looking back at the skin ecology, physical and biological factors harmonize to provide a variety of habitats, and furthermore, you will be able to think about the delicate balance between humans and microorganisms. When the balance of the symbiotic relationship is disrupted, skin breakdown and infection occur at the same time.

Accordingly, the present inventors isolated and identified the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) from the skin of companion animals, inoculated into MRS medium using this, and culturing to prepare a fermented product , found that it can induce harmful skin-microbiome changes in companion animals ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergy, moisturizing and skin barrier strengthening effects. The present invention has been completed.

Republic of Korea Patent Publication No. 10-2173646 Republic of Korea Patent Publication No. 10-2154927 Republic of Korea Patent Publication No. 10-0926047

An object of the present invention is to provide a novel strain having skin-microbiome change induction (reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergic, moisturizing and skin barrier strengthening effects. have.

Another object of the present invention is to provide a method for producing a fermented product and a fermented product prepared therefrom, comprising inoculating a novel strain into a medium formulated to maximize efficacious activity, and culturing.

Another object of the present invention is to provide a cosmetic composition comprising a fermented product of a novel strain as an active ingredient.

In order to achieve the above object, the skin-microbiome (skin-microbiome) change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergy, moisturizing and Streptococcus having a skin barrier strengthening effect Thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) is provided.

In the present invention, the strain is characterized in that it is derived from companion animal skin.

In addition, the present invention provides a method for producing a fermented product, comprising the steps of inoculating a Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) into a medium formulated to maximize efficacious activity, and culturing. .

In the present invention, the medium is characterized in that it contains Maltose and Glutamine.

In addition, in the present invention, the culture is characterized in that it is carried out at 35 ~ 37 ℃ for 90 ~ 130 hours.

In addition, the present invention provides a fermented product fermented with Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P).

In addition, the present invention provides a cosmetic composition comprising the fermented product as an active ingredient.

In the present invention, the cosmetic composition is a skin-microbiome (skin-microbiome) change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergic, moisturizing, and Streptococcus having a skin barrier strengthening effect Thermophilus Breezytail_AIR21 strain (Accession No.: KCCM13005P) It is characterized in that the use selected from the group consisting of effects.

Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) fermented product provided by the present invention is skin-microbiome (skin-microbiome) change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, Since it is excellent in anti-allergy, moisturizing and skin barrier strengthening effects, it has excellent effects as a cosmetic composition when applied to a cosmetic base as an active ingredient.

1 shows the nucleotide sequence of the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) according to the present invention.
Figure 2 shows the reduction of S. aureus and the increase of lactic acid bacteria when the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) according to the present invention is treated on the skin.
3 is a graph showing the results of the anti-allergic effect of the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) fermented product according to the present invention.
4 is a graph showing the skin barrier strengthening effect of the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) fermented product according to the present invention.
5 is a graph showing the moisturizing effect results of the Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) fermented product according to the present invention.

Therefore, in the present invention, skin-microbiome change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergy, moisturizing and skin barrier strengthening effects from microorganisms isolated from companion animals By screening the strains that have, excellent strains were selected, and as a result of 16s rRNA sequencing, it was confirmed that it was a novel microorganism having a nucleotide sequence similar to that of Streptococcus thermophilus. Accordingly, the Streptococcus thermophilus_Breezytail_AIR21 strain was named and deposited at the Korean Culture Center of Microorganisms, and was given an accession number KCCM13005P.

Accordingly, the present invention relates to a Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) (hereinafter abbreviated as 'Breezytail_AIR21').

At this time, the Breezytail_AIR21 strain is characterized in that it is derived from companion animals.

In addition, the present invention relates to a method for producing a fermented product, comprising inoculating the Breezytail_AIR21 strain into a medium formulated to maximize efficacy and culturing.

The medium used for the culture is characterized in that it contains Maltose and Glutamine. Among them, MRS medium (proteose peptone No.3 10g, beef extract 10g, yeast extract 5g, dextrose 20g, Polyoxyethylene sorbitan monooleate 1g, ammonium citrate 2g, maganessium sulfate 0.1g, manganesse selfate 0.05g, dipotassium phosphate 2g, sodium acetate 5g/ L, pH 6.5) and fermented using a medium to which maltose 0.5g/L and Glutamin 0.25g/L were added as the basis, the yield of the fermented product can be most effectively improved and anti-allergic and microbiome changes can be minimized. It is preferable in terms of

The Breezytail_AIR21 strain was added to MRS broth containing Maltose and Glutamine from 1.0x10 ⁷ to The ^{step of inoculating and culturing at a level of 1-3% by making the number of bacteria 1.0x10 9} is characterized in that the stationary culture is performed in anaerobic conditions at 32 to 37° C. for 90 to 130 hours. If the culture temperature and time are out of the above range, the culture conditions of the fermented strain are not suitable, so skin-microbiome change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergic, It is not preferable because it cannot exhibit moisturizing and skin barrier strengthening effects, as well as strain growth.

