KR102329921B1 - Composition for Improving Skin Conditions Extracts of Swallow's Nest Fermented by Novel Strain of Lactobacillus sp. as Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for improving skin condition, comprising a hydrobatidae nest fermented extract using a novel Lactobacillus brevis strain as an active ingredient. The hydrobatidae nest fermented extract of the present invention has an excellent effect in improving skin condition because the content of Ν-acetylneuraminic acid is increased and the activity of antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration is improved, and thus can be usefully used in fields of cosmetics and foods. In addition, the composition of the present invention is very safe to the human body, and also has excellent stability.

Description

Composition for Improving Skin Conditions Extracts of Swallow's Nest Fermented by Novel Strain of Lactobacillus sp. as Active Ingredient}

The present invention relates to a composition for improving skin condition comprising, as an active ingredient, a bird's nest ferment extract using a new strain of the genus Lactobacillus, and more particularly, having antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration activities. It relates to a composition for improving skin condition.

Some of the substances that play an important role in the process for maintaining homeostasis of living organisms are physiologically active substances derived from various living organisms. Until now, many studies have been conducted on numerous physiologically active substances, and among them, research on substances having physiological activity isolated from microorganisms, plants, or animals, etc. is a very important field in life science and medicine.

Conventionally developed vitamin C, retinoic acid, transforming growth factor (TGF), etc. are known to be effective in skin wrinkle improvement, whitening, antioxidant, etc., and as whitening ingredients, kojic acid, arbutin ( It is well known as a substance that inhibits tyrosinase enzyme activity such as arbutin).

In addition, in recent years, the development of cosmetics that increase the functionality acting on the skin by introducing physiologically active substances obtained from natural products into cosmetics to further strengthen the inherent defense function of the skin itself has been actively developed.

Therefore, there is an urgent need for the development of natural materials that are safe to the living body, have stable active ingredients, and are more effective than those with existing skin whitening, antioxidant, moisturizing, and skin regenerating effects above all else.

On the other hand, in the Bonchogangmok, the nest of swallowtails is introduced, “It makes the skin shiny and it is beneficial to the blood, making it lively.” In addition, swallow's nest is excellent for nourishing effects, and the Empress Dowager of China used a mixture of swallow's nest and pearl powder for skin beauty to maintain her beauty and youthfulness. Swallow's nest contains protein that nourishes the skin, making dry skin softer, and contains vitamin C, vitamin E, arbutin, and collagen that give elasticity to tired skin. Proteoglycan, a major component contained in swallow's nest, plays an important role in binding or supporting individual cells, such as the entire body, skin, and organs, and has an excellent skin regeneration effect. .

The collection of bird's nest is conducted under strict supervision to the extent that only authorized collectors are allowed to collect it, and the annual amount is also limited, and it is used as a very expensive material. Regarding the efficacy of extracts extracted from swallow's nest, antiviral action, whitening effect, etc. are known.

In addition, as a prior art related to swallow's nest, Patent Application No. 10-2015-0053874 'Functional cosmetic composition containing swallow's nest extract as an active ingredient' is Although it is disclosed that it is effective and can be used in a functional cosmetic composition, compared to using a combination of expensive raw materials, the effect is not sufficient, and there is a disadvantage in that economical efficiency is lowered. In addition, Patent Publication No. 10-2018-0086062, 'Composition for improving skin condition of acne skin containing sea swallow's nest complex extract as an active ingredient', developed by the inventors, and Patent Registration No. 10-1754645, 'Bird's nest complex extract' A composition for improving skin wrinkles comprising a fermented extract as an active ingredient, a patch for improving skin wrinkles using the same, and a mask pack' technology are disclosed. However, the use of expensive swallowtail nests as raw materials for cosmetics or food is limited to use in small amounts due to economic problems, and this has a disadvantage in that the functional effect is not sufficient.

Under this background, the present inventors have made intensive research efforts to improve the functionality of bird's nest, which is an expensive functional material, and increase its utility value. The content of neuraminic acid is increased by 31.9 times, and antioxidant, anti-inflammatory, and blood circulation improvement, moisturizing, whitening, and skin regeneration activities are remarkably increased, confirming that it has an excellent effect in improving the skin condition, and the present invention was completed.

KR 10-2018-0086062 (2018.07.30) KR 10-1754645 (2017.06.30)

Therefore, the main object of the present invention is to contain, as an active ingredient, a bird's nest extract fermented with a new strain SDCM 1105 of the genus Lactobacillus, which has excellent effects in antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and promoting regeneration of damaged skin. To provide a composition that

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a cosmetic composition comprising, as an active ingredient, a bird's nest extract fermented with a new strain of Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP).

