KR102065905B1 - Composition for anti-aging comprising Bacillus pumilus strain or culture solution of the strain - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising a Bacillus pumilus strain or a culture medium thereof. The cosmetic composition according to one aspect includes a Bacillus pumilus strain or a culture medium thereof, and thus has an excellent skin aging prevention or amelioration effect, a skin wrinkle prevention or alleviation effect, or a skin regeneration effect.

Description

Composition for preventing or improving aging comprising a Bacillus pumilus strain or a culture thereof {Composition for anti-aging comprising Bacillus pumilus strain or culture solution of the strain}

It relates to a composition for preventing or improving aging comprising a Bacillus pumilus strain or a culture thereof.

In the field of functional cosmetics, there is a great interest in products that delay skin aging to help improve wrinkles of the skin, products that protect the skin from ultraviolet rays and the external environment, and products that help to whiten the skin. Among these, as the aging society enters, efforts to develop wrinkle improvement cosmetics for delaying skin aging are continuing. In general, wrinkles and decreased skin elasticity, sagging and dryness of aging skin are mostly due to changes in substrate proteins present in the dermis. Since the dermis plays a role in supporting the strength and shape of the skin in the skin, if morphological changes occur in this area as aging progresses, it plays a crucial role in wrinkle formation and skin sagging. Matrix metalloproteinases (MMPs) are representative enzymes that degrade these matrix proteins, and MMPs activity in the skin is increased in a single UV irradiation, and it is a major factor in promoting skin aging by remarkably degrading collagen and other matrix proteins in the skin. do.

Photo aging is considered the biggest aging factor of skin aging. There are many products to prevent photo aging, but photo aging prevention technology using natural materials is limited in use and there are many problems in verifying efficacy. There is this.

On the other hand, probiotics (probiotics) is a general term for microorganisms that have a beneficial effect on the human body refers to the microorganisms (benefit) to our body. Most probiotics known to date are known as lactic acid bacteria. Although probiotics have been reported to produce effective effects through various beneficial effects on the human body, studies on the interaction between the microorganisms of Bacillus and skin have been insufficient.

Accordingly, the present inventors have confirmed that the microorganism of Bacillus or its culture can be usefully used to improve skin condition by alleviating skin aging, and completed the present invention.

One aspect provides a cosmetic composition for preventing or improving aging comprising Bacillus pumilus strain or a culture thereof as an active ingredient.

Another aspect provides a method of making a Bacillus pumilus strain or a culture thereof.

One aspect provides a cosmetic composition for preventing or improving skin aging comprising Bacillus pumilus strain or a culture thereof as an active ingredient.

As used herein, the term "cosmetic composition" means an article used in the human body to clean and beautify the human body to add charm and brighten the appearance, or to maintain or promote the health of the skin and hair.

The cosmetic composition may be for preventing or improving skin wrinkles. The cosmetic composition may be for increasing skin elasticity. The cosmetic composition may be for preventing or improving skin aging or pigmentation. The cosmetic composition may be for skin regeneration. The thin cosmetic composition may be for promoting the formation or regeneration of skin tissue. Bacillus fumilus strains or cultures thereof are superior to other Bacillus strains, such as Bacillus subtilis or sperm extracts known to have anti-aging effects, and are effective in preventing or improving skin aging or wrinkles and preventing or improving skin regeneration. Since the effect is excellent, the composition comprising the Bacillus pumilus strain or its culture may be usefully used in cosmetic compositions.

The term "Bacillus pumil Ruth (Bacillus pumilus)" herein is a Gram-positive are commonly found in soil, aerobic, spore forming bacterium. The Bacillus pumilus may be separated from food, soil, sea, and the like.

The strain may be a Bacillus pumilus CX-UV strain (Accession Number: KCCM12274P).

