KR102226187B1 - Lactobacillus iners AHC2030 and Fermented Product Manufactured Using Thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus iners AHC2030 strain, and a fermented material produced by using the same. Skin-microbiome change induction (Cutibacterium acnes) of a novel Lactobacillus iners AHC2030 strain (deposition number KCCM1275OP) is reduced, and Staphylococcus aureus is reduced. In addition, lactic acid bacteria are increased. Also, antioxidant, whitening, wrinkle alleviation, skin density, and skin elasticity improvement effects are excellent. Thus, when the Lactobacillus iners AHC2030 strain is applied as an active ingredient to a cosmetic material base, the strain has an excellent effect as a cosmetic composition.

Description

Lactobacillus iners AHC2030 strain and fermented product manufactured using the same {Lactobacillus iners AHC2030 and Fermented Product Manufactured Using Thereof}

The present invention relates to a Lactobacillus Enus AHC2030 strain and a fermentation product prepared using the same.

The skin's ecosystem provides a variety of habitats for microorganisms, and a wide range of microorganisms live there. People who are hosts have a symbiotic relationship with them, and are known to have many positive effects on the host. The skin consists of various types of habitats such as depressions and specialized crevices, and helps a wide distribution of microorganisms to grow. Basically, the skin forms a physical film and helps to defend against potential hazards and toxic substances from the outside. The skin becomes a point of connection with the external environment and is also a collection of various microorganisms (fungi, bacteria, viruses and small larvae). In accordance with the choice of physical and chemical functions, microbes adapt to specialized niches to prepare habitats. In general, the skin is cold, acidic, and remains dry. Structurally, the epidermis forms the 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 keratinocytes. The epidermis has a shape called "brick and mortar structure". The skin tissue goes through a continuous self-healing process, and the squames that have gone through the end of the differentiation process are constantly being removed from the skin tissue.

Meanwhile, bacteria that obtain energy by fermenting sugars and produce a large amount of lactic acid are collectively referred to as lactic acid bacteria. Lactobacillus live in the intestine, coexist with the digestive system of the human body, and play a role in decomposing fiber and complex proteins into important nutrients. In addition, by keeping the intestinal environment acidic, it inhibits the growth of harmful bacteria, improves diarrhea and constipation, synthesizes vitamins, and inhibits blood cholesterol. Lactobacillus has the property of being able to strongly bind to the intestinal mucosa and epithelial cells, so it helps a lot in intestinal function, and by promoting the activity of macrophages and spleen, it has the effect of secreting and promoting substances involved in the immune response. In addition, it has an immune-modulating function, so it is effective against atopy and allergy-related diseases.

In the field of cosmetics, lactic acid bacteria culture medium has been commercialized in 1955 and has been used up to the present. Mainly , Streptococcus genus, Lactobacillus genus, Lactococcus genus, Leuconostoc genus, and Bifidobacterium genus ( Bifidobacterium ), etc., direct fermentation in culture broth, such as filtration or extraction, etc., and inoculation into active materials and fermentation, followed by filtering or extraction, etc. are being actively conducted.

Among them , the technology that is applied to various uses using the genus Lactobacillus is Korean Patent Publication No. 10-1846796 ``Cosmetics for improving elasticity or moisturizing containing whey obtained by fermentation with Korean lactic acid bacteria'', Korea Registered Patent Publication No. 10-1492003 「New Lactobacillus Plantarum HY7714 with skin wrinkle improvement and moisturizing effect and a product containing it as an active ingredient」 and Korean Registered Patent Publication No. 10-1998067 「Bifidobacterium genus GFC-B09 and fermented products manufactured using the same” and the like are disclosed.

Looking at the skin ecological environment again, physical and biological factors are well harmonized to provide a variety of habitats, and furthermore, it will be possible to think about a delicate balance between humans and microorganisms. When the balance of symbiotic relationships is broken, skin breakdown and infection occur at the same time.

Accordingly, the present inventors have separated and identified a Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P), and using this to prepare a fermented product through the step of inoculating and culturing the MRS medium, skin-micro Induction of changes in skin-microbiome ( reduction of Cutibacterium acnes and reduction of Staphylococcus aureus , increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density And it was discovered that the effect of improving skin elasticity can be provided, and the present invention was completed.