The present inventors have made diligent research efforts to increase the usability of the material by further increasing the functionality for improving the skin condition of the expensive swallow's nest. As a result of a study on fermentation using strains, the content of Ν-acetylneuraminic acid in the fermented bird's nest ferment extract increased by 31.9 times when fermented with Tibetan mushrooms (Kefir grain), and antioxidant and anti-inflammatory properties , blood circulation improvement, moisturizing, whitening, and skin regeneration activities were improved, and it was confirmed that there was an excellent effect in improving the skin condition.

As used herein, the term "bird's nest" refers to a nest built by Collocalia fuciphaga, a type of sea swallow, and the nest is constructed by weaving seaweed and fish fins as main materials with secretions secreted from the salivary glands of the swallowtail. . The geumsayeon makes a nest by secreting undigested food when it builds a nest on a coastal cliff.

As used herein, the term "improving skin condition" indicates antioxidant activity, improves symptoms of inflammatory skin disease, reduces the total amount of melanin pigment to exhibit a whitening effect, replenishes moisture to the skin, and helps improve skin blood circulation. , is used in the meaning of promoting the regeneration of skin tissue or skin cells.

The term " Lactobacillus sp. SDCM 1105" strain of the present invention is a novel Lactobacillus sp. strain newly isolated and identified by the present inventors from Tibetan mushrooms (Kefir grain), in the present specification "Lactobacillus SDCM 1105"" or "SDCM 1105" is used synonymously with " Lactobacillus sp SDCM 1105".

As used in the present invention, the term "extract" is an extract obtained by extraction treatment of sea swallowtail fermented with a new strain of Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP), a diluted or concentrated solution of the extract, and obtained by drying the extract. It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a dried product, a prepared or purified product of the extract, or a mixture thereof. The extract of the present invention may be prepared and used in the form of a dry powder after extraction.

The cosmetic composition of the present invention is characterized in that it has antioxidant activity. In a preferred embodiment of the present invention, it was confirmed that the concentration-dependent increase in antioxidant activity of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus according to the present invention, and excellent antioxidant that is equal to or greater than that of ascorbic acid as a control. It was confirmed that the performance was shown (see FIG. 3).

In addition, the cosmetic composition of the present invention is characterized in that it suppresses the production of nitric oxide (NO) and suppresses the expression of inflammatory cytokines IL-1β and TNF-α genes. In a preferred embodiment of the present invention, it was confirmed that the fermented bird's nest ferment extract had an excellent effect on the inhibition of nitric oxide production (see FIG. 5 ), and concentration-dependently inhibited the production of LPS-induced IL-6 and TNF-α proteins. It could be confirmed that (see FIGS. 6 and 7).

In addition, the cosmetic composition of the present invention is characterized in that it exhibits skin moisturizing activity by promoting the synthesis of hyaluronic acid. In a preferred embodiment of the present invention, the amount of hyaluronic acid (Hyaluronan) production increases in a concentration-dependent manner by the treatment of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus, and in the 10% concentration range, it is further increased than the positive control group treated with retinoic acid It was confirmed that the hyaluronic acid production promoting effect was shown (see FIG. 8).

In addition, the cosmetic composition of the present invention is characterized in that it promotes the expression of vascular endothelial growth factor (VEGF) factor to exhibit skin blood circulation improvement activity. In a preferred embodiment of the present invention, the experimental group treated with the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus was 0.1%, 1%, and 10% of the VEGF factor at concentrations of 0.1%, 1%, and 10% compared to the control group (Con) that only gave out scratches. It was confirmed that the expression increased by 21.95%, 107.59%, and 431.95%, respectively (see FIG. 9 ).

In addition, the cosmetic composition of the present invention is characterized in that it exhibits skin whitening activity by inhibiting the production of melanin pigment. In a preferred embodiment of the present invention, the experimental group treated with the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus is 0.1%, 1%, and 10% concentration compared to the α-melanocyte stimulating hormone alone treatment group (Con) It was confirmed that the inhibition of melanin pigment production by 7.73%, 27.26%, and 69.48%, respectively (see FIG. 10).

In addition, the cosmetic composition of the present invention is characterized in that it promotes skin regeneration. In a preferred embodiment of the present invention, it was confirmed that skin regeneration was stimulated in a concentration-dependent manner in the NHDF cells treated with the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus (see FIG. 11 ).

The cosmetic composition of the present invention is harmless to the human body by using lactic acid bacteria derived from edible Tibetan mushrooms (Kefir grain) and fermented edible swallow's nest, and does not damage the quality of cosmetic materials due to its addition Therefore, it is suitable for use in a cosmetic composition. In particular, since it has functions related to antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration activity, it is preferably included in a functional cosmetic composition.

The cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, mask pack, It may be prepared in any one formulation selected from soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser, but is not limited thereto.

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

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

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

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

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

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

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

In the cosmetic composition of the present invention, the Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP) fermented sea swallow's nest extract is specifically, by dry weight, to be included in an amount of 0.0001% to 50% by weight of the total weight of the cosmetic composition. and, more specifically, may be included in an amount of 0.0005 wt% to 10 wt%. Within the above range, there is an advantage of exhibiting excellent efficacy for antioxidant, anti-inflammatory, skin wrinkle and blood circulation improvement, moisturizing, whitening, and skin regeneration activity, and there is an advantage in that the formulation of the composition is stabilized.

According to another aspect of the present invention, the present invention provides a food composition comprising, as an active ingredient, a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP).

The bird's nest extract fermented with the new Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP) of the present invention exhibits excellent effects on antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration, and is a food for improving skin condition It can be usefully used as a composition. The effect on the anti-oxidation, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration of the bird's nest extract fermented with the Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP) of the present invention is as described above. The food composition may be used in the form of health functional food, but is not limited thereto.

The food composition of the present invention may be included in the form of a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP), and their processed products. In addition, the composition may include a food pharmaceutically acceptable food additive in addition to the active ingredient.

In the present invention, "food supplementary additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverages, etc., but the above examples are not limited to the type of food supplementary additive of the present invention.

The food composition of the present invention may include a health functional food. In the present invention, "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful for the human body. Here, the term “functionality” refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological actions with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art.

In addition, the dosage form of the health functional food may also be manufactured without limitation as long as it is a dosage form recognized as a health functional food. The food composition of the present invention can be prepared in various forms, and unlike general drugs, it uses edible lactic acid bacteria and edible sea swallow's nest as raw materials, and has the advantage of not having side effects that may occur when taking food for a long period of time, and is portable. It is excellent and can be taken as an adjuvant to enhance its effects on antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration activities.

There is no limitation on the form that the health functional food of the present invention can take, and it may include any food in a conventional sense, and may be used in combination with terms known in the art, such as functional food. In addition, the health functional food of the present invention can be prepared by mixing known additives with other suitable auxiliary ingredients that may be included in the food according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and There are vitamin complexes and the like, and it can be prepared by adding to juice, tea, jelly, juice, etc. prepared by using the bird's nest extract fermented with Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP) as a main component according to the present invention. . Also included are foods used as feed for animals.

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides a composition for improving skin condition comprising, as an active ingredient, a bird's nest ferment extract using a new strain SDCM 1105 of the genus Lactobacillus.

(2) The fermented bird's nest ferment extract of the present invention has an increased content of Ν-acetylneuraminic acid, and has improved antioxidant, anti-inflammatory, blood circulation improvement, moisturizing, whitening, and skin regeneration activity, which is excellent for improving skin condition. As it is effective, it can be usefully used in cosmetics and food fields.

(3) In addition, the composition of the present invention is not only very safe for the human body, but also has excellent stability.

1 is a graph showing the results of high-performance liquid chromatography analysis of a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP).
Figure 2 is a human keratinocyte (HaCaT), dermal fibroblast (HDF) and mouse melanoma cells (B16F10) cells according to the treatment with a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP). It is a graph showing the measurement of viability.
3 is a graph showing the change in antioxidant activity according to the treatment with a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP).
FIG. 4 is a graph showing the cytoprotective effect measured by MTT assay after treatment with the Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP) fermented sea swallow's nest extract to RAW 264.7 cells.
FIG. 5 is a graph showing changes in NO production after treatment with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP) fermented bird's nest extract to RAW 264.7 cells.
FIG. 6 is a graph showing changes in the expression level of IL-1β protein by ELISA after treatment with a bird's nest extract fermented with Lactobacillus new strain SDCM 1105 (Accession No. KCTC 14200BP) to RAW 264.7 cells. .
FIG. 7 is a graph showing changes in the expression level of TNF-α protein measured by ELISA method after treatment of a bird's nest extract fermented with Lactobacillus new strain SDCM 1105 (Accession No. KCTC 14200BP) to RAW 264.7 cells. .
8 is a human keratinocyte (HaCaT) fermented with a new strain SDCM 1105 (Accession No. KCTC 14200BP) of the genus Lactobacillus, after treatment with human keratinocytes (HaCaT), the change in the amount of hyaluronic acid (Hyaluronan) synthesis is measured by the ELISA method. This is the graph shown.
9 is a human keratinocyte (HaCaT) treated with a fermented bird's nest extract fermented with a new strain of Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP), followed by measuring the change in the expression level of the VEGF factor by ELISA. It is a graph.
10 is a graph showing the change in the amount of melanin produced after treatment with the Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP) to mouse melanoma cells (B16F10) cells with a fermented bird's nest extract.
11 is a photograph taken of the results of cell staining in the plate using a cell stain solution after treating NHDF cells with a bird's nest extract fermented with Lactobacillus genus new strain SDCM 1105 (Accession No. KCTC 14200BP); This is a graph showing quantification.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1. Preparation of lactic acid bacteria fermented bird's nest extract

