Composition for skin moisturization and wrinkle alleviation, comprising bacteriophage against intestinal microorganisms

Abstract

The present invention relates to a composition for skin moisturization and wrinkle alleviation, the composition comprising a bacteriophage against intestinal microorganisms. The composition increases collagen production, increases moisture content, is highly safe, and is evaluated to have an excellent feeling of use in terms of spreadability, absorption, fragrance, etc., and thus can be effectively used as a cosmetic composition for skin moisturization and wrinkle alleviation.

Description

Composition for skin moisturizing and wrinkle improvement containing bacteriophage for intestinal microorganisms

[0001] The present invention relates to a composition for skin moisturizing and wrinkle improvement containing a bacteriophage for intestinal microorganisms, or a filler containing a peptide derived from a bacteriophage for intestinal microorganisms, and a method for producing the same.

[0002] Bacteriophages are a type of virus that infect bacteria, commonly referred to as phages. Bacteriophages are simple organisms with a core of genetic material composed of nucleic acid, surrounded by a protein coat. The nucleic acid can be either single-stranded or double-stranded DNA or RNA. Bacteriophages require a host for survival, and it is known that all bacteria possess specific phages.

[0003] Bacteriophages invade their hosts, complete replication, and then express a suite of enzymes necessary to degrade the bacterial cell wall. These enzymes attack the murein or peptidoglycan layers of the cell wall, which provide rigidity and mechanical strength, thereby destroying the cell wall.

[0004] Bacteriophages were discovered in 1915 by the British bacteriologist Twort, who was studying the phenomenon of Staphylococcus (Micrococcus) colonies dissolving into transparent substances. In addition, in 1917, the French bacteriologist d'Herelle discovered that the filtrate of the stool of a dysentery patient contained a substance that had the effect of dissolving Shigella disentriae. Through his research on this, he independently discovered bacteriophages, and named them bacteriophages because they eat bacteria.

[0005] Since then, bacteriophages for various pathogens such as Shigella, Typhi, and Cholera have been continuously discovered, and research on bacteriophages of Escherichia coli was focused on by Delbruck of the California Institute of Technology and European scientists who immigrated to the United States during World War II. After the discovery of penicillin by Flemming in 1950, research on bacteriophages continued only in some Eastern European countries due to the widespread use of antibiotics. However, after 2000, due to the continued misuse of antibiotics, the frequency of pathogenic bacteria with multi-drug resistance has increased, and research on bacteriophages has been conducted again, mainly in developed countries, due to the possibility of developing them as alternatives to antibiotics.

[0006] However, while the bacteriophage technology up to now has been applied to antibiotics targeting bacteria for infectious diseases, the inventors of the present invention have made efforts to provide a composition for skin moisturizing and wrinkle improvement using bacteriophage.

[0007] Against this backdrop, the inventors of the present invention have completed the present invention by manufacturing a composition for skin moisturizing and wrinkle improvement containing a bacteriophage for intestinal microorganisms, and confirming that the composition increases collagen production, increases skin moisture content, has excellent safety, and is also excellent in terms of usability evaluations such as spreadability, absorbency, and fragrance, and can be usefully used for skin moisturizing and wrinkle improvement.

[0008] The purpose of the present invention is to provide a cosmetic composition for skin moisturizing and wrinkle improvement, which comprises a bacteriophage, a bacteriophage metabolite or a combination thereof deposited under the accession number KCTC 15445BP as an active ingredient.

[0009] Another object of the present invention is to provide a method for improving skin moisturizing, comprising a step of applying the composition to the skin of a subject.

[0010] Another object of the present invention is to provide a method for improving skin wrinkles, comprising a step of applying the composition to the skin of a subject.

[0011] Another object of the present invention is to provide a biodegradable polymer filler comprising a bacteriophage-derived peptide deposited under accession number KCTC 15445BP as an active ingredient.

[0012] Another object of the present invention is to provide a method for improving skin wrinkles, comprising a step of applying the filler to the skin of a subject other than a human.

[0013] Another object of the present invention is to provide a method for producing a biodegradable polymer filler, comprising the steps of: i) preparing a peptide derived from a bacteriophage; and ii) dispersing a mixture containing biodegradable polymer microparticles and a biocompatible carrier in the peptide.

[0014] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined solely by the scope of the claims.

