KR20120068367A - Manufacturing with psychrophile cultivated fermentation cosmetic composition - Google Patents

Abstract

PURPOSE: A fermentation cosmetic composition containing fermentation prepared from psychrophiles is provided to ensure antioxidation, anti-wrinkling, whitening, and moisturization. CONSTITUTION: A cosmetic composition contains a fermentation prepared from pshychrophiles. The cosmetic composition further contains a fermentation prepared by fermenting natural medicinal materials, gruits, vegetables, or oriental medicinal materials at low temperature using psychrophiles. The psychrophiles are Arthorobcter agilis, Arthrobacter globiformis, Arthrobacter psychrolactophilus, Arthrobacter rhombi, Arthrobacter sulfonivorans, Bacillus macroides, Bacillus psychrodurans, Colwellia aestuarii, Colwellia piezophila, Flavobacterium frigidarium, Flavobacterium segetis, Lacinutrix mariniflava, Pseudoalteromonas arctica, Pseudoalteromonas citrea, and Pseudoalteromonas halopla.

Description

Fermentation cosmetic composition using low temperature microorganism {MANUFACTURING WITH PSYCHROPHILE CULTIVATED FERMENTATION COSMETIC COMPOSITION}

The present invention relates to a fermentation cosmetic composition using a metabolite obtained by low-temperature fermentation of natural medicinal substances, fruits, vegetables and the like using microorganisms that grow well at low temperatures.

As human skin ages, the activity of skin cells decreases internally, thereby reducing the biosynthesis of skin constituent proteins, and thickening of the skin occurs due to external factors such as ultraviolet rays and elasticity decreases. At the same time, various skin colors such as blemishes, freckles, and blotch are changed, making the skin dull and rough. Therefore, the most active field of research to alleviate the skin aging phenomenon is the study on the improvement and whitening of skin wrinkles. Therefore, a lot of research continues on the active ingredient having the skin wrinkle improvement and whitening effect.

The cause of skin aging has not been elucidated precisely, but the well-known mechanisms so far are as follows.

First, the collagen tissue of the dermis is weakened, collagen fibers are reduced by ultraviolet rays, stress, etc., and the skin tissue is loosened and elasticity is lost.

Second, it is a phenomenon that occurs when the metabolism of the epidermis decreases due to the degradation of cellular function of the epidermis, and the exfoliation does not occur well.

Third, free radicals generated by ultraviolet rays in sunlight are highly reactive reactive oxygen species, which cause cell damage and death by oxidizing or denaturing key biological components such as sugars, proteins, DNA, and cell membrane lipids. In particular, it has been found to be the biggest cause of cell aging in the skin. Free radicals are elements or molecules with one or more unpaired electrons that occur due to oxygen reduction, ultraviolet irradiation, or phagocytosis during the breathing process of living organisms. Properties to cause cell damage. Therefore, continuous reaction of these free radicals causes damage to skin cells and promotes aging.

Fourth, the inflammatory response caused by external harmful environment such as ultraviolet rays, stress, and the activity of immune cells due to aging is reduced, so the skin's immune function regulation is not normal, thereby promoting skin aging.

Fifth, due to the reduction of moisture through the stratum corneum, the skin is dried and the surface is rough, and the skin is lost to the gloss and dull skin is promoted. The skin barrier not only protects against various stimuli (chemicals, air pollutants, dry environment, UV rays, etc.) from the outside, but also protects against excessive release of moisture through the skin. The function can be maintained only when the configured stratum corneum is normally formed. The stratum corneum of the epidermis contains 10 to 20% of moisture and is present on the outermost side of the skin to inhibit evaporation of moisture from the body while preventing excess moisture from penetrating. It is known that the sebum component and the epidermal lipid, which are the lipid membrane components of the keratinocyte layer, maintain the moisture loss at an appropriate level. The stratum corneum layer contains a hydrophilic, water-retaining substance called Natural Moisturizing Factor (NMF), which helps the skin to maintain flexibility and maintain proper moisture. However, when the moisture content of the stratum corneum is less than 10%, the skin becomes rough and the body loses its protective function, causing aging. Recently, due to changes in the environment and living patterns, stress and environmental pollution caused by social life, frequent face-washing caused by makeup habits, moisture in the stratum corneum decreases, the skin becomes dry, the surface becomes rough, and the skin loses luster. Phenomenon such as dull appearance occurs, the problem of skin moisturizing is becoming increasingly important.