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

Example 1. Preparation of lactic acid bacteria fermented bird's nest extract

1-1. Identification of strain isolation

Tibetan mushrooms (Kefir grain) 1g was quantified, 0.85% NaCl 9mL was added to mix thoroughly and diluted, and then smeared on BCP solid medium, and cultured at 37°C for 1 to 3 days. After culturing, a single colony that turned yellow was isolated and cultured in MRS (OXOID, UK) under the same culture conditions.

For molecular biological identification of the cultured strain, 16S rRNA sequencing was performed using primers 518F and 805R, followed by sequence analysis. As a result of finding out the homology with the 16S rRNA nucleotide sequence of the strains registered in the NCBI GeneBank database, it was found that the Lactobacillus brevis showed a similarity of 99.52% with the ATCC14869 strain, and the isolated lactic acid bacteria was Lactobacillus genus (Lactobacillus brevis). sp.) was named SDCM 1105 strain, and was deposited with the Korea Biological Resources Center (KCTC) on June 3, 2020 and was given an accession number KCTC 14200BP.

1-2. Production of lactic acid bacteria fermented bird's nest extract

20-fold sterilized distilled water was added to 100 g of the bird's nest raw water, and sterilized MRS medium was added at a concentration of 5.2% (w/v). The sample prepared in this way was inoculated with the new strain SDCM 1105 of the genus Lactobacillus at a concentration of 1% (v/v), incubated at 37° C. for 24 hours, and then inactivated by heating the strain. Thereafter, the medium was centrifuged to collect the supernatant, and then filtered through a 0.45um filter to prepare a bird's nest extract (Preparation Example).

20 times of sterilized distilled water was added to 100 g of the bird's nest raw water, and without fermentation, extraction was performed at 80° C. for 3 hours. After centrifuging the extract thus extracted, the supernatant was filtered through a 0.45um filter to prepare a bird's nest extract (Comparative Example).

Example 2. Changes in composition of sea swallowtail extract according to strain fermentation

A high-performance liquid chromatography analysis was performed to compare the content of index components of the swallow's nest fermented with SDCM 1105 of the Lactobacillus genus of the present invention prepared in Example 1-2 and the non-fermented sea swallow's nest.

For high-performance liquid chromatography analysis, ACQUITY UPLC I-Class (Waters, Xevo TQ-S micro with ESI probe MassLynx™ software, version 4.1) was used. HPLC analysis conditions are as follows. As a column, Unison UK-Amino (Imtakt, 100x2.1 mm, 3 μm) was used, and the column temperature was set to 30°C. As the mobile phase, water containing 0.1% formic acid and acetonitrile (gradient mode) [A:B=Water:ACN (Gradient, 17min, 0.1% F.A.)] were used. Each of the fermented bird's nest extract and the unfermented sea swallow's nest extract was diluted 10-fold and dissolved in a mobile phase solvent, then injected in a volume of 3uL and measured at a flow rate of 0.4ml/min (see FIG. 1).

[Table 1]

As a result of the measurement, Ν-acetylneuraminic acid (N-Acetylneuraminic acid, Neu5Ac, formula C ₁₁ H ₁₉ NO _{9 ,} CAS NO.: 131-148-6) was measured to be increased by 31.9 times.

Example 3. Cytotoxicity evaluation of fermented bird's nest ferment extract

The cytotoxicity to human keratinocytes (HaCaT), dermal fibroblasts (HDF) and mouse melanoma cells (B16F10) of the bird's nest extract fermented with the new Lactobacillus genus SDCM 1105 of the present invention prepared in Example 1 tested.