[0015] The terminology used herein is for the purpose of describing embodiments only and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise. As used herein, the terms "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components in addition to the mentioned components. Like reference numerals refer to like components throughout the specification, and "and / or" includes each and any combination of one or more of the mentioned components. Although "first", "second", etc. are used to describe various components, these components are not limited by these terms. These terms are only used to distinguish one component from another. Therefore, it should be understood that a first component mentioned below may also be a second component within the technical spirit of the present invention.

[0016] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those skilled in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0017] The present invention provides a cosmetic composition for skin moisturizing and wrinkle improvement, comprising a bacteriophage, a bacteriophage metabolite or a combination thereof deposited under accession number KCTC 15445BP as an active ingredient.

[0018] The bacteriophage of the present invention is a bacteria-specific virus that infects a specific bacterium and inhibits and suppresses the growth of the bacterium, and refers to a virus containing single or double-stranded DNA or RNA as genetic material.

[0019] The bacteriophage of the present invention has excellent lytic activity against the intestinal microorganism strains.

[0020] The bacteriophage of the present invention was deposited by the inventors at the Biological Resource Center of the Korea Research Institute of Bioscience and Biotechnology on May 18, 2023, under the accession number KCTC 15445BP. In a specific embodiment of the present invention, the inventors selected a bacteriophage that lyses intestinal microorganisms and deposited the isolated bacteriophage at the Korea Research Institute of Bioscience and Biotechnology.

[0021] The above intestinal microorganisms may be strains selected from the group consisting of Escherichia coli, Aeromonas hydrophila, Bacillus cereus, Lactobacillus sp., Bifidobacterium sp., Clostridium sp., Staphylococcus sp., Acinobacterium sp., Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter sp., but are not limited thereto.

[0022] The lysis of the present invention refers to a phenomenon in which the cell wall or cytoplasmic membrane of a bacteria is destroyed and the bacteria are dissolved, and refers to a phenomenon in which the bacteria are killed.

[0023] It was confirmed that the composition of the present invention is non-cytotoxic.

[0024] The composition of the present invention can increase collagen production and increase skin moisture content. The composition can improve skin wrinkles and moisturizing effects.

[0025] The improvement of the present invention means alleviating symptoms of aging, dryness or inflammation of the skin by using the composition of the present invention.

[0026] The active ingredient of the composition of the present invention may be included in an amount of 0.01 to 90 wt% based on the total weight of the cosmetic composition, but is not limited thereto. The active ingredient may be included in an amount of 0.01 to 80 wt%, 0.01 to 70 wt%, 0.01 to 60 wt%, 0.01 to 50 wt%, 0.01 to 40 wt%, 0.01 to 30 wt%, 0.01 to 20 wt%, 0.01 to 15 wt%, 0.01 to 10 wt%, or 0.01 to 5 wt%, but is not limited thereto.

[0027] The cosmetic composition of the present invention may have a formulation selected from the group consisting of, but not limited to, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

[0028] The above cosmetic composition may additionally include conventional adjuvants and carriers such as components commonly added to cosmetic compositions, such as stabilizers, solubilizers, water-soluble vitamins, oil-soluble vitamins, polymeric peptides, polymeric polysaccharides, sphingolipids, pigments, and fragrances.

[0029] In addition, examples of the compounding ingredients that may be added include oil components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc. The compounding ingredients that may be added may be compounded in an amount of 0.01 to 5 wt%, more specifically 0.01 to 3 wt%, based on the total weight of the cosmetic composition, but are not limited thereto.

[0030] In addition, the present invention provides a method for improving skin moisturizing, comprising a step of applying the composition to the skin of a subject.

[0031] The moisturizing or skin moisturizing effect of the present invention refers to increasing the skin's moisture content and maintaining its moist state. The skin moisturizing effect can help improve wrinkles and increase skin elasticity.

[0032] In one specific embodiment of the present invention, it was confirmed that the composition of the present invention increases the moisture content of the skin to 60 A.U or more (Fig. 2).

[0033] The subject of the present invention includes, but is not limited to, mammals or livestock, including humans. Livestock refers to useful animals domesticated and improved by humans to coexist with humans, and includes, but is not limited to, pigs, cows, chickens, horses, ducks, or dogs.