As described above, the cultured strain is filtered under pH 4.0-5.0 conditions, and then the filtered strain is cultured at 20-30° C. for 7 days to prepare a fermented product.

In addition, the fermented product of the present invention Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) induces skin-microbiome changes ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, It has anti-allergic, moisturizing and skin barrier strengthening effects, so it is very effective as a cosmetic composition.

Accordingly, it relates to a cosmetic composition comprising a fermented product of Streptococcus thermophilus_Breezytail_AIR21 (Accession No.: KCCM13005P) as an active ingredient.

The cosmetic composition is a skin-microbiome (skin-microbiome) change induction ( reduction of S. aureus , increase in lactic acid bacteria), anti-inflammatory, anti-allergic, moisturizing and skin barrier strengthening effect selected from the group consisting of can

The cosmetic composition for skin-microbiome change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergic, moisturizing, and skin barrier strengthening effects of the present invention is an external skin ointment, cream, and softening agent. Longevity, nourishing lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, It may have a formulation selected from the group consisting of nourishing cream, eye cream, moisture cream, hand cream, foundation, nourishing essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto. does not The composition of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by those skilled in the art.

When the formulation of the present invention is a paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc acid may be used as a carrier component. can

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

In the cosmetic composition for whitening and wrinkle improvement of the present invention, the fermented product may be contained in an amount of 0.01 to 90% by weight, preferably 3 to 15% by weight, based on the total weight of the composition. Skin-microbiome (skin-microbiome) change induction ( reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergy, moisturizing and skin barrier strengthening effect if included in an effective amount that can provide the effect is not limited thereto.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it is for those of ordinary skill in the art to which the present invention pertains that the scope of the present invention is not limited by these examples according to the gist of the present invention. it will be self-evident

<Example 1> Isolation of Streptococcus thermophilus_Breezytail_AIR21 strain

After a 12-month-old Maltese puppy's back skin was swapped with gauze, shaken in MRS medium, and ^{0.1 mL of a sample diluted 10 -4} times (volume) with MRS agar medium (Peptospecial 10 g, beef extract 10 g, yeast extract 5g, Glucose 20g, Triammonium Citrate 2g, sodium acetate 5g, maganessium sulfate 0.2g, manganesse selfate 0.05g, dipotassium phosphate 2g, Agar 15g, Tween80 1g/L, pH 6.2±0.2, MBcell (Cat.No. MB-M1025) ) and incubated for 2 days at 37°C in an anaerobic incubator. The formed colonies were separated and cultured in pure water, and the formed single colony was isolated after culturing in an anaerobic incubator for 24 hours. Among the isolated strains, the Breezytail_AIR21 strain with excellent skin-microbiome change induction (reduction of S. aureus , increase of lactic acid bacteria), anti-inflammatory, anti-allergic, moisturizing and skin barrier enhancement was selected.

<Example 2> Selection strain 16s rRNA sequencing analysis

SEQ ID NO: 1: 27F (5' AGA GTT TGA TCM TGG CTC AG 3') and SEQ ID NO: 2: 1492R (5' TAC GGY TAC CTT GTT ACG ACT T 3'), which are the gDNA and universal primers of the finally selected Breezytail_AIR21 strain Using 16s DNA sequencing analysis, it was confirmed that it was a strain with high homology to Streptococcus thermophillus, and was named as Streptococcus thermophilus_Breezytail_AIR21 strain (SEQ ID NO: 3), and International Microorganisms It was deposited with the Korea Center for Microbial Conservation (KCCM), a patent depository institution, and was given an accession number KCCM13005P.