Cytotoxicity was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Methyl thiazol tetrazolium (MTT) assay is used to measure cell viability, which converts MTT into a purple-producing formazan stain. Each cell was cultured for 24 to 72 hours by adding a bird's nest extract fermented with Lactobacillus genus SDCM 1105 (Test Example) and an unfermented sea swallow's nest extract (Comparative Example) by concentration, and the concentration of MTT was 0.1mg/mL was added to each well. Thereafter, the three types of cells were cultured at 37° C. for 3 hours in the _{presence of 5% CO 2} and 95% O ₂ to induce a reduction reaction. The substrate-containing medium was removed and 500 μl of DMSO was added to each well to dissolve the formazan crystals. In addition, the plate was shaken with an orbital shaker at room temperature for 30 minutes. Absorption of 100 μL aliquots was quantified by measuring absorption at 595 nm using a microplate reader (Molecular Devices E09090; San Francisco, CA, USA). For cytotoxicity, relative cell viability (%) was calculated based on the 100% viability of the untreated group in which only cells were cultured without treatment of the specimen. In addition, all experiments were repeated three times.

As a result of the experiment, as shown in FIG. 2 , the treatment of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus was performed on human keratinocytes (HaCaT), dermal fibroblasts (HDF) and mouse melanoma cells (B16F10). It was shown not to be toxic to 3 types of cells. Based on these results, it was confirmed that the bird's nest extract fermented with SDCM 1105, a new strain of the genus Lactobacillus of the present invention, does not show toxicity to the human body and can be safely used.

Example 4. Antioxidant activity of fermented bird's nest ferment extract

Sang S et al. using the reducing power of DPPH (1,1-diphenyl-2-picrylhydrazyl, Sigma, USA) (Sang S et al., J. Agric. Food Chem. 50(8), p2459-2463, 2002) According to the method described, the antioxidant activity of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus according to the present invention was measured. DPPH is widely used to measure the hydrogen donating ability of antioxidants through absorbance measurement because DPPH is decolorized to dark purple by removing radicals when reacting with substances with antioxidant activity.

A bird's nest extract fermented with SDCM 1105 of a new strain of Lactobacillus genus SDCM 1105 dissolved in 160 μL of a 0.2 mM DPPH solution dissolved in methanol and 40 μL of distilled water (Test Example) and an unfermented bird nest extract (Comparative Example) were respectively 0.1, 1, and After incubation for 30 minutes at 37°C in a 96-well microplate at a concentration of 10% (v/v), the amount of reduced DPPH was measured at 520 nm using a spectrophotometer (Molecular Devices FilterMax F5; San Francisco, CA, USA). The absorbance was measured and the antioxidant activity was compared. For the comparative experiment, ascorbic acid was used as a positive control. In addition, all experiments were repeated three times.

As a result of the measurement, it was found that the antioxidant activity of the bird's nest extract that was not fermented was weak, but the concentration-dependent increase of the antioxidant activity of the seabird's nest extract fermented with SDCM 1105, a new strain of the genus Lactobacillus according to the present invention. It was observed to exhibit superior antioxidant activity equal to or greater than that of ascorbic acid as a control group (see FIG. 3 ).

Example 5. Anti-inflammatory activity of fermented bird's nest ferment extract

5-1. cytoprotective effect

The cytoprotective effect of the Lactobacillus genus SDCM 1105-treated sea bird's nest extract on the growth of the LPS-treated macrophage, RAW 264.7 cells, was confirmed in the following way.

RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea), and 10% FBS, 100 μg/mL penicillin in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum). And 100 μg/mL streptomycin was inoculated with the cells in a medium, and cultured at _{37° C., 5% CO 2 condition.}

The RAW 264.7 cells ^{were inoculated into a 96-well plate at a concentration of 1×10 6} cells/mL (in DMEM) and cultured for 24 hours. was simultaneously treated with a sample diluted to 0.1, 1, and 10% (v/v) concentrations, respectively, and a new medium containing LPS (1 μg/mL) known as an endotoxin, and cultured for 24 hours. Thereafter, the concentration of the MTT reagent was treated to be 0.1 mg/mL, and after 3 hours, the formazan generated by the MTT treatment was dissolved in DMSO in the cells to measure the absorbance at 595 nm. For the comparative experiment, 1 μg/mL of dexamethasone treated group was used as a positive control group. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 4 , it was found that both the bird's nest extract treated group fermented with the new strain SDCM 1105 of the genus Lactobacillus had a better cell protection effect than that of the bird's nest extract that was not fermented. In addition, it was found that the bird's nest extract fermented with the new strain SDCM 1105 of the Lactobacillus genus of the present invention had a more excellent cell protective effect than the positive control group treated with dexamethasone.

5-2. Inhibition of nitric oxide production

In order to confirm the anti-inflammatory effect of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus, the inhibition rate of nitric oxide (NO), a representative cytotoxic substance involved in inducing inflammation, was measured.