[0034] Application of the present invention includes methods of administering or applying to the skin of a subject, such as application using fingers, brushes, cotton balls, pads, sprays, sponges, cotton swabs, roll-ons, etc. Those skilled in the art can readily determine the appropriate method of application.

[0035] In addition, the present invention provides a method for improving skin wrinkles, comprising a step of applying the composition to the skin of a subject.

[0036] The composition, object and application of the present invention are as described above.

[0037] The wrinkle improvement of the present invention means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already formed.

[0038] In one specific embodiment of the present invention, it was confirmed that the composition of the present invention increased collagen production more than when treated with L-ascorbic acid, a positive control substance (Fig. 1).

[0039] The collagen of the present invention is a major matrix protein produced by fibroblasts of the skin and exists in the extracellular matrix. Its important functions are known to include mechanical strength of the skin, resistance of connective tissue and cohesion of tissue, support of cell adhesion, and induction of cell division and differentiation (during organism growth or wound healing).

[0040] Collagen decreases with age and photoaging caused by ultraviolet irradiation, and collagen depletion is accelerated by the activity of collagenase, an enzyme that breaks down collagen. This is known to be closely related to the formation of wrinkles on the skin.

[0041] The composition of the present invention, which includes a bacteriophage for intestinal microorganisms as an active ingredient, can improve skin wrinkles by increasing collagen production and improve skin moisturizing effect by increasing moisture content.

[0042] In addition, the present invention provides a biodegradable polymer filler comprising a bacteriophage-derived peptide deposited under accession number KCTC 15445BP as an active ingredient.

[0043] The term "bacteriophage" in the present invention is as described above.

[0044] The peptide of the present invention may be derived from a bacteriophage, and the peptide may be 1000 to 1200 Da, and more specifically, may be 1098.04 Da.

[0045] The filler of the present invention can improve skin wrinkles and increase skin elasticity.

[0046] The wrinkle improvement of the present invention means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already formed.

[0047] The increase in elasticity of the present invention means restoring decreased skin elasticity by producing new collagen in addition to the effect of contracting sagging skin tissue.

[0048] The filler of the present invention can be used for filling biological tissues, improving wrinkles through filling wrinkles, remodeling the face, restoring or increasing the volume of soft tissues, etc. The filler can be administered in a dosage form suitable for the above-mentioned purposes, and is preferably an injection, but is not limited thereto.

[0049] In addition, the present invention provides a method for improving skin wrinkles, comprising a step of applying the filler to the skin of a subject.

[0050] The improvement of skin wrinkles of the present invention means suppressing or inhibiting the formation of wrinkles on the skin or alleviating wrinkles that have already been formed.

[0051] The subject of the present invention includes, but is not limited to, mammals or livestock, including humans. Livestock refers to useful animals domesticated and improved by humans to coexist with humans, and includes, but is not limited to, pigs, cows, chickens, horses, ducks, or dogs.

[0052] The above application includes a method of administering the filler to the skin of a subject, such as by injecting the filler into the skin using a syringe needle or the like.

[0053] Additionally, the present invention provides a method for increasing skin elasticity, comprising a step of applying the filler to the skin of a subject.

[0054] The terms object and application of the present invention are as described above.

[0055] The increase in skin elasticity of the present invention means restoring decreased skin elasticity by producing new collagen in addition to the effect of contracting sagging skin tissue.

[0056] The collagen of the present invention is a major matrix protein produced by fibroblasts of the skin and exists in the extracellular matrix. Its important functions are known to include mechanical strength of the skin, resistance of connective tissue and cohesion of tissue, support of cell adhesion, and induction of cell division and differentiation (during organism growth or wound healing).

[0057] Collagen decreases with age and photoaging caused by ultraviolet irradiation, and collagen depletion is accelerated by the activity of collagenase, an enzyme that breaks down collagen. This is known to be closely related to a decrease in skin elasticity.

[0058] In addition, the present invention provides a method for producing a peptide polymer filler, comprising the steps of: i) preparing a peptide derived from a bacteriophage; and ii) dispersing a mixture containing biodegradable polymer microparticles and a biocompatible carrier in the peptide.

[0059] The term bacteriophage of the present invention is as described above.