Korean Registered Patent Publication No. 10-1846796 Korean Registered Patent Publication No. 10-1492003 Republic of Korea Patent Publication No. 10-1998067

The object of the present invention is to induce changes in skin-microbiome ( Cutibacterium acnes and Staphylococcus aureus decrease, increase of lactic acid bacteria), antioxidant, whitening, It is to provide a novel strain having an effect of improving wrinkles, skin density, and skin elasticity.

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

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 present invention is a skin-microbiome skin-microbiome (skin-microbiome) change induction ( Cutibacterium acnes ) reduction and Staphylococcus aureus (Staphylococcus aureus ) reduction, increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) having the effect of improving skin density and skin elasticity is provided.

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

In addition, the present invention provides a method for producing a fermented product comprising the step of inoculating and culturing a Lactobacillus iners AHC2030 strain (accession number: KCCM12750P) in a medium formulated to maximize potency activity. do.

In the present invention, the medium is characterized in that it contains Maltose, Lactose, Glycine and Inulin.

In addition, in the present invention, the culture is characterized in that it is carried out for 90 to 130 hours at 32 ~ 37 ℃.

In addition, the present invention provides a fermented product fermented with Lactobacillus iners AHC2030 strain (accession number: KCCM12750P).

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 (skin-microbiome) change induction ( Cutibacterium acnes ) reduction and Staphylococcus aureus (Staphylococcus aureus) of It is characterized in that it is a use selected from the group consisting of the effect of reducing, increasing lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity improvement.

The fermented product of Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) provided from the present invention is a skin-microbiome change induction (cutibacterium acnes ) reduction and staphylo As it has excellent effects on reducing Staphylococcus aureus , increasing lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity, it is used as a cosmetic composition when applied to a cosmetic base as an active ingredient. The effect is excellent.

Figure 1 shows the nucleotide sequence of the Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) according to the present invention.
2 is a reduction of Cutibacterium acnes and Staphylococcus aureus when the fermented product of the Lactobacillus iners AHC2030 strain according to the present invention is treated on the skin, lactic acid bacteria It shows the increasing change of.
Figure 3 is a graph showing the antioxidant effect results of the fermentation of the Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) according to the present invention.
4 is a graph showing the results of anti-wrinkle effect of fermentation of Lactobacillus iners AHC2030 strain (accession number: KCCM12750P) according to the present invention.
5 is a graph showing the results of anti-wrinkle effect of fermentation of Lactobacillus iners AHC2030 strain (accession number: KCCM12750P) according to the present invention.
Figure 6 is a graph showing the results of the whitening effect of the fermentation of Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) according to the present invention.
7 is a graph showing the results of the anti-inflammatory effect of fermentation of Lactobacillus iners AHC2030 strain (accession number: KCCM12750P) according to the present invention.

Thus, in the present invention, skin-microbiome skin-microbiome change induction ( Cutibacterium acnes ) and Staphylococcus aureus (Staphylococcus) from microorganisms isolated from female skin in their twenties. aureus ), increase of lactobacillus), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity improvement effect, screening strains to select excellent strains, 16s rRNA sequencing analysis, Lactobacillus Enus ( Lactobacillus) iners) . Accordingly, it was named as Lactobacillus iners AHC2030 strain and deposited with the Korean Culture Center of Microorganisms.

Accordingly, the present invention relates to a Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number: KCCM12750P) (hereinafter abbreviated as'AHC2030').

At this time, the AHC2030 strain is characterized in that it is derived from the skin of a woman in her twenties.

In addition, the present invention relates to a method for producing a fermented product comprising the step of inoculating and culturing the AHC2030 strain in a medium formulated to maximize efficacy and activity.

The medium used for the culture is characterized in that it contains Maltose, Lactose, Glycine and Inulin. 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 5g/ L, pH 6.5), fermented with a medium containing 0.5g/L of Maltose, 1.0g/L of Lactose, 0.5g/L of Glycine, and 1.0g/L of Inulin, the fermentation yield is most effectively improved. It is preferable in terms of being able to make changes in skin flora.