First, RAW 264.7 cells, a macrophage cell line, were purchased from the Korea Cell Line Bank (KTCC, Seoul, Korea) and used in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (Fetal bovine serum) with 10% FBS, 100 μg/ The cells were inoculated in a medium supplemented with mL penicillin and 100 μg/mL streptomycin, and cultured at _{37° C., 5% CO 2 conditions.}

Specifically, the RAW 264.7 cells ^{were inoculated into a 96-well plate at a concentration of 1×10 6} cells/mL (in DMEM) and cultured for 24 hours, followed by fermentation with the new strain SDCM 1105 of the Lactobacillus genus prepared in Example 1-2. A sample diluted with 0.1, 1, and 10% (v/v) concentrations of the treated bird's nest extract, respectively, and a fresh medium containing LPS (1 μg/mL) known as endotoxin were simultaneously treated and cultured for 24 hours. After that, mix 100 μL of cell culture supernatant and 100 μL of Griess reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphtylethylenediamine in 2.5% (v/v) phosphoric acid] in a 96-well plate for 10 minutes. After reacting, the amount of nitric oxide produced was measured by measuring absorbance at 540 nm using an ELISA reader. The concentration of the produced nitrite (nitrite) was calculated using a standard curve in which sodium nitrite (sodium nitrite) was dissolved in DMEM medium. The nitric oxide production inhibitory activity of each sample was confirmed based on the difference in the amount of nitrite produced in the LPS-treated control group and the LPS-untreated control group. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 5 , it was confirmed that the nitric oxide production inhibitory effect of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus was superior to that of the bird's nest extract that was not fermented.

Specifically, compared to the LPS (1 μL/mL) treatment group, the positive control group reduced nitric oxide production by 33.76%, and the fermented bird's nest extract (Hotwater BN) treated group showed a decrease in nitric oxide production at 0.1% and 1% concentrations, respectively. decreased by 7.13% and 9.8%. In contrast, in the group fermented with the Lactobacillus genus new strain SDCM 1105 of the present invention, nitric oxide production was observed to decrease by 8.58%, 12.62%, and 30.23% at 0.1%, 1%, and 10% concentrations, respectively. and it was confirmed that the fermented bird's nest ferment extract of the present invention exhibited an excellent effect in inhibiting nitric oxide production (see FIG. 5).

5-3. Inhibition of expression of inflammatory factors

In relation to the regulation of the expression of inflammatory factors, IL-1β and TNF-α genes, the effect of the bird's nest extract fermented with the new Lactobacillus genus SDCM 1105 was verified.

^{First, RAW 264.7 cells were inoculated into a 24 well cell culture dish at a concentration of 1×10 5} cells/mL in DMEM (Dulbeccos modified Eagles medium) medium, which is a dedicated medium containing 10% fetal bovine serum (FBS), and then at 37°C. , 5% CO ₂ Incubated for 24 hours under humid conditions. Thereafter, after removing the medium and diluting with serum-free DMEM medium, the fermented bird's nest extract and non-fermented seabird's nest extract were used by concentration (0.1%, 1%, and 10%) with the new strain SDCM 1105 of Lactobacillus genus. was treated with 1 mL of a medium containing LPS (1 μg/mL) known as an endotoxin and a sample diluted with , and cultured for 24 hours.

After incubation, the supernatant was taken and centrifuged at 7,500rpm, 5min, 4℃ conditions, and IL-6 and TNF-α antibodies (R&D Systems, Inc., Minneapolis, MN, USA) were used to ELISA method using IL- 6 and the expression level of TNF-α protein was confirmed. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIGS. 6 and 7 , it was found that the Lactobacillus genus new strain SDCM 1105 fermented bird's nest extract inhibited the LPS-induced production of IL-6 and TNF-α protein in a concentration-dependent manner.

Through the above results, the sea bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus has a remarkable effect in inhibiting the production of IL-6 and TNF-α, which are inflammatory mediators, compared to the sea swallow's nest extract that is not fermented. , it was confirmed that it effectively blocks the inflammatory reaction and effectively improves the condition of the inflammatory skin.

Example 6. Skin moisturizing activity

In order to verify the skin moisturizing effect of the bird's nest extract fermented with the new strain SDCM 1105 of the Lactobacillus genus of the present invention, the effect on promoting the synthesis of hyaluronic acid (Hyaluronan) was tested.

Hyaluronic acid (HA, Hyaluronan, Hyaluronic acid) is mainly synthesized by epidermal keratinocytes and dermal fibroblasts, and is a substance that plays an important role in creating an environment in which cells can function normally by combining with water. A decrease in hyaluronic acid in the skin is one of the direct causes of a decrease in skin elasticity and a decrease in moisture content. Therefore, in this experimental example, the amount of hyaluronic acid produced in human keratinocytes was confirmed.