[0060] The biodegradable polymer microparticles of the present invention may be at least one selected from the group consisting of poly-L-lactic acid, poly-D-lactic acid, polydioxanone, polylactic acid, poly-ε-caprolactone, polyglycolic acid, copolymers thereof, and mixtures thereof, and may preferably be poly-L-lactic acid, but are not limited thereto.

[0061] The biocompatible carrier of the present invention may be at least one selected from the group consisting of hyaluronic acid, alginic acid, carboxymethylcellulose, dextran, collagen, gelatin, and elastin, preferably hyaluronic acid, but is not limited thereto.

[0062] The poly-L-lactic acid described above is a polymeric synthetic material with biocompatibility and biodegradability as a support. The hyaluronic acid described above is a biosynthetic natural substance abundant in animal skin, capable of forming a molecular framework to maintain the shape and tone of epidermal tissue.

[0063] The filler manufactured by the manufacturing method of the present invention is advantageous in that it can achieve an excellent effect of preserving biodegradable polymer microparticles without being phagocytosed by macrophages even after injection into the skin while stably maintaining injection force.

[0064] The present invention provides a composition for skin moisturizing and wrinkle improvement comprising a bacteriophage for intestinal microorganisms, and a biodegradable polymer filler for skin wrinkle improvement comprising a peptide derived from a bacteriophage for intestinal microorganisms and a method for producing the same. The composition increases collagen production, increases moisture content, has high safety, and is excellent in terms of usability evaluations such as spreadability, absorbency, and fragrance, and thus can be usefully used as a cosmetic composition for skin moisturizing and wrinkle improvement. The filler significantly improves skin wrinkles and has an excellent effect of promoting skin elasticity, and thus can be usefully used for skin regeneration.

[0065] Figure 1 is a diagram showing the measurement of collagen content after using SHC-100.

[0066] Figure 2 is a diagram showing the measurement of moisture content to confirm improvement in skin hydration after using SHC-100.

[0067] Figure 3 is a graph showing the change in skin moisture content after using SHC-100.

[0068] Figure 4 is a diagram showing the results of HPLC analysis for bacteriophage-derived peptides.

[0069] Figure 5 is a diagram showing the results of MS analysis performed for mass spectrometry of bacteriophage-derived peptides.

[0070] Figure 6 is a diagram comparing skin wrinkles before and two weeks after injection of a biodegradable polymer filler containing a bacteriophage-derived peptide (sample 15).

[0071] Figure 7 is a comparison of skin wrinkles before and two weeks after injection of a biodegradable polymer filler containing a bacteriophage-derived peptide (sample 19).

[0072] Hereinafter, the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes modifications of technical concepts equivalent thereto.

[0073] Example 1. Preparation of test substances

[0074] The test substance SHC-100 containing bacteriophage as an active ingredient was diluted to a concentration of 0.01 to 10% and used as a test substance in the experiment.

[0075] 1-1. Sampling of target bacteria and phages

[0076] Samples collected from the natural environment and intestinal microbiota cultures were added together at a ratio of 1:100 to 1,000 and cultured with shaking at 30 to 37°C for 3 to 4 hours. The intestinal microbiota cultures were inoculated into the centrifuged supernatant at a ratio of 1:100 and cultured with shaking. The soft agar overlay method was performed with this supernatant.

[0077] The above soft agar overlay method was screened by observing the lysis of bacteriophages using host cells grown on top agar (attached to a solid medium using 0.4% agar).

[0078] 1-2. Identification of bacteriophages

[0079] 1-2-1. Morphological analysis of bacteriophages

[0080] To analyze the DNA pattern of bacteriophage, the genomic DNA of bacteriophage was extracted, cut with restriction enzymes, and then analyzed by electrophoresis.

[0081] Additionally, for structural protein analysis of bacteriophage, 1x10 9 A purified bacteriophage solution at a concentration of PFU / ml and a 5X SDS sample solution were mixed, boiled at 95°C for 5 minutes, and then subjected to 10% SDS-PAGE.

[0082] Example 2. Method for evaluating intracellular collagen production

[0083] 2-1. Cell line preparation

[0084] Cell line information is as follows. The cell line is human fibroblast CCD-986SK, the source is ATCC CRL-1947, and the Lot No. is 70016637.

[0085] 2-2. Cell line storage

[0086] Approximately 1 mL was dispensed into culture medium containing 10% (v / v) DMSO, frozen, and then transferred to a liquid nitrogen storage container for preservation.