^{The AHC2030 strain was 1.0x10 7} to MRS liquid medium to which Maltose, Lactose, Glycine and Inulin were added. The ^{step of inoculating and culturing at a level of 1 to 3% by making the number of bacteria 1.0x10 9} is characterized in that the stationary culture is performed at 32 to 37°C for 90 to 130 hours under anaerobic conditions. If the culture temperature and time are out of the above range, the culture conditions of the fermentation strain are not appropriate, leading to sufficient skin-microbiome change ( reduction of Cutibacterium acnes and Staphylococcus au Reus ( Reduction of Staphylococcus aureus ), increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity improvement effects, as well as not being able to exhibit, it is not preferable because the strain does not grow.

As described above, the cultured strain is filtered under the conditions of pH 3.8 to 4.2, and then the filtered strain is aged at 20 to 30° C. for 10 to 16 days to prepare a fermented product.

In addition, the fermented product of the present invention Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number KCCM1275OP) induced changes in skin-microbiome ( Cutibacterium acnes ) and Staphylococcus Staphylococcus aureus (Reduction of Staphylococcus aureus), increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity improvement effect is very excellent as a cosmetic composition.

Accordingly, the present invention relates to a cosmetic composition comprising a fermented product fermented with Lactobacillus iners AHC2030 strain (accession number KCCM12750P) as an active ingredient.

The cosmetic composition is a skin-microbiome change induction ( reduction of Cutibacterium acnes and reduction of Staphylococcus aureus , increase of lactic acid bacteria), antioxidant, whitening , Wrinkle improvement, skin density and skin elasticity improvement effect may be a use selected from the group consisting of.

Induction of skin-microbiome change of the present invention ( reduction of Cutibacterium acnes and reduction of Staphylococcus aureus , increase of lactic acid bacteria), antioxidant, whitening, Cosmetic compositions for improving wrinkles, skin density and skin elasticity include skin ointments, creams, softening lotion, nutrient lotion, pack, essence, hair tonic, shampoo, conditioner, hair conditioner, hair treatment, gel, skin lotion, Skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, eye cream, moisture cream, hand cream, foundation, nutrition essence, sunscreen, soap, cleansing foam, cleansing lotion, It may have a formulation selected from the group consisting of cleansing cream, body lotion, and body cleanser, but is not limited thereto. The composition of each of these formulations may contain various bases and additives necessary and appropriate for 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, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc acid may be used as a carrier component. I can.

When the formulation of the present invention is a powder or 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, additionally 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, a solvating agent or an emulsifying agent is used as a carrier component, such as 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, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracant, and the like may be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linoline derivatives or ethoxylated glycerol fatty acid esters may be used.

In the cosmetic composition 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, but preferred skin-microbiome (skin-microbiome) induction of change ( reduction of Cutibacterium acnes and reduction of Staphylococcus aureus , increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin It is not limited thereto as long as it includes an effective amount capable of providing an elasticity improvement effect.

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

<Example 1> Lactobacillus iners Isolation of AHC2030 strain

After swapping the skin of a woman in her twenties with gauze and shaking it in MRS medium, ^{0.1 mL of a sample diluted 10 -4} times (volume) was added to MRS agar medium (Peptospecial 10g, beef extract 10g, 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)) Incubated in an incubator at 37° C. for 2 days. The formed colonies were separated and cultured in pure water, cultivated in an anaerobic incubator for 24 hours, and then a single colony formed was isolated. Among the isolated strains, skin-microbiome change induction ( reduction of C. acne and reduction of S. aureus , increase of lactic acid bacteria), antioxidant, whitening, wrinkle improvement, skin density and skin elasticity improvement effect The excellent AHC2030 strain was selected.

<Example 2> Selected strain 16s rRNA sequence analysis

The gDNA of the finally selected AHC2030 strain and the universal primer of 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') were used. Using 16s DNA sequencing analysis, it was confirmed that it is a strain having high homology with Lactobacillus iners (SEQ ID NO: 3), and was named as Lactobacillus iners AHC2030 strain (Lactobacillus iners AHC2030) strain, and an international microbial patent It has been deposited with the Korean Microorganism Conservation Center (KCCM), a depository institution, and has been assigned the accession number KCCM12750P.