As used herein, the term "skin aging" refers to the type and intangible changes that appear on the skin with age, such as thinning of the epidermis, number of cells or blood vessels in the dermis, DNA damage repair ability, cell replacement cycle, Wound recovery, skin barrier function, epidermis moisture retention, sweat secretion, sebum secretion, vitamin D production, physical damage protection, chemical removal ability, immune response, sensory function, reduction of body temperature control. The Bacillus pumilus strain or its culture may be for improving skin aging caused by exogenous factors or endogenous factors. The exogenous factor refers to various external factors such as ultraviolet light, and the endogenous factor is also referred to as a chronological factor and mainly refers to a factor generated by the passage of time. That is, the skin aging is a natural aging phenomenon that occurs as the proliferation of skin cells decreases with age as well as premature aging symptoms induced by external stimuli caused by ultraviolet rays, pollution, tobacco smoke, chemicals, and the like. The concept includes all wrinkles, reduced elasticity, skin sagging and dry phenomenon. Wrinkles also include those in which stimulation caused by changes in internal and external factors causes wrinkles by changing the components of skin tissue.

The aging may be photoaging. The term "photoaging" is a phenomenon caused by external environmental factors, the most representative of which is ultraviolet rays. Ultraviolet rays cause damage to biological components such as proteolytic enzyme activation, substrate cleavage and abnormal crosslinking, and the repetition of this mechanism leads to apparent skin aging.

As used herein, the term "wrinkle" refers to a state in which the elasticity of the skin is lost and loosened, for example, the skin may be folded. Pigmentation refers to a state in which the amount of pigment in the body is abnormal or abnormal in the place where the pigment appears, for example, it may be a blemish and freckles. The "prevention or improvement of skin wrinkles" may mean any action of preventing or improving wrinkles by increasing the expression of factors related to wrinkles or increasing the total amount of collagen.

As used herein, the term “improvement” may mean any action that at least reduces a parameter, eg, the degree of symptoms, associated with the alleviation or treatment of a condition.

As used herein, the term "prevention" may mean any action that inhibits or delays skin photoaging, pigmentation, or wrinkles by application of the composition.

As used herein, the term "culture liquid" refers to a culture supernatant obtained by culturing a strain, a concentrate thereof, or a culture supernatant obtained by removing the strain from a lyophilizate or culture medium, a concentrate or a lyophilisate thereof. The culture medium may contain useful active ingredients such as proteins produced during the process of culturing Bacillus pumilus strains.

The culture may be a suspension culture for about 45 to 50 hours at about 25 to 30 ℃, but is not limited thereto. The culture temperature and incubation time may be appropriately selected in a range in which the strain exhibits an improvement or prevention effect of aging.

The composition may be to inhibit the expression of MMP-1 (Matrix metalloproteinase-1), specifically may be to inhibit the expression of the MMP-1 gene or the activity of the MMP-1 protein to exhibit a skin aging inhibitory effect.

The composition may be to increase collagen synthesis. The composition may be to increase the expression of collagen, for example type 1 collagen gene. Specifically, by increasing the expression of the type 1 collagen gene to restore the collagen production may be to exhibit the effect of inhibiting skin aging or wrinkle improvement.

The composition may comprise culture in an effective amount. The effective amount may be appropriately selected depending on the individual. The severity of the condition, the age, weight, health, sex of the individual, sensitivity to the extract of the individual, time of administration, route of administration and rate of excretion, duration of administration, and other physiological and other physiological and other factors, including other compositions in combination or concurrently with the composition It may be determined according to factors well known in the medical field.

Specifically, the culture solution is 0.001% to 80% by weight, for example 0.01% to 60% by weight, 0.01% to 40% by weight, 0.01% to 30% by weight, 0.01% by weight relative to the total weight of the cosmetic composition % To 20%, 0.01% to 10%, 0.01% to 5%, 0.05% to 60%, 0.05% to 40%, 0.05% to 30%, 0.05% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt% %, 0.1% to 10% by weight, or 0.1% to 5% by weight.

Ingredients included in the cosmetic composition may include ingredients commonly used in cosmetic compositions in addition to the composition as an active ingredient, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments and flavorings. It may include.