[0087] The cells were thawed and used before the test, and thawing and culture were performed jointly with other tests in relation to test application.

[0088] 2-3. Cell culture conditions

[0089] Cells were cultured in a CO2 cell incubator at a temperature of 37 ± 1 ℃, a CO2 concentration of 5%, and a humidity of 90% or higher.

[0090] 2-4. Cell culture medium

[0091] 2-4-1. Full badge

[0092] The complete badge composition is as shown in Table 1 below.

[0093]

[0094] 2-4-2. Serum-free medium

[0095] The composition of serum-free medium is as shown in Table 2 below.

[0096]

[0097] 2-5. Cytotoxicity test

[0098] 2-5-1. Military composition

[0099] The test substance was diluted into five concentrations and performed as shown in Table 3 below.

[0100]

[0101] 2-5-2. Cell division

[0102] 1 x 10 cells in a 96 well plate 4 (100 μL / well) and cultured in a 37°C, 5% CO2 incubator for 24 hours.

[0103] 2-5-3. Test substance processing

[0104] The medium was replaced with serum-free medium containing test substances at different concentrations and cultured for 24 hours in a 37°C, 5% CO2 incubator.

[0105] 2-5-4. MTT assay

[0106] MTT solution was dispensed at 200 μL / well and incubated for 2 hours in a 37°C, 5% CO2 incubator. After incubation, the medium was removed, 150 μL / well of DMSO solution was added, and extraction was performed on a plate shaker for 20 minutes.

[0107] 2-5-5. Analysis of cell viability results

[0108] The absorbance was measured at 540 nm using an ELISA reader, and the cell viability was calculated according to the following formula.

[0109]

[0110] 2-6. Intracellular collagen production test

[0111] 2-6-1. Military composition

[0112] The test substance was diluted into three concentrations and performed as shown in Table 4 below.

[0113]

[0114] 2-6-2. Cell division

[0115] 5 x 10 cells in a 48 well plate 4 (1 mL / well) was dispensed and cultured in a 37 ℃, 5% CO2 incubator for 24 hours.

[0116] 2-6-3. Test substance treatment

[0117] The medium was replaced with a serum-free medium containing test substances and positive control substances, and cultured for 24 hours in a 37°C, 5% CO2 incubator.

[0118] 2-6-4. Protein quantification

[0119] The cell culture solution was recovered and the protein content was measured according to the BCA protein assay reagent kit (Thermo scientific PIERCE, USA) using a BSA standard curve.

[0120] 2-6-5. Collagen Quantification

[0121] The cell culture medium was recovered and tested according to the Procollagen type I Peptide EIA kit (TAKARA, Japan), and the content was measured according to the collagen standard curve.

[0122] 2-7. Results Production

[0123] The intracellular collagen production rate was converted into collagen content in terms of protein content and calculated according to the following formula.

[0124]

[0125] 2-8. Statistical processing

[0126] The results of each experiment were statistically analyzed using the SPSS statistical program (ver. 19.0). First, Levene's test was performed, and then a one-way ANOVA test was performed to confirm the significance between the experimental groups. After that, a post-hoc test (Duncan's new multiple range test for homogeneous variances and Dunnett's T3 test for heterogeneous variances) was performed depending on the homogeneity of variances. The confidence interval was set at 95%.

[0127] Example 3. Method for measuring and evaluating skin hydration

[0128] 3-1. Environmental conditions

[0129] The experiment was conducted under constant temperature and humidity conditions where the temperature was maintained at 20 to 22°C and the humidity was maintained at 42 to 51%.

[0130] 3-2. Measurement site and procedure

[0131] The measurement site was designated as the subject's face. Upon visiting the center, the subject was cleansed with the same cleanser used at the center and then allowed to rest for 30 minutes under constant temperature and humidity conditions. To ensure objective and accurate measurements, the same examiner performed each measurement.

[0132] 3-3. How to measure skin hydration

[0133] To assess the moisture content of the cheek area of ​​the subject's face, a corneometer was used. The corneometer measures capacitance and displays the difference in dielectric constant between water and the measured substance as a measurement value. The corneometer probe was placed in contact with the skin, and a sensor was used to measure three consecutive times, and the average value was used as the measurement value.