<Example 3> Preparation of fermented product of AHC2030 strain

In order to prepare the fermented product of the isolated AHC2030 strain, Maltose, Lactose, Glycine and Inulin were added to the MRS medium to prepare the AHC2030 strain, and then 1% was inoculated with the number of bacteria of ^{1.0×10 9.} There was no oxygen supply to maintain the anaerobic state after inoculation, and stationary culture was carried out at 37°C for 120 hours. After the cultivation was completed, it was confirmed that the pH was formed to 4.0 (error 0.2), and when the dark brown color was confirmed visually, the cultivation was completed and a 0.2 µm filter (Millipore) sanitization was performed. When the filter was completed, the fermented product of the AHC2030 strain was prepared through a two-week aging period at room temperature.

<Comparative Example 1> Preparation of fermented product using MRS general medium of AHC2030 strain

Comparative Example 1 was prepared in the same manner as in Example 3, except that only the general MRS medium was used and Maltose, Lactose, Glycine, and Inulin were not added to prepare a fermented product using the general medium of the isolated AHC2030 strain. .

<Experimental Example 1> Subject and Skin-Microbiome Sample Collection

The samples used in this experiment were tested on subjects who obtained prior consent from the GP Life Science Research Institute. The fermented product of the AHC2030 strain was used as the probiotics used in the test, and the culture solution from which the cells were removed after cultivation was applied to the skin of the subject's face, and skin samples were collected for a total of 72 hours and used for analysis. The method was used, and a skin sample was collected by scrubbing with gauze. The collection of microorganisms on the skin consisted of pre-treatment, post-treatment, non-treatment (rest) and re-treatment groups.

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

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

The supernatant obtained by suspending the sample collected from the skin in 0.85% NaCl was centrifuged (17,000 rpm/m) to take the cells, and after washing the cells once in sterilized physiological saline, FastDNA SPIN KIT (MP Biomedical, France) was used. Then, DNA was extracted.

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

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

As a result, as can be seen in Figure 2, depending on the AHC2030 strain fermentation treatment, the reduction of representative harmful strains, Cutibacterium acnes and Staphylococcus aureus , and the increase of the beneficial strain, lactic acid bacteria Confirmed. Therefore, it was confirmed that the increase of beneficial dermatophytes depending on the fermentation of the AHC2030 strain was treated.

<Experimental Example 4> Antioxidation test: DPPH radical scavenging activity

An antioxidant efficacy test was conducted using the fermented product of Example 3 (AHC2030 strain fermented product) and Comparative Example 1. Free radical refers to free radicals, and the oxygen we breathe creates energy and is a substance with a high oxidative power that is thousands of times higher in the process of being reduced to water. It is produced in the metabolic process of cells in the body of animals and plants, or it is generated by stress, photostimulation, and bacterial penetration. When an excessive amount of active oxygen is present in the body, it indiscriminately attacks normal cells and becomes the main culprit of various diseases and aging. In addition, it acts as a pathological factor showing an immune response sensitive to external stimuli. Whether the extract of the present invention exhibits antioxidant activity, thereby reducing inflammation, was confirmed by the free radical scavenging ability of DPPH, and DPPH inhibition by diluting the fermented products of Example 3 and Comparative Example 1 at a concentration of 50 (㎍/㎖), respectively. The degree was evaluated. After diluting the fermented products of Example 3 and Comparative Example 1 to a concentration of 50 (㎍ / ㎖), 0.1M DPPH 250 µl (1,1-diphenyl-2-picrylhydrazyl) solution was mixed at 4° C. for 30 minutes. Reacted. When the reaction was over, each sample was placed in a 96 well plate and absorbance was measured at 520 nm using an ELISA reader. At this time, ascorbic acid was used as a positive control.

In order to confirm the DPPH radical scavenging activity, it was calculated using Equation 1 below.