<Example 3> Preparation of fermented product of Breezytail_AIR21 strain

In order to prepare a fermented product of the isolated Breezytail_AIR21 strain, Maltose and Glutamine were added to the MRS medium, and then, the Breezytail_AIR21 strain was inoculated with 1% of the strain so that the number of bacteria was ^{1.0x10 9 .} After inoculation, there was no oxygen supply to maintain anaerobic conditions, and stationary culture was performed at 37° C. for 120 hours. After the culture was completed, it was confirmed that the pH was 4.0 (error 0.2), and when it was visually confirmed that the color was dark brown, the culture was finished and a 0.2 μm filter (Millipore) was sterilized. When the filter was completed, a fermentation product of Breezytail_AIR21 strain was prepared after a one-week aging period at room temperature.

<Comparative Example 1> Preparation of fermented product using MRS general medium of Breezytail_AIR21 strain

Comparative Example 1 was prepared in the same manner as in <Example 3>, except that only general MRS medium was used and maltose and glutamine were not added to prepare a fermentation product using the normal medium of the isolated Breezytail_AIR21 strain.

<Experimental Example 1> Skin-Microbiome sample collection

The samples used in this experiment were tested on dogs with prior consent from the dog owner at the GFC Life Science Research Institute. As the probiotics used in the test, the fermented product of Breezytail_AIR21 strain was used. After culturing, the culture solution from which the cells were removed was applied to the back of the dog, and skin samples were collected for a total of 72 hours and used for analysis. The collection method was the cotton swab method. skin samples were collected by scrubbing using gauze. The collection of microorganisms on the skin consisted of pre-treatment, post-treatment, untreated (resting period) and re-treatment groups.

Skin samples were collected using sterile water and a scraper based on references, and all work was carried out in a clean bench. This study was confirmed to be a reason for exemption from the IRB deliberation criteria.

<Experimental Example 2> Genomic DNA extraction and DNA amplification of microorganisms resident on the skin

The supernatant of the sample collected from the skin was suspended in 0.85% NaCl and centrifuged (17,000 rpm/m) to collect the cells and wash the cells once in sterile physiological saline, using FastDNA SPIN KIT (MP Biomedical, France) and DNA was extracted.

<Experimental Example 3> Analysis of skin flora 16s metagenome through NGS analysis

16s metagenome analysis was performed for the analysis of the flora using the extracted DNA. For NGS analysis, Thermofisher's Ion torrent S5 Plus system and 16s metagenome kit were used. The analyzed 16s metagenome sequence was diagrammed after analysis using Thermofisher's ion repoter DB and software.

As a result, as can be seen in Figure 2, depending on the Breezytail_AIR21 strain fermented product treatment, it was confirmed that the reduction of the representative harmful strain S. aureus and the increase of the beneficial strain lactic acid bacteria. Therefore, it was confirmed that beneficial skin flora was increased depending on the Breezytail_AIR21 strain fermented product treatment.

<Experimental Example 4> Anti-allergy test: histamine inhibitory activity

Beta-hexosaminidase (β-hexosaminidase) is a substance constituting the granules of mast cells, and the secretion of histamine by degranulation of mast cells is proportional to the amount of beta-hexosaminidase released. Therefore, the degranulation and histamine secretion inhibitory effects of the deodeok saponin fraction were confirmed by measuring the release amount of beta-hexosaminidase in RBL-2H3 cells, a mast cell line of a rat. After suspending RBL-2H3 cells in DMEM containing 10% FBS, 2X10 ⁵ cells were dispensed per well in a 24 well plate, and then sensitized with 0.5 μg/ml DNP-IgE per well and in a 5% CO2 incubator. Incubated overnight. Afterwards, the cells in each well were washed twice with Siraganian buffer (119 mM NaCl, 5 mM KCl, 5.6 mM glucose, 0.4 mM MgCl2, 25 mM HEPES, 40 mM NaOH, 1 mM CaCl2, 0.1% BSA, pH 7.2) and then 37 It was pre-reacted with Siraganian buffer at ℃ for 10 minutes, and reacted again for 10 minutes after addition of the test substance. Thereafter, the cells were treated with antigen (DNP-BSA, 10 μg/ml) at 37°C for 30 minutes to make them degranulated, placed on ice for 10 minutes to terminate the reaction, and 20 μl of the supernatant was taken and transferred to a 96-well plate. After adding 1 mM p-nitrophenyl-N-acetyl- β-D-glucosaminide and incubating for 1 hour, stop solution (0.1 M Na2CO3/NaHCO3) was added, and absorbance was measured at 405 nm by ELISA and quantified.

3 shows the histamine inhibitory activity results for the fermented product prepared in Example 3 and Comparative Example 1 of the present invention.

As a result, the fermented product obtained in Example 3 compared to Comparative Example 1 had remarkably excellent histamine inhibitory ability, and it was confirmed that the stimulation relief effect was improved in a concentration-dependent manner.