[0134] The unit is AU (Arbitrary Unit), and an increase in the result value indicates an improvement in skin hydration.

[0135] 3-4. Survey Evaluation

[0136] The subjects used the test product (test substance) and 4 weeks later completed a validity questionnaire on the items requested by the client.

[0137] 3-5. Compliance Assessment

[0138] Compliance evaluation was conducted by distributing the product and a compliance diary to the subjects, so that the subjects could directly record whether they used the test product.

[0139] In this regard, compliance was assessed as follows. If compliance was less than 80%, it was excluded from the test data.

[0140]

[0141] 3-6. Safety Assessment

[0142] While using the test product, we checked whether any adverse reactions were reported by all subjects.

[0143] 3-7. Side effect evaluation

[0144] All adverse reactions were recorded, and at each visit, the subject's skin was examined through a questionnaire and visual assessment by a specialist, and recorded in an individual case report form.

[0145] The number of subjects, incidence rate, and pattern of adverse reactions for each category were recorded based on severity, causality, related measures, and outcome. Reasons for discontinuation or withdrawal from the study were reviewed and recorded in the case report.

[0146] Experimental Example 1. Cytotoxicity Evaluation

[0147] When SHC-100 (test substance) was treated at concentrations of 0.65%, 1.25%, 2.5%, 5%, and 10%, cell viability rates of 96.98 ± 0.34%, 92.45 ± 1.61%, 8.00 ± 0.52%, 7.45 ± 0.13%, and 7.78 ± 0.20% were observed at each concentration.

[0148] As a result of the cytotoxicity test, a cell viability of more than 70% was observed at a concentration of 1.25% or less, and the highest concentration of the sample used in the intracellular collagen production test using CCD-986SK cells was selected as 1.25%.

[0149] Experimental Example 2. Evaluation of Intracellular Collagen Production

[0150] When treated with SHC-100 (test substance) at concentrations of 0.5%, 1%, and 1.25%, collagen contents of 192.90 ± 7.07%, 181.45 ± 3.02%, and 119.81 ± 9.09% were measured at each concentration, respectively. Meanwhile, when treated with 200 μM of L-ascorbic acid, a positive control substance, it was observed that intracellular collagen production increased by 138.58 ± 4.11% compared to the control group (Fig. 1).

[0151] Through this, it was confirmed that SHC-100 (test substance) significantly increased collagen production in the intracellular collagen production test.

[0152] Experimental Example 3. Skin Compliance and Moisturizing Evaluation

[0153] 3-1. Test subject information

[0154] Information on subjects who participated in the application test of test substance SHC-100 is as shown in Tables 5 to 7 below.

[0155]

[0156]

[0157]

[0158] As shown in Table 5, the test subjects were 22 women, aged 41.6 ± 11.2 years. As shown in Table 6, the age distribution of the subjects included 5 subjects in their 20s (22.7%), 5 subjects in their 30s (22.7%), 6 subjects in their 40s (27.3%), and 6 subjects in their 50s (27.3%). Table 7 shows the skin types of each subject.

[0159] 3-2. Compliance Assessment

[0160] All subjects who completed the test were evaluated as having a compliance rate of 90% or higher with the test product (SHC-100) (Table 8).

[0161]

[0162] 3-3. Moisturizing Evaluation

[0163] The results of measuring the moisture content of the test product before, after 2 weeks of use, and after 4 weeks of use using a corneometer are shown in Table 9, Figures 2 and 3.

[0164]

[0165] As a result of confirming with the nonparametric Friedman test and the parametric Repeated measures ANOVA according to the normality test, the AU value increased statistically significantly after 2 and 4 weeks of use compared to before use.

[0166] This suggests that skin hydration improves over time after using the test product (SHC-100).

[0167] Experimental Example 4. Evaluation of Effectiveness and Usability

[0168] To evaluate the efficacy of the test product after use, a questionnaire evaluation was conducted on 22 registered test subjects (Table 10).

[0169]

[0170] As a result, it was confirmed that the test product was effective in improving skin, such as improving wrinkles and brightening skin tone.

[0171] Additionally, a questionnaire evaluation was conducted on 22 registered test subjects to evaluate the product usability (Table 11).