[Equation 1]

Free radical scavenging ability (%)=[100-(absorbance of sample treated group/absorbance of untreated group X 100)]

division Free radical scavenging activity (%) Positive control 70.32±1.31 Example 3 (fermented product of AHC2030 strain) 88.05±0.69 Comparative Example 1 58.14±0.28

As a result, as shown in Table 1 and Figure 3, Example 3 (AHC2030 strain fermented product) was confirmed to be superior to the DPPH radical scavenging ability compared to Comparative Example 1.

This result was judged to be an effect due to the difference in the growth amount of the strain and the amount of effective postbiotics produced by the substances added during the fermentation process.

<Experimental Example 5> Anti-wrinkle efficacy test: MMP-1 inhibition and PIP Assay

Using the fermented products of Example 3 and Comparative Example 1, MMP-1 (collagenase) inhibition and PIP (Procollagen type 1 C-Peptide) production were evaluated.

(1) MMP-1 inhibition

In order to confirm the anti-wrinkle activity by diluting the fermented products of Example 3 and Comparative Example 1 at a concentration of 50 (µg/ml), respectively, an assay experiment for MMP-1 was performed. The inhibitory activity of MMP-1 expressed by UVA irradiation was confirmed through real-time PCR, and it was confirmed whether it affects the inhibitory activity of MMP-1 by preparing and treating each sample treatment concentration.

division MMP-1 inhibitory activity (%) Positive control (EGCG) 58.03±1.42 Negative control 1.10±0.39 Example 3 (fermented product of AHC2030 strain) 72.58±0.40 Comparative Example 1 47.36±0.65

(2) Procollagen Type Ⅰ C-peptide(PIP) Assay

The effect of the fermented products of Example 3 and Comparative Example 1 on the procollagen synthesis of human dermal fibroblasts was investigated. Each of the fermented products of Example 3 and Comparative Example 1 was diluted to a concentration of 50 (µg/ml), treated, and cultured for 24 hours, and then 100µl of each treated culture solution was added to a 96well plate of Procollagen Type IC-peptide (PIP) kit. They were dispensed each and put in a 37°C incubator and allowed to react for 2 hours. Then, after washing with D-PBS, 100 µl of each antibody-POD conjugate solution was dispensed and placed in an incubator at 37° C. and allowed to react for 1 hour. After washing with D-PBS again, 100 μl of each of the substrate solutions (3,3',5,5'-tetrametylbenzidine) was dispensed and reacted at room temperature for 15 minutes. After 15 minutes, absorbance was measured at 450 nm using a multi-reader.

division PIP generation (ng/ml) Control 100±0.3 Positive control (EGCG) 165±3.9 Example 3 (fermented product of AHC2030 strain) 155±4.4 Comparative Example 1 121±6.2

As a result, as shown in Table 3 and FIGS. 4 to 5, it was confirmed that Example 3 had superior MMP-1 inhibitory activity and collagen production activity compared to Comparative Example 1. This result was judged to be an effect due to the difference in the growth amount of the strain and the amount of effective postbiotics produced by the substances added during the fermentation process.

<Experimental Example 6> Whitening efficacy test: Measurement of melanin production

Using the fermented products of Example 3 and Comparative Example 1, the amount of melanin production was measured to confirm the effect on the production of melanin related to skin whitening.

B16F10 melanoma cells were counted equally at a rate of 2.0x10 ⁵ cells/well per dish in a 60Φ Petri dish using a hemacytometer, and then cultured for 24 hours at 37°C and 5% carbon dioxide. After culturing for 24 hours, the obtained cell culture solution was removed, and each Example and Comparative Example were taken together with α-MSH 50 nM for inducing melanin production, mixed with a medium, dispensed, and reacted for 60 hours, and then the culture solution (supernatant) Collected. In order to measure the amount of extracellular melanin produced, 1 ml of the culture solution (supernatant) was reacted for 60 hours, and the absorbance was measured at 450 nm using an ELISA plate reader. At this time, arbutin was used as a positive control.

In order to confirm the amount of melanin production, it was calculated using Equation 2 below.