First, human keratinocytes were inoculated with 1 mL of a culture solution in a 24-well cell culture dish at a concentration of about 1×10 ⁵ cells/mL, and then cultured at 37° C., 5% CO ₂ environment for 24 hours. Thereafter, including the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus prepared in Example 1-2, and the bird's nest extract without fermentation (0.1, 1, and 10% (v/v), respectively) The medium was replaced and cultured for 24 hours. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4°C and 7,500 rpm, and the expression level of Hyaluronan (Hyaluronan kit, R&D Systems, Inc., Minneapolis, MN, USA) was performed using an ELISA method. was confirmed. For the comparative experiment, a normal control group (Nor, 100% DMEM) and a sample untreated group (Veh, 10% DW (solvent) + 90% DMEM medium) were used, and the retinoic acid (RA, retinoic acid) treated group was positive. It was used as a control. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 8, in the test group treated with the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus of the present invention, it was confirmed that the amount of hyaluronic acid (Hyaluronan) increased in a concentration-dependent manner, It was observed that the production amount of hyaluronic acid increased by 16.52%, 98.17%, and 251.24% at concentrations of 0.1, 1, and 10%, respectively, compared to the sample untreated group. In addition, in the test group treated with the fermented bird's nest extract at a concentration of 10%, a more increased hyaluronic acid production (192.31% increase) was measured than in the retinoic acid treatment group.

Through the above results, it was confirmed that the seabird's nest extract fermented with SDCM 1105, a new strain of the genus Lactobacillus of the present invention, effectively increased the production of hyaluronic acid in skin cells, so that it could be very effectively used for skin moisturizing purposes.

Example 7. Skin blood circulation improvement activity

In relation to the regulation of expression of vascular endothelial growth factor (VEGF) factor, which is a blood circulation improving factor, the effect of the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus was verified.

First, put 1 mL of DMEM culture solution in a 24-well cell culture dish, and inoculate human keratinocytes (HaCaT) cells at a ^{concentration of about 1.2x10 5} cells/mL, 37°C, 5% CO ₂ environment for 24 hours. incubated during Thereafter, the blue bird's nest extract fermented with the new Lactobacillus genus SDCM 1105 prepared in Example 1-2 was replaced with a medium containing samples diluted to 0.1, 1, and 10% (v/v) concentrations, 24 time incubation. Thereafter, the culture medium was harvested and centrifuged for 5 minutes at 4° C., 7,500 rpm, and VEGF antibody (Human VEGF kit, R&D Systems, Inc., Minneapolis, MN, USA) was used by ELISA method. A change in the expression level of the protein was confirmed. At this time, all groups except for the untreated normal control group (Nor) were treated with the sample after scratching. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 9 , the expression of VEGF factor was decreased in the treated group (Con) with only scratch compared to the untreated normal control group (Nor), and the VEGF factor was also treated in the group treated with the fermented sea swallow chip extract. A pattern of overall decrease in expression was observed. In contrast, the expression of VEGF factor was increased in a concentration-dependent manner in the experimental group treated with the Lactobacillus genus new strain SDCM 1105 of the present invention, which was fermented, 0.1%, 1 compared to the control group (Con) with only scratch. %, and at 10% concentrations, it was observed that the expression of VEGF factor increased by 21.95%, 107.59%, and 431.95%, respectively.

Through the above results, the fermented bird's nest extract fermented with the new strain SDCM 1105 of the Lactobacillus genus of the present invention can be effectively used to improve skin blood circulation by promoting the expression of VEGF factors that promote blood vessel formation and increase microvascular permeability. could confirm that there was

Example 8. Skin whitening activity

In order to verify the whitening effect of the bird's nest extract fermented with the new Lactobacillus genus SDCM 1105 of the present invention, the method reported in the prior literature (Hosoi et al., 1985) was modified to measure the melanin content. First, after inoculating mouse melanoma cells and culturing for 24 hours, 0.1, 1, and 10% (v / v) ) concentration and α-melanocyte stimulating hormone (α-MSH, alpha melanocyte stimulating hormone) 100nM concentration was treated and cultured for 72 hours. Thereafter, the medium was removed, washed twice with PBS (phosphate buffer saline), and 200 μL of 1N NaOH was added to dissolve melanin, and the melanin content was measured at 405 nm absorption wavelength using a microplate reader. For a comparative experiment, an arbutin 50 μg/mL treatment group was used as a positive control group. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 10 , it was confirmed that the group fermented with the new strain SDCM 1105 of the Lactobacillus genus of the present invention inhibited the production of melanin pigment in a concentration-dependent manner, and was not fermented in all sections. It showed a superior effect of inhibiting the production of melanin pigment compared to the untreated swallow's nest treatment group.