[0172]

[0173] As a result, the test product was found to have excellent spreadability, moisture, and absorbency, and a high preference for fragrance, providing an overall very good user experience.

[0174] Experimental Example 5. Safety Evaluation

[0175] There were no adverse reactions observed while using the test product (SHC-100).

[0176] 5-1. Evaluation of adverse reactions by a dermatologist

[0177] The test subjects confirmed that no adverse reactions such as allergic contact dermatitis or irritant contact dermatitis occurred after using the test product.

[0178] 5-2. Reporting of adverse skin reactions by test subjects

[0179] As a result of checking with the test subjects, no specific symptoms related to adverse skin reactions were reported after using the test product.

[0180] 5-3. Cases of side effects

[0181] There were no cases of side effects after using the test product.

[0182] Through this, it was confirmed that the test product SHC-100, which includes a composition containing a bacteriophage for intestinal microorganisms of the present invention, improves wrinkles by increasing collagen production, and the skin moisturizing evaluation results showed a significant increase after use of the product, and that it was also excellent in safety.

[0183] Manufacturing Example 1. Sampling and identification of bacteriophages

[0184] 1-1. Sampling of target bacteria and phages

[0185] Samples collected from the natural environment and intestinal microbiota cultures were added together at a ratio of 1:100 to 1,000 and cultured with shaking at 30 to 37°C for 3 to 4 hours. The intestinal microbiota cultures were inoculated into the centrifuged supernatant at a ratio of 1:100 and cultured with shaking. The soft agar overlay method was performed with this supernatant.

[0186] The above soft agar overlay method was screened by observing the lysis of bacteriophages using host cells grown on top agar (attached to a solid medium using 0.4% agar).

[0187] 1-2. Identification of bacteriophages

[0188] To analyze the DNA pattern of bacteriophage, genomic DNA of bacteriophage was extracted, digested with restriction enzymes, and analyzed by electrophoresis. In addition, to analyze the structural proteins of bacteriophage, 1x10 9 A purified bacteriophage solution at a concentration of PFU / ml and a 5X SDS sample solution were mixed, boiled at 95°C for 5 minutes, and then subjected to 10% SDS-PAGE.

[0189] Manufacturing Example 2. Production and identification of bacteriophage-derived peptides

[0190] After growing the bacteriophage at 37°C with shaking for 4.5 hours, 1 / 6 volume of a solution (20% w / v polyethylene glycol-8,000 and 2.5 M sodium chloride) was added to precipitate the phage in the supernatant overnight at 4°C. The precipitate was then centrifuged, and the supernatant was recovered.

[0191] Thereafter, the supernatant was analyzed using HPLC equipment (flow rate: 1 mL / min, UV detection: 214 nm) (Fig. 4), and the eluate from the outlet of the chromatography column was injected into a mass spectrometer to analyze the mass of the bacteriophage-derived peptide (Fig. 5). As a result, it was confirmed that the bacteriophage-derived peptide of the present invention had a mass of 1098.04 Da.

[0192] The process of synthesizing the above peptides on resin begins with the attachment of the first amino acid (AA) to the C-terminal residue (carboxyl group), followed by peptide construction at the N-terminal end. The amino acid is attached to a peptide sequence supported by the alpha-amino group, and the reactive side chains are protected by the tert-butoxycarbonyl (Boc) and fluoren-9-ylmethyloxycarbonyl (Fmoc) groups. The carboxyl group of the amino acid must be activated to bind to the resin. Once the amino acid is attached, the resin is filtered and washed to remove byproducts and excess reagents.

[0193] The N-protecting group of the new amino acid couple is removed. Boc is removed with an acid such as trifluoroacetic acid (TFA), and Fmoc is removed with a base such as piperidine. The resin is washed again to remove byproducts and excess reagents.

[0194] Synthetic peptide coupling requires the activation of the C-terminal carboxylic acid of the incoming amino acid using a carbodiimide, such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). These coupling reagents react with the carboxyl group to form a highly reactive O-acylisorean intermediate, which is rapidly displaced by nucleophilic attack of the deprotected primary amino group at the N-terminus of the growing peptide chain to form the nascent peptide bond.

[0195] Carbodiimides form reactive intermediates that can lead to racemization of amino acids. Therefore, reagents that react with O-acylisourea intermediates, including 1-hydroxybenzotriazole (HOBt), which forms less reactive intermediates to reduce the risk of racemization, can be added.