[Equation 2]

Melanin production amount (%) = (absorbance of sample treatment group/absorbance of control group) X100

. division Melanin contents (% of control) Positive control (Arbutin) 24.40±0.30 Negative control 89.63±1.88 Example 3 (fermented product of AHC2030 strain) 25.19±0.96 Comparative Example 1 28.54±0.93

As a result, as shown in Table 4 and FIG. 6, both Example 3 and Comparative Example 1 were found to have low melanin production, and thus it was confirmed that the melanin production inhibitory activity was excellent. These results imply that the fermented product of the AHC2030 strain (accession number: KCTC12750P) strain has a whitening effect.

<Experimental Example 7> Anti-inflammatory efficacy test: Nitric Oxide (NO) inhibitory activity

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

To a type of RAW 264.7 cell of macrophages using a DMEM medium containing 1% penicillin / scrap Toma who and 10% FBS (fetal bovineserum) in 24 Well plate after the frequency division by the same coefficient by 1.0x10 ⁴ cell / well , 37 ℃ and incubated for 24 hours at 5% carbon dioxide conditions. After mixing Example 3 and Comparative Example 1 with the medium at a concentration of 50 (㎍ / ㎖) to RAW 264.7 cells cultured for 24 hours, 1 ml was added to each well and reacted in an incubator under conditions of _{37° C. and 5% CO 2 for 24 hours} Made it. At this time, LPS (Lipo polysaccharide), which is an inflammation-inducing factor expressing NO, was treated at a concentration of 1 μg/ml and reacted for 24 hours at 37° C. and 5% carbon dioxide. Next, after separately taking only the supernatant of each well, 100 ml of the culture solution is taken into a 96 well plate using a Nitric Oxide detection kit in each well, and 50 µl of Griess reagent A (N-1-Naphthylethylenediamine (NEDHC)) and 50 µl of Griess reagent B. 50 µl of (Sulfanilamide) was added and reacted for 10 minutes each, and absorbance was measured at 540 nm using an ELISA plate reader. The concentration of NO in the cell culture was calculated by comparing it with the standard curve for each concentration of sodium nitrite (NaNO2).

division NO concentration (μM) Positive control 14.4±1.09 Negative control 70.1±0.82 Example 3 (fermented product of AHC2030 strain) 13.6±1.24 Comparative Example 1 26.8±0.90

As a result, as shown in Table 5 and FIG. 7, Example 3 was confirmed to have a lower concentration of nitric oxide generation compared to Comparative Example 1, and thus it was confirmed that the effect of inhibiting formation of nitric oxide was excellent.

These results indicate that the AHC2030 strain (accession number: KCTC12750P) strain fermented product has an anti-inflammatory effect.

<Experimental Example 8> Antibacterial efficacy test: MIC test

The degree of antibacterial activity was confirmed using the fermented products of Examples and Comparative Examples.

1. Preparation of the evaluation medium

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

2. MIC test

After preparing an independent nutrient broth sterilized at 121° C. for 15 minutes, each standard test strain was inoculated in an amount of ^{3×10 5 cfu/ml.} Example 3 and Comparative Example 1 were typically set to test concentrations of 1%, 0.5%, 0.25%, 0.125%, 0.0625%, 0.03125%, 0.015625%, 0.0078125%, and inoculated and cultured using single colony of the test strain. . After the culture was completed, the absorbance (600 nm) was measured to confirm the result. The antimicrobial effect was confirmed at the lowest concentration in which no microorganism was detected.

Classification (Unit: %) Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Candida albicans Example 3 (fermented product of AHC2030 strain) 0.25 0.125 0.125 0.50

As a result, as shown in Table 6, the AHC2030 strain culture was classified as E. coli (Escherichia coli , ATCC 8739), Staphylococcusaureus , ATCC 6538P, which are four publicly disclosed standard test strains (see the Ministry of Food and Drug Safety and MIC test guide line), It was confirmed that the inhibitory effect on Pseudomonas aeruginosa (ATCC 9027) and Candida albicans (ATCC 10231) was confirmed, and the antibacterial effect was excellent.