Specifically, compared to the α-melanocyte-stimulating hormone-treated group (Con) alone, the bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus of the present invention showed melanin production at concentrations of 0.1%, 1%, and 10%. 7.73%, 27.26%, and 69.48% inhibition was observed, respectively, and it was confirmed that the 10% concentration treatment group had a better melanin production inhibitory effect than the positive control group (arbutin treatment group reduced by 50.18%).

Through the above results, it was confirmed that the sea Swallow's nest extract fermented with SDCM 1105, a new strain of the genus Lactobacillus of the present invention, effectively inhibits melanin production in skin cells and can be effectively used for skin whitening related purposes.

Example 9. Skin regeneration activity

The skin wound regeneration activity was evaluated according to the treatment of the bird's nest extract fermented with the Lactobacillus genus new strain SDCM 1105 of the present invention.

First, NHDF (normal human dermal fibroblast) cells were seeded in a 24-well culture dish using an insert for wound production, and then cultured for 24 hours. After 24 hours, the fermented bird's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus was injected at concentrations of 0.1 and 1% (v/v), respectively, and the cell regeneration effect was confirmed.

After cell staining, it was confirmed with a microscope, and compared and quantitatively measured before the extract treatment. For the comparative experiment, allantoin 10ug/ml treatment group was used as a positive control group. In addition, all experiments were repeated three times.

As a result of the experiment, referring to FIG. 11 , it was confirmed that skin regeneration was stimulated in a concentration-dependent manner in the NHDF cells treated with the bird's nest extract fermented with SDCM 1105 of the Lactobacillus genus of the present invention. Specifically, the group treated with the new Lactobacillus genus SDCM 1105 fermented bird's nest extract showed superior cell regeneration effect in all concentration ranges compared to the non-fermented bird's nest treatment group. Cell proliferation improved by 56.02% and 74.57%, respectively, was observed at % and 1% (v/v) concentrations, whereas 96.75% and 112.88% and 112.88 by fermented bird's nest extract at 0.1% and 1% (v/v) concentrations, respectively. % improved cell proliferation was observed. In addition, 105.23% cell proliferation was observed in the allantoin 10 μg/mL treatment group compared to the control group, indicating the same level of skin regeneration promoting effect as the fermented bird's nest extract. In addition, in the morphological shape of the regenerated skin cells, it was observed that cell proliferation and differentiation were smoother and the cell density in the wound area was higher than that of the skin cells of the untreated group (see FIG. 11 ). ).

Based on the above results, when the sea swallow's nest extract fermented with the new strain SDCM 1105 of the genus Lactobacillus of the present invention is applied to damaged skin, the regenerated skin tissue does not differ from the surrounding normal skin tissue. It was found that it has the effect of promoting the regeneration of skin cells while preventing the skin wounds from fading.

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

Korea Institute of Bioscience and Biotechnology KCTC14200BP 20200603

Claims (8)

Contains as an active ingredient, a bird's nest extract fermented with a new strain of Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP),
The fermented bird's nest extract exhibits skin blood circulation improvement activity by promoting the expression of vascular endothelial growth factor (VEGF) factor,
Characterized in promoting skin wound regeneration,
A cosmetic composition for improving skin blood circulation and regenerating skin wounds. According to claim 1,
The composition is a cosmetic composition for improving skin blood circulation and regenerating skin wounds, characterized in that it has antioxidant activity. According to claim 1,
The composition suppresses the production of nitric oxide (NO), and suppresses the expression of inflammatory cytokines IL-1β and TNF-α genes. A cosmetic composition for improving skin blood circulation and regenerating skin wounds. According to claim 1,
The composition promotes the synthesis of hyaluronic acid, and a cosmetic composition for improving skin blood circulation and regenerating skin wounds, characterized in that it exhibits skin moisturizing activity. delete According to claim 1,
The composition is a cosmetic composition for improving skin blood circulation and regenerating skin wounds, characterized in that it exhibits skin whitening activity by inhibiting the production of melanin pigment. delete Contains as an active ingredient, a bird's nest extract fermented with a new strain of Lactobacillus genus SDCM 1105 (Accession No. KCTC 14200BP),
The fermented bird's nest extract exhibits skin blood circulation improvement activity by promoting the expression of vascular endothelial growth factor (VEGF) factor,
Characterized in promoting skin wound regeneration,
A food composition for improving skin blood circulation and regenerating skin wounds.

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