[0196] Furthermore, side reactions with carbodiimides have led to the investigation of other coupling agents, including benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) and 2-(1H-benzotriazol-1-yl)-1,3,3-tetramethyluronium hexafluorophosphate (HBTU), both of which require an activating base to mediate amino acid coupling.

[0197] After successive cycles of amino acid deprotection and coupling, any remaining protecting groups must be removed from the initial peptide. These groups are cleaved by acidolysis, and the chemicals used for cleavage vary depending on the intended protection.

[0198] Strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr), or trifluoromethane sulfonic acid (TFMSA) are used to cleave the Boc and Bzl groups, whereas relatively weak acids such as TFA are used to cleave the Fmoc and tBut groups.

[0199] A properly performed cleavage removes the N-terminal protecting group of the last amino acid added, the C-terminal protecting group (chemical or resin) of the first amino acid, and all side-chain protecting groups. As with deprotection, a scavenger is involved in this step, reacting with the free protecting group.

[0200] Manufacturing Example 3. Manufacturing of hyaluronic acid (HA) and polylactic acid (PLLA) containing peptides.

[0201] As shown in Table 12 below, Examples 1 to 6 were prepared containing 1 ppm (100 parts by weight) or 10 ppm (1000 parts by weight) of bacteriophage-derived peptide.

[0202]

[0203] The contents of the above-mentioned manufactured examples 1 to 6 were injected into a syringe container at 1 to 10 cc, and then packaged through a sterilization process.

[0204] Experimental Example 6. Evaluation of Wrinkle Improvement Effect

[0205] A panel test was conducted on the effect of improving skin wrinkles using the fillers of Examples 1 to 6 and the control group (without peptide).

[0206] First, 30 healthy women aged 35 to 50 were divided into 6 groups of 5 each, and checked before and 2 weeks after injection (Figs. 6 and 7).

[0207] Additionally, the degree of improvement in skin wrinkles was assessed through a subject questionnaire. The improvement in skin wrinkles and increased elasticity was assessed on a three-point scale: "no improvement," "slight improvement," and "significant improvement" compared to before use. The results are shown in Table 13 below.

[0208] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Control group No improvement 14 Slight improvement 23121 Significant improvement 432543

[0209] As shown in FIG. 6, FIG. 7 and Table 13 of the present invention, it was confirmed that the biodegradable polymer filler containing the bacteriophage-derived peptide of the present invention as an active ingredient has an excellent effect of improving skin wrinkles and enhancing skin elasticity.

[0210] While the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments, but can be manufactured in various different forms. Those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

[0211] [Accession number]

[0212] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0213] Accession number: KCTC15445BP

[0214] Date of acceptance: 20230518

[0215] [Correction pursuant to Rule 91, July 13, 2023]

Claims

1. A cosmetic composition comprising a bacteriophage, a bacteriophage metabolite or a combination thereof deposited under the deposit number KCTC 15445BP as an active ingredient.

2. In paragraph 1, A cosmetic composition characterized in that the above bacteriophage lyses intestinal microorganisms.

3. In paragraph 2, A cosmetic composition characterized in that the intestinal microorganism is a strain selected from the group consisting of Escherichia coli, Aeromonas hydrophila, Bacillus cereus, Lactobacillus sp., Bifidobacterium sp., Clostridium sp., Staphylococcus sp., Acinobacterium sp., Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter sp.

4. In paragraph 1, A cosmetic composition characterized in that the composition increases collagen production.

5. In paragraph 1, A cosmetic composition characterized in that the composition increases moisture content.

6. In paragraph 1, A cosmetic composition characterized in that the composition improves skin moisturizing and wrinkles.

7. In paragraph 1, A cosmetic composition, characterized in that the above-mentioned effective ingredient is contained in an amount of 0.01 to 90 wt% based on the total weight of the cosmetic composition.

8. In paragraph 1, A cosmetic composition characterized in that the composition has a formulation selected from the group consisting of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleanser, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray.

9. A method for improving skin moisturizing, comprising a step of applying the composition of paragraph 1 to the skin of a subject other than a human.

10. A method for improving skin wrinkles, comprising a step of applying the composition of paragraph 1 to the skin of a subject other than a human.

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