<Production Example 1> Preparation of cosmetics containing fermented product of AHC2030 strain

(1) Manufacture of lotion

A lotion was prepared by a conventional method from the composition shown in Table 7 below.

ingredient Content (% by weight) AHC 2030 fermented product 3.00 Hydroxyethylene cellulose (2% aqueous solution) 12.00 Xanthan gum (2% aqueous solution) 2.00 1,3-butylene glycol 6.00 glycerin 4.00 Sodium hyaluronate (1% aqueous solution) 5.00 Purified water 68.00 Sum 100.00

(2) lotion manufacturing

A lotion was prepared by a conventional method from the composition shown in Table 8 below.

ingredient Content (% by weight) AHC 2030 fermented product 3.00 Ascorbic acid-2-magnesium phosphate salt 1.00 Water-soluble collagen (1% aqueous solution) 1.00 Sodium citrate 0.01 Citric acid 0.05 1,3-butylene glycol 3.00 Purified water 91.94 Sum 100.0

As described above, specific parts of the present invention have been described in detail, and it will be apparent to those of ordinary skill in the art that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Korea Microorganism Conservation Center (KCCM) KCCM12750P 20200617

<110> GFC Life Science Co., Ltd <120> Lactobacillus iners AHC2030 and Fermented Product Manufactured Using Thereof <130> p20218 <160> 3 <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> 1461 <212> DNA <213> Artificial Sequence <220> <223> Lactobacillus iners AHC2030 <400> 3 ccgagcgcgt caactgcagt cgagcgagtc tgccttgaga tcggagtgct tgcactctgt 60 gaaacaagat acaggctagc ggcggacggg tgagtaacac gtgggtaacc tgcccaagag 120 atcgggataa cacctggaaa cagatgctaa taccggataa caacagatga tgcctatcaa 180 ctgtttaaaa gatggttctg ctatcactct tggatggacc tgcggtgcat tagctagttg 240 gtagggtaac ggcctaccaa ggcgatgatg catagccgag ttgagagact gatcggccac 300 attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360 tggacgcaag tctgatggag caacgccgcg tgagtgaaga agggtttcgg ctcgtaaagc 420 tctgttgttg gtgaagaagg acaggggtag taactgacct ttgtttgacg gtaatcaatt 480 agaaagtcac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggatttat tgggcgtaaa gcgagtgcag gcggctcgat aagtctgatg tgaaagcctt 600 cggctcaacc ggagaattgc atcagaaact gtcgagcttg agtacagaag aggagagtgg 660 aactccatgt gtagcggtga aatgcgtaga tatatggaag aacaccggtg gcgaaggcgg 720 ctctctggtc tgttactgac gctgaggctc gaaagcatgg gtagcgaaca ggattagata 780 ccctggtagt ccatgccgta aacgatgagt gctaagtgtt gggaggtttc cgcctctcag 840 tgctgcagct aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatccatag ccagtctaag agattagatg ttcccttcgg 1020 ggactatgag acaggtggtg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttgtcatta gttgccagca ttaagttggg cactctaatg 1140 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggac ggtacaacga gaagcgaccc tgtgaaggca 1260 agcggatctc tgaaagccgt tctcagttcg gattgcaggc tgcaactcgc ctgcatgaag 1320 ctggaatcgc tagtaatcgc aaatcagcac gttgcggtga atacgttccc gggccttgta 1380 cacaccgccc gtcacaccat gagagtctgt aacgcccgaa gccggcgggt aaccgaaagg 1440 atcagcctca agcgacgtgg g 1461

Claims (7)

A novel Lactobacillus Enus AHC2030 (Lactobacillus) that has the effect of reducing Cutibacterium acnes , reducing Staphylococcus aureus , antioxidant, whitening, wrinkle improvement, skin density and skin elasticity. iners AHC2030) strain (accession number KCCM12750P).
According to claim 1, wherein the strain is Lactobacillus iners AHC2030 (Lactobacillus iners AHC2030) strain (accession number KCCM12750P), characterized in that the skin is derived.
A method for producing a fermented product comprising the step of inoculating and culturing a Lactobacillus iners AHC2030 strain (accession number KCCM12750P) in a medium containing Maltose, Lactose, Glycine and Inulin.
The method of claim 3, wherein the cultivation is performed at 32 to 37°C for 90 to 130 hours.
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