Summarizing these aging mechanisms, internal factors such as the passage of time and external factors such as ultraviolet rays work together to damage the extracellular matrix of the dermis, resulting in the breakdown of proteins such as collagen, which make up the substrate. It is lowered and wrinkles are generated. With the enforcement of the Cosmetics Act in July 2000, research and development on functional cosmetics has been further developed, and a lot of research has been conducted in conjunction with the increasing desire for consumers to have young and elastic skin in accordance with the aging society. have.

Low temperature microorganisms grow well at 0 ° C, with optimum growth temperatures of 15 ° C or below, and peak temperatures usually around 20 ° C. Low temperature microorganisms are well separated in the Antarctic and Arctic regions. In the Arctic and Antarctic, 90% of the ocean is below 5 degrees, making it a good habitat for low temperature microorganisms. Enzymes, transport systems, and protein synthesis work well at low temperatures. Cell membranes of low temperature microorganisms contain a lot of unsaturated fatty acids to maintain semi-fluidity even in cold environments.

Most products that are fermented cosmetics are fermented products fermented with herbs, fruits, vegetables and natural medicinal plants through microorganisms that are active at room temperature, and are sensitive to temperature when developing fermentation materials using microorganisms that produce excellent metabolites. It was very hard to get the skin effect you wanted.

In view of this, the present inventors have carried out a manufacturing method applying a culture method of low temperature microorganisms which are sensitive to temperature, thereby leading to the development of a new fermentation cosmetic composition having excellent effects on the skin and low side effects.

Provided is a composition comprising an extracellular polysaccharide (exopolysaccharide), which is a metabolite of low temperature microorganisms living at low temperatures, which has very excellent cell efficacy and improves various skin aging phenomena when applied directly to human skin.

It provides a cosmetic composition comprising a fermentation product of low temperature microorganisms to produce a fermentation product at a temperature of 3 ℃ to 15 ℃, in particular a cosmetic composition comprising a fermentation product of high temperature microorganisms active at a temperature of 4 or 10 ℃. do. Low temperature microbial fermentation cosmetic composition according to an embodiment of the present invention is a herbal, natural medicinal material. It may include metabolites fermented by adding one or more components selected from vegetables, fruits, grains and seaweeds to the medium.

According to one embodiment of the present invention, a low temperature microorganism having high activity at 4 or 10 ° C is used. The activity is not particularly limited as long as the activity is high at the temperature and the fermentation can be performed well. As an active microorganism at 4 ° C., it is known as Arthrobacter agilis, Arthrobacter globiformis, Arthrobacter psychrolactophilus, and Acrobacter romby (Arthrobacter rhombi), Arthrobacter sulfonivorans, Bacillus macroides, Bacillus psychrodurans, Colwellia aestuarii, Colwellia piezophylla (Colwellia piezophila), Flavoacterium frigidarium, Flavobacterium segetis, Lacinutrix mariniflava, Pseudoalteromonas Artica, Pseudoalteromonas arctic Pseudoalteromonas citrea, Pseudoalteromonas elyakovii, Pseudoaltermonas halo Pseudoalteromonas haloplanktis, Pseudomonas veronii, Pseudomonas viridiflava, Psychrobacter aquimaris, Pychrobacter aquimaris, Psychrobacta psicotica arcicosactica , Psychromonas profunda, Stenotrophomonas maltophilia, Thalassomonas sediminis, Vibrio logei, Zoogloea ramigera One or more selected from the group consisting of can be used.

As microorganisms highly active at 10 ° C., Acinetobacter johnsonii, Algibacter lectus, Alteromonas hispanica, Alteromonas macleodii, Al Alteromonas tagae, Arthrobacter globiformis, Arthrobacter nicotianae, Arthrobacter protophormiae, Athrobacter sulfos Arthrobacter sulfurous, Bacillus aquimaris, Bacillus baekryungensis, Bacillus cereus, Bacillus firmus, Bacillus fuformus cocillus, Bacillus fusiformis Bacillus koguryoae, Bacillus megaterium, Covetia marina, Exigiobacterium oxidase Exiguobacterium oxidotolerum, Gillisia illustrilutea, Glaciecola mesophila, Halomonas marina, Maribacter forsetii, Paracoccus homiensis (Paracoccus homiensis), Photobacterium leiognathi, Photobacterium phosphoreum, Photobacterium profundum, Planococcus donhaensis, Planococcus Planococcus maritimus, Pseudoalteromonas denitrificans, Pseudoalteromonas ganghwensis, Pseudoalteromonas nigrifaciens, Pseudoalteroensus Pseudomonas putida, Pseudomonas syringae, Pseudomonas tolaasii, Pseudovibrio denitrificans, Psychrobacter marincola, Psychrobacter okhotskensis, Psychrobacter urativorans, and Schwanella prigiliana Shewanella frigidimarina, Shewanella gelidimarina, Staphylococcus saprophyticus, Sulfitobacter dubius, Vibrio cyclotripicus, Vibrio cyclitrophicus One or more microorganisms selected from the group consisting of Vibrio gigantis, Vibrio pomeroyi, Vibrio splendidus, Vibrio tasmaniensis can be used.