<Experimental Example 5> Anti-inflammatory efficacy test: Nitric Oxide (NO) inhibitory activity

Using the fermented products of Example 3 and Comparative Example 1, the degree of inhibition of nitric oxide was measured to confirm the anti-inflammatory activity.

RAW 264.7 cells, a type of macrophages, were equally counted and dispensed at a rate of ^{1.0x10 4} cells/well in a 24 Well plate using DMEM medium containing 1% penicillin/scraptomycin and 10% FBS (fetal bovineserum). Incubated for 24 hours at 37° C. and 5% carbon dioxide. In RAW 264.7 cells cultured for 24 hours, Example 3 and Comparative Example 1 were mixed with the medium at a concentration of 50 (㎍ / ㎖), 1ml each was added to each well, and reacted for 24 hours in an incubator at _{37 ℃ and 5% CO 2 conditions} made it At this time, LPS (Lipo poly saccharide), an inflammation-inducing factor expressing NO, was treated together at a concentration of 1 μg/ml and reacted at 37° C. and 5% carbon dioxide condition for 24 hours. Next, after taking only the supernatant from each well, 100 ml of the culture medium was placed in a 96 well plate using a Nitric Oxide detection kit in each well, 50 μl of Griess reagent A (N-1-Naphthylethylenediamine (NEDHC)) and 50 μl of Griess reagent B After adding 50 μl of (Sulfanilamide), each reacted for 10 minutes, absorbance was measured at 540 nm using an ELISA plate reader. The concentration of NO in the cell culture medium was calculated by comparing it with a standard curve for each concentration of sodium nitrite (NaNO2).

division NO production concentration (μM) positive control 12.10.98 negative control 81.01.02 Example 3 17.70.86 Comparative Example 1 43.50.88

As a result, as shown in Table 1, Example 3 was confirmed to have a lower nitric oxide production concentration compared to Comparative Example 1, confirming that the nitric oxide production inhibitory effect was excellent.

These results mean that there is an anti-inflammatory effect on the Breezytail_AIR21 strain (Accession No.: KCCM13005P) fermentation.

<Experimental Example 6> Antibacterial efficacy test: MIC test

The degree of antibacterial activity was confirmed using the fermented products of Example 3 and Comparative Example 1.

1. Preparation of Evaluation Medium

The paper disc method was used as the evaluation method, and the harmful standard test strains (the Ministry of Food and Drug Safety and MIC test guide line) Escherichia coli (ATCC 8739), Staphylococcus aureus, ATCC were used for the evaluation. 6538P), Pseudomonas aeruginosa (ATCC 9027), and Candida albicans (ATCC 10231) were purchased from the Korea Microorganism Resource Center (KCTC, Korea) and the Korea Microorganism Conservation Center (KCCM, Korea) and used. TSA medium (Tryptic Soy Agar) was used for culturing E. coli, Staphylococcus aureus and Pseudomonas aeruginosa among the standard test strains, and YM medium (Yeast extract Media) was used for culturing Candida albicans.

2. MIC test

After preparing an independent nutrient broth sterilized at 121°C for 15 minutes, each standard test strain was inoculated at an amount of ^{3X10 5 cfu/ml.} Example 3 and Comparative Example 1 were typically adjusted to test concentrations of 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125%, and inoculated using a single colony of the test strain and cultured . After the incubation was completed, the absorbance (600 nm) was measured to confirm the result. The antibacterial effect was confirmed at the lowest concentration in which no microorganisms were detected.

Category (Unit: %) coli Staphylococcus aureus Pseudomonas aeruginosa candida albicans Example 3 0.125 0.125 0.125 0.25 Comparative Example 1 0.125 0.5 0.5 -

As a result, as shown in Table 2, Example 3 is E. coli (Escherichia coli, ATCC 8739), Staphylococcus aureus (ATCC 6538P), Pseudomonas aeruginosa, which are four standard test strains (refer to the Ministry of Food and Drug Safety and MIC test guide line). (Pseudomonas aeruginosa, ATCC 9027) and Candida albicans (Candida albicans, ATCC 10231) was confirmed that the inhibitory effect was significantly higher than that of Comparative Example 1, confirming that the antibacterial effect was excellent.