In the composition, the strain or its culture solution is a lotion (skin lotion), skin softener, skin toner, astringent, lotion, milk lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence , Nutritional essences, packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, suspensions, gels, powders, pastes, mask packs or sheets or aerosol compositions. Compositions of such formulations may be prepared according to conventional methods in the art. The cosmetic composition may further include preservatives, stabilizers, surfactants, solubilizers, moisturizers, emollients, ultraviolet absorbers, preservatives, fungicides, antioxidants, pH adjusters, organic and inorganic pigments, fragrances, coolants or limiters, etc. have. The blending amount of the additional components such as the moisturizing agent can be easily selected by those skilled in the art within the range that does not impair the object and effect of the present invention, the blending amount is 0.001 to 5% by weight, specifically 0.01 to 3 based on the total weight of the composition Weight percent.

In addition, the composition may be formulated as an external skin preparation.

In the present specification, the external skin agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, drug-containing bandage, lotion, or a combination thereof. The external skin preparations are commonly used in skin external preparations such as cosmetics and medicines, for example, aqueous components, oily ingredients, powder components, alcohols, humectants, thickeners, ultraviolet absorbers,

It can be suitably blended with a whitening agent, preservative, antioxidant, surfactant, perfume, colorant, various skin nutrients, or a combination thereof as necessary. The external skin preparations include metal blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, caffeine, tannin, belafamil, licorice extract, glablidine, and caline. Fruit water extract, various herbal medicines, drugs such as tocopherol acetate, glytilinic acid, tranexamic acid and derivatives thereof or salts thereof, vitamin C, magnesium ascorbic acid phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, Sugars, such as trehalose, etc. can also be mix | blended suitably.

Another aspect provides a method of ameliorating a skin of a subject comprising applying the composition to the subject. The composition is as described above.

Specifically, the method may be a method for preventing or improving skin aging of an individual or preventing or improving skin wrinkles.

The terms “applying”, “administering”, “introducing” and “transplanting” are used interchangeably and result in an individual by a method or route that results in at least partial localization of the composition to the desired site according to one embodiment. It can mean the placement of a composition according to one embodiment into it. Administration of any of the cultures or culture components of the composition according to one embodiment may be by any suitable route to deliver to the desired location in the surviving individual.

The subject may be a mammal, for example a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be a subject in need of skin condition improvement, for example skin aging improvement, or skin wrinkle improvement.

Another aspect includes the steps of culturing a Bacillus pulilus strain; Centrifuging the cell culture medium obtained after the culture to remove the cells and recovering the supernatant; It provides a method for producing a Bacillus pumilus strain culture comprising a; and filtering the recovered supernatant to remove the remaining cells.

The Bacillus fumilus strain is the same as described above.

In the method, the Bacillus pumilus strain may be deposited with the accession number KCCM12274P.

The culture may be a suspension culture for about 45 to 50 hours at about 25 to 30 ℃, but is not limited thereto.

Bacillus pumilus strain prepared by the above method or a culture thereof may increase the expression of MMP-1 to strengthen the skin barrier or prevent aging, increase or decrease the expression of type 1 collagen type 1 collagen By restoring the expression, it is possible to prevent skin aging or prevent or improve skin wrinkles. Skin barrier reinforcing, skin moisturizing, anti-aging, skin wrinkle improvement, or skin regeneration of such Bacillus fumilus strain or its culture is very excellent compared to other Bacillus genus strains. Therefore, the Bacillus pumilus strain prepared by the above method or its culture may be used for various purposes such as cosmetics, external skin preparations.

According to the cosmetic composition according to one aspect, including the Bacillus pumilus strain or a culture thereof has excellent skin aging prevention or improvement, or skin wrinkle prevention or improvement, or skin regeneration effect.

1 is a graph evaluating the efficacy of the Bacillus pumilus strain or its culture to inhibit the expression of MMP-1 increased by UV treatment. CON: control group not treated with UVB; UV: UVB treated group; CM: medium treatment group to which the sperm extract was added after UVB treatment; NASA1: Culture solution treated group of Bacillus pumilus strain after UVB 15 sec treatment; NASA4: culture solution treatment group of Bacillus pumilus strain after UVB 30 sec treatment; RL: Bacillus sp. Treatment group after UVB treatment
Figure 2 is a graph evaluating the efficacy of Bacillus pumilus strain or its culture to restore the expression of collagen type 1 reduced by UV treatment. CON: control group not treated with UVA; UVA: UVA treated group; CM: medium treated group to which sperm extract was added after UVA treatment; FM: group treated with culture solution + fungal extract of Bacillus pumilus strain after UVA treatment; FMP: cultured group of Bacillus pumilus strain after UVA treatment; PNA: Bacillus sp. Treatment group after UVA treatment.