The cosmetic composition may be in the form of external skin ointment, softening lotion, nourishing lotion, cream, gel, essence, pack, or emulsion, oil, water, surfactant, moisturizer, lower alcohol, thickener, chelating agent, pigment as necessary It may further include an additive selected from the group consisting of a preservative, and a fragrance.

The cosmetic composition according to the present invention can be applied as various cosmetics having high safety to the skin while having better antioxidant, cell activity effect, wrinkle, elasticity improvement effect, whitening effect and moisturizing effect.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, these examples are only provided to aid the understanding of the present invention, the present invention is not limited to these examples.

Example

Example  1: Low temperature microbial culture conditions

Incubating fermented natural products at 4 ° C. and 10 ° C., respectively, using Pseudoalteromonas denitrificans (Table 1) and Staphylococcus saprophyticus (Table 2) as low temperature microorganisms. It was. Media composition is shown in Tables 1 and 2 below. Table 1 shows TSA (TW80) medium. pH was fixed at 7.0.

Raw material Composition ratio Enzymatic Digest of Casein 15.0 g Enzymatic Digest of Soybean Meal 5.0 g Sodium Chloride 5.0 g Agar 20.5 g Tween 80 5 g Lecithin 0.7 g Natural product extract 1.0 g

Raw material Composition ratio Eptone 5.0 g Yeast extract 1.0 g FePO4-4H2O 0.01 g Agar 15.0 g Aged seawater 750 mL Natural product extract 1.0 g Purified water 250 mL

Example  2: freedom Radical Scatters  exam

The free radical scavenging ability of the composition fermented with low temperature microorganisms was determined by the 1,1-diphenyl-2-pycryl-hydrazyl (DPPH) method as follows (Blois). , Nature 181, 1190, 1958). DPPH is a relatively stable free radical which, when present in the radical state, exhibits maximum absorption at 517 nm and loses absorbance when erased. DPPH was used as a product of Sigma, Inc. and dissolved in methyl alcohol at a concentration of 0.15 mM.

First, the low temperature microbial fermentation product prepared in Example 1, the fermentation product fermented at room temperature (25 ° C.) using low temperature microorganisms, and ascorbic acid, respectively, were added to each well of a 96-well plate. Μl was added. 100 μl of DPPH solution was added thereto, and the mixture was left at room temperature for 30 minutes, and then absorbance at 517 nm was measured using a Micro plate reader (BioTek EL-340). At this time, methyl alcohol was used as a control.

The concentration of the sample when the absorbance of the sample was half the absorbance of the control group was expressed as IC ₅₀ (Inhibitory Concentration 50), and the results are shown in Table 3 below.

sample IC ₅₀ (%, w / v) Methyl alcohol 0.0025 Room temperature (25 ℃) fermented metabolites 0.001 Low Temperature (4 ℃) Fermentation Metabolites 0.0030 Low Temperature (10 ° C) Fermentation Metabolites 0.0035 IC ₅₀ (Inhibitory Concentration 50)-50% cell killing concentration

Referring to Table 3, the low temperature fermentation metabolite used in the cosmetic composition of the present invention was found to be a very powerful free radical scavenger.

Example  3: cell proliferation Activity  Measure

For the composition comprising the microorganism-derived trihalose or extracellular polysaccharide, the effect of cell proliferation activity was measured as follows. First, human fibroblasts were cultured to perform the MTT reduction method (3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide reduction method), and the formazan product wasopropyl alcohol. Extraction was performed and the relative absorbance at 570 nm was measured and compared. At this time, a medium containing 10% FBS (fetal bovine serum) was used as a positive control, and a medium without FBS was used as a negative control, and a culture medium without a sample was used as a control. Using the following equation 1 to calculate the viability of the cell numerical value is shown in Table 4 below. The cell proliferation activity of Table 4 was determined by five repeated tests.

sample Proliferation rate (%) Control group (no addition) 100 Room temperature (25 ℃) fermented metabolites 95 Low Temperature (4 ℃) Fermentation Metabolites 120 Low Temperature (10 ° C) Fermentation Metabolites 118

Referring to Table 4, it was found that the low temperature microbial fermentation broth used in the cosmetic composition of the present invention has an excellent cell activity effect by promoting skin cell proliferation.