<Experimental Example 7> Skin barrier improvement test: Filaggrin production increase effect

In order to measure the skin barrier function improvement effect of Example 3 and Comparative Example 1, the ability to increase the production of filaggrin, a material that plays an important role in the differentiation of the stratum corneum of the epidermis and the formation of a natural moisturizing factor, was confirmed by RT-PCR. . Human epidermal keratinocytes were treated with Dulbecco's Modified Eagle's Medium (DMEM), 10% Fetal bovine serum (FBS), and 1% Penisilin-Streptomycin in 24 ^{wells at 2x10 5} /well at 37°C, 5% CO ₂ . Conditions were incubated for 24 hours. When human epidermal keratinocytes were confluenced by 80% or more, the serum-free medium was changed and cultured, and the samples of Example 3 and Comparative Example 1 were respectively treated with the cells by concentration. After that, additional culture was performed for 24 hours, and the expression of the Filaggrin gene was confirmed by real-time PCR. For RNA isolation and cDNA synthesis, Nucleospin RNA (Cat.no MN740955-50) was used. Cells from which the medium was removed were washed with DBPS (Dulbecco's Phosphate-Buffered Saline), and RNA Isolation was performed according to the Nucleospin RNA manual. After the isolated RNA was synthesized using a cDNA Synthesis kit (Gendepot), the synthesized cDNA was used as a template to analyze the Filaggrin gene expression and 2X IQ Sybr Green SuperMIX (Bio-Rad) was used. . The expression rate was normalized with housekeeping genes (beta-actin). For qRT-PCR conditions, polymerase activation and DNA denaturation were performed at 95°C for 3 minutes, amplication was performed at 94°C for 15 seconds, denaturation and annealing were performed at 62°C for 30 seconds, and extension was performed at 72°C for 30 seconds. 30 cycles were performed under the condition of seconds. The primers used for RT-PCR of Filaggrin are SEQ ID NO: 4: For (5'- GCTGAAGGAACTTCTGGAAAAG-3') and SEQ ID NO: 5: Revers (5'-GCCAACTTGAATACCATCAGAAG-3'), beta-actin qRT- The primers used for PCR are the same as SEQ ID NO: 6: For (5'-GTGGGGCGCCCCAGGCACCA-3') and SEQ ID NO: 7: Revers (5'-CTCCTTAATGTCACGCACGATTC-3').

As a result, as shown in FIG. 4 , in Comparative Example 1, the ability to increase the production of Filaggrin was not confirmed. On the other hand, when 1% of the fermented product of Example 3 was treated, the ability to increase the production of Filaggrin was 202%. Therefore, it was confirmed that the skin barrier function was increased by the fermentation conditions of Example 3 compared to Comparative Example 1.

<Example 8> Moisturizing efficacy test: aquaporin (Aguaporin) production increase effect

In order to measure the moisturizing effect of Example 3 and Comparative Example 1, the ability to increase the production of aquaporin, a membrane transfer protein that regulates the entry and exit of water into cells, was confirmed by a Western blot test. HaCaT cells, which are human-derived keratinocytes, were cultured in an incubator at _{37° C. and 5% CO 2} condition using DMEM medium containing 10% Fetal bovin serum (FBS) and 1% Penicillin/streptomycin. ^{The cultured HaCaT cells were aliquoted to a concentration of 1.2×10 6} in a 100 mm plate, and then cultured in an incubator at 37° C. and 5% CO ₂ conditions and attached to the plate. After 24 hours, it was replaced with serum free media not containing FBS and cultured for 24 hours. Then, the fermented products of Example 3 and Comparative Example 1 were treated with media containing each concentration and cultured in an incubator for 3 hours, and then the cells were recovered and protein was extracted with 50 μl of RIPA buffer. The extracted protein was quantified by BCA assay. After diluting the protein amount of each sample to 25 μg, electrophoresis was performed on an 8% SDS gel. After electrophoresis, it was transferred to a PVDF membrane, and blocking was performed with 5% Bovine serum albumin solution. After reacting overnight with the primary antibody (Rabbit anti-aquaporin 3 antibody), the remaining antibody was washed with TBS-T buffer to remove it, and then reacted with the secondary antibody (Goat anti-rabbit IgG-HRP). After removing the remaining antibody, the ECL solution causing the color reaction was treated with Membrane and then photographed through the ChemiDoc gel imaging system and quantified through the Image Lab program. At this time, retinoic acid was used as a positive control.