It will be described in more detail through the following examples. However, these examples are provided to illustrate one or more embodiments illustratively and the scope of the present invention is not limited to these examples.

Example 1 Bacillus pumilus Bacillus pulilus)  Isolation and Identification of Strains

The soil and dry reeds of the reed forest, which are known to have a high distribution of Bacillus subtilis, are collected, and 100 g of the mixed sample of soil and reeds is suspended in sterile distilled water at 100 rpm for 1 hour, and 10 ml of the supernatant is 1/10, 1/100. , Diluted to 1/1000 and inoculated in R2A, TSA, NA medium. After culturing for 48 hours in a 25 ℃ incubator, only colonies of white and beige rounded colonies were selected for pure separation culture. The isolated colonies were subjected to pure separation culture once more, and commissioned an analysis of colonized plates by Macrogen to identify 30 species selected.

As a result, two species Bcillus The homology with pumilus was found to be greater than 98%, with the remainder being microbes of different species or strains. Among the two identified Bacillus pumillus , the species with much higher UV resistance were selected. The isolated strain was named Bacillus pumilus CX-UV, which was deposited on June 14, 2018 at the Korea Microbial Conservation Center, and was given accession number KCCM12274P.

Example  2. Bacillus Pumilus  Strains and Bacillus Pumilus  Preparation of Strain Culture

In order to cultivate Bacillus pumilus CX-UV strain, a medium containing R2 medium component as a main component was used. 5 g of R2 medium was suspended in 1 liter of water, dissolved, and sterilized at 121 ° C. for 15 minutes to prepare a medium. At this time, the medium was prepared a solid medium containing agar, and a liquid medium containing no agar. First, a Bacillus pumilus CX-UV strain was plated in a solid medium and incubated at 28 ° C. for 3 days, and the cultured Bacillus pumilus CX-UV strain was subjected to a 30 second exposure to UV light. One UV treated colony was inoculated into 1 liter of liquid medium. The inoculated liquid medium was suspended in culture (100 rpm) for 48 hours at 28 ° C. After incubation, the supernatant was removed only after centrifugation (7000 rpm, 30 minutes) through a centrifuge, and filtered through a 0.2 μm filter. Sterilization was performed.

Experimental Example 1.Efficacy evaluation of MMP-1 in culture of Bacillus pumilus strain

It was confirmed how the culture medium of Bacillus pumilus strain prepared in Example 1 affects the activity and expression of MMP-1.

In order to evaluate the effect of MMP-1 inhibition at the tissue level, an experiment was conducted using EpiDermFT ™ (MatTek, USA), a three-dimensional artificial skin tissue.

Epidermidis - After stabilizing the EpiDermFT ™ containing both the dermis for a primary to 24 hours in culture medium to remove the medium and fermentation of the back for the remaining experimental groups except the UVB non-adjustable sagun review of UVB of 60 mJ / cm ² Bacillus pumil loose strain Lysates or comparative strains were treated. Thereafter, the sperm extract, known to have an effect of improving skin aging or wrinkles, was replaced with a medium added by concentration, and further cultured for 12 hours. Then, the culture medium obtained by culturing artificial skin tissue was collected and used to measure the amount of protein contained in the medium by removing suspended solids by centrifugation. The amount of MMP-1 protein contained in the culture medium was measured by the guided method using the MMP-1 ELISA Kit (EMD Millipore, USA). The results of measuring the expression change of MMP-1 is shown in Figure 1, the experimental group is as follows.

CON: control group not treated with UVB; UV: UVB treated group; CM: medium treatment group to which the sperm extract was added after UVB treatment; NASA1: cultured group of Bacillus pumilus strain after 15 sec UVB treatment; NASA4: culture solution treatment group of Bacillus pumilus strain after UVB 30 sec treatment; RL: Bacillus subtilis strain treated group after UVB treatment.