Example  4: Collagen Life Performance Measurement

Human-derived fibroblasts isolated from human skin tissue were cultured in DMEM medium containing 10% FBS and antibiotics (antibiotics) to seed cells (1 × 10 ⁴ cells) in 96 well plates. . The next day, the cells were washed once with PBS, then changed into FBS-free DMEM medium, and each cytokine was incubated for two more days. The finished cell cultures were collected and subjected to EIA assay (TaKaRa Bio Inc.) for procollagen type I to determine the respective collagen synthesis concentrations. EIA analysis was performed following the method of Kit provided by the company. Synthetic concentration was calculated with the results of the synthetic concentration, and the results are shown in Table 5 below.

division Collagen Synthesis Growth Rate (%) Control group (no addition) Room temperature (25 ℃) fermented metabolites 100 Low Temperature (4 ℃) Fermentation Metabolites 130 Low Temperature (10 ° C) Fermentation Metabolites 135 Vitamin C (final concentration 52.8 ㎍ / mL) 125

Referring to Table 5, it was confirmed that the collagen synthesis increased the effect of the low temperature microbial fermentation carried out in the present invention, the fermentation product of the low-temperature microorganism fermented at room temperature (25 degrees) was not effective. The collagen synthesis effect promotes the synthesis of collagen when applied to the skin, and has effects on wrinkle improvement, regeneration, skin elasticity improvement, stretch marks, scar reduction, and moisturizing effects.

Example 5 : Measurement of melanin synthesis inhibitory effect

The composition containing the microorganism-derived trihalose or extracellular polysaccharide was added to the culture solution of melanocytes, which are melanocyte synthetic cells, to test the melanin synthesis inhibitory effect at the cellular level. The cell cultures were collected by centrifugation at 1,500 g for 5 minutes, washed with PBS buffer, and then extracted with 200 μL of extraction buffer (1% Nonidet P-40, 0.01% SDS, 0.1M Tris-HCl pH 7.2, protease inhibitor cocktail). Extraction was carried out at 4 ° C. for 1 hour. The extract was centrifuged at 15,000 rpm for 5 minutes and the resulting precipitate was treated with 1N NaOH and then transferred to a 96 well plate. Relative melanin content was determined by measuring absorbance at 480 nm with an ELISA reader (PowerWave X340, Bio-tech Instruments, Inc.). Three replicates of one concentration were performed and the results were averaged. Intracellular melanogenesis inhibitory ability of the composition was evaluated as the melanin production inhibitory ability as a relative melanin content by obtaining a ratio of absorbance values measured for each case containing no sample as a control. The total protein amount of each well was quantified through a BCA ^TM (Bicinchoninic acid) assay kit, and the amount of melanin synthesized by the total protein amount was compared and evaluated. 6 is shown.

division Melanin synthesis inhibition rate (%) Control group (no addition) - Room temperature (25 ℃) fermented metabolites - Low Temperature (4 ℃) Microbial Fermentation Metabolites 41.5 Low Temperature (10 ℃) Microbial Fermentation Metabolites 40.5 Arbutin (final concentration 1 mg / mL) 39.5

When the low-temperature microbial fermentation composition was treated, it was confirmed that the low-microbial fermentation at room temperature and the control of melanin synthesis were superior to the control.

Example  5. allergy  Test( LLNA )

To determine whether allergens of trihalose or extracellular polysaccharides derived from microorganisms are induced, an experimental method using ethanol as a carrier medium was selected. [Kimber I (1990): Identification of contact allergens using the murine local lymph node assay, J. Appl. Toxicol. 10 (3); 173-180]. 50 μl in both ears of the mouse (Balb / c) for 3 days, the low temperature microbial fermentation product prepared in Example 1, the fermentation product at room temperature (25 ° C.) using the low temperature microorganism, and acetone: olive oil 4: as a control. After applying a mixture of 1 ratio, auricular lymph nodes were isolated from the rats. Lymph nodes were pulverized into single cells, incubated for 24 hours with the addition of the radioisotope [3H] -methyl thymidine, followed by