As a result, as shown in FIG. 5 , in Comparative Example 1, the ability to increase production of aquaporin, a moisturizing factor, was not confirmed. On the other hand, in Example 3, 0.5% treatment showed aquaporin production enhancing ability of 105.34%, and 1% treatment showed aquaporin production enhancing ability of 150.92%. Therefore, it was confirmed that the moisturizing effect was increased by the fermentation conditions of Example 3 compared to Comparative Example 1.

As a specific part of the present invention has been described in detail above, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center (KCCM) KCCM12750P 20210609

<110> Breezytail Inc. <120> Streptococcus thermophilus_Breezytail_AIR21 and Fermented Product Manufactured Using Thereof <130> p21063 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Universal primer 27F <400> 1 agagtttgat cmtggctcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Universal primer 1492R <400> 2 tacggytacc ttgttacgac tt 22 <210> 3 <211> 1432 <212> DNA <213> Artificial Sequence <220> <223> Streptococcus thermophillus_Breezytail_AIR21 <400> 3 cccagcgctg caactgcagt agaagctgaa agaggagctt gctcttcttg gatgagttgc 60 gaacgggtga gtaacgcgta ggtaacctgc cttgtagcgg gggataacta ttggaaacga 120 tagctaatac cgcataacaa tggatgacac atgtcattta tttgaaaggg gcaattgctc 180 cactacaaga tggacctgcg ttgtattagc tagtaggtga ggtaatggct cacctaggcg 240 acgatacata gccaacctga gagggtgatc ggccacactg ggactgagac acggcccaga 300 ctcctacggg aggcagcagt agggaatctt cggcaatggg ggcaaccctg accgagcaac 360 gccgcgtgag tgaagaaggt tttcggatcg taaagctctg ttgtaagtca agaacgggtg 420 tgagagtgga aagttcacac tgtgacggta gcttaccaga aagggacggc taactacgtg 480 ccagcagccg cggtaatacg taggtcccga gcgttgtccg gatttattgg gcgtaaagcg 540 agcgcaggcg gtttgataag tctgaagtta aaggctgtgg ctcaaccata gttcgctttg 600 gaaactgtca aacttgagtg cagaagggga gagtggaatt ccatgtgtag cggtgaaatg 660 cgtagatata tggaggaaca ccggtggcga aagcggctct ctggtctgta actgacgctg 720 aggctcgaaa gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg 780 atgagtgcta ggtgttggat cctttccggg attcagtgcc gcagctaacg cattaagcac 840 tccgcctggg gagtacgacc gcaaggttga aactcaaagg aattgagggg ggcccgcaca 900 agcggtggag catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat 960 cccgatgcta tttctagaga tagaaagtta cttcggtaca tcggtgacag gtggtgcatg 1020 gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaaccccta 1080 ttgttagttg ccatcattca gttgggcact ctagcgagac tgccggtaat aaaccggagg 1140 aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca 1200 atggttggta caacgagttg cgagtcggtg acggcgagct aatctcttaa agccaatctc 1260 agttcggatt gtaggctgca actcgcctac atgaagtcgg aatcgctagt aatcgcggat 1320 cagcacgccg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga 1380 gtttgtaaca cccgaagtcg ggaggtaacc tttgagccgc cgctaaggac gg 1432 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin primer For <400> 4 gctgaaggaa cttctggaaa ag 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Filaggrin primer Revers <400> 5 gctgaaggaa cttctggaaa ag 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin primer For <400> 6 gtggggcgcc ccaggcacca 20 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> beta actin primer Revers <400> 7 ctccttaatg tcacgcacga ttc 23

Claims (7)

Staphylococcus aureus ( Staphylococcus aureus ) A novel Streptococcus thermophilus_Breezytail_AIR21 strain having a reduction, anti-inflammatory, anti-allergic, moisturizing and skin barrier strengthening effect (Accession No.: KCCM13005P).
The Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) according to claim 1, wherein the strain is derived from the skin of a companion animal.
Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) inoculated into a medium containing maltose and glutamine, and a method for producing a fermented product comprising the steps of culturing.
The method according to claim 3, wherein the culturing is performed at 35 to 37° C. for 90 to 130 hours.
Fermented product of Streptococcus thermophilus_Breezytail_AIR21 strain (Accession No.: KCCM13005P) in a medium containing Maltose and Glutamine.
A cosmetic composition comprising the fermented product of claim 5 as an active ingredient.
The cosmetic according to claim 6, wherein the cosmetic composition is selected from the group consisting of Staphylococcus aureus reduction, anti-inflammatory, anti-allergic, moisturizing and skin barrier strengthening effects. composition.

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