As shown in FIG. 1, MMP-1 expression was increased in the UVB irradiated group (UV), whereas the experimental group (CM) recovered and cultured in a medium to which sperm extract was added after UVB treatment was expressed in MMP-1 compared to the UV treated group. This slightly decreased. The group treated with the culture solution of Bacillus pumilus strain after UVB treatment (NASA1, NASA4) showed significantly lower MMP-1 expression than these, compared with the group treated with other Bacillus strains (RL). Lower MMP-1 expression level was shown.

Therefore, it was confirmed that the expression of MMP-1 whose expression was increased by UV irradiation was reduced by the treatment of Bacillus pumilus strain. Through the above results, it can be seen that the Bacillus pumilus strain exhibits an anti-aging effect by inhibiting the activity of MMP-1.

Experimental Example  2. Bacillus Pumilus  Evaluation of Efficacy of Type 1 Collagen in Cultures of Strains

It was confirmed how the culture medium of Bacillus pumilus strain prepared in Example 1 affects the expression of type 1 collagen.

In order to evaluate the effect of increasing the type 1 procollagen at the tissue level, the experiment was conducted under the same conditions except that UVA was treated instead of UVB using the same tissue as in Experimental Example 1. Finally, the procollagen type I C-peptide (PIP) EIA kit (Takara-bio Inc., Japan) was used to measure the content of procollagen type 1 in the culture medium. The measurement results are shown in FIG. 2, and the experimental group is as follows.

CON: control group not treated with UVA; UVA: UVA treated group; CM: medium treated group to which sperm extract was added after UVA treatment; FM: group treated with culture solution + fungal extract of Bacillus pumilus strain after UVA treatment; FMP: culture solution treatment group of Bacillus pumilus strain after UVA treatment; PNA: Bacillus sp. Treatment group after UVA treatment.

As shown in FIG. 2, since the content of type 1 procollagen in the culture medium of the UVA irradiated group was reduced compared to the non-irradiated group (CON), it was confirmed that type 1 procollagen was inhibited by UV treatment. In addition, in the treatment group treated with the culture solution of Bacillus pumilus strains, the content thereof was significantly increased, it was confirmed that the collagen expression inhibited by ultraviolet light was recovered by the Bacillus pumilus strains. This effect was significantly higher than the group treated with Bacillus subtilis, another strain of the genus Bacillus.

Therefore, it was confirmed that the culture solution of Bacillus pumilus strain inhibited the expression of MMP-1 gene and MMP-1 activity in the cells after UV treatment and restored the expression of type 1 collagen reduced by ultraviolet light. It can be used for alleviating, improving and preventing skin aging.

Korea Microorganism Conservation Center (overseas) KCCM12274P 20180614

Claims (9)

Bacillus pulilus ( Bacillus pulilus ) strain or a cosmetic composition comprising the culture solution thereof as an active ingredient. The cosmetic composition of claim 1, wherein the Bacillus pumilus strain has been deposited with accession number KCCM12274P. The cosmetic composition according to claim 1, which is for preventing or improving skin aging or for preventing or improving skin wrinkles. The cosmetic composition according to claim 3, wherein the skin aging is photoaging. The cosmetic composition of claim 1, wherein the Bacillus pumilus strain or its culture is contained in an amount of 0.01 wt% to 10 wt% based on the total weight of the cosmetic composition. The cosmetic composition according to claim 1, which suppresses the expression of Matrix metalloproteinase-1 (MMP-1). The cosmetic composition according to claim 1, wherein the cosmetic composition increases the expression of the type 1 collagen gene. Culturing Bacillus pulilus strains;
Centrifuging the cell culture medium obtained after the culture to remove the cells and recovering the supernatant; And
Filtering the recovered supernatant to remove remaining cells; a method for producing a Bacillus pumilus strain culture comprising the. The method of claim 8, wherein the culture is suspended incubated at 25-30 ° C. for 45-50 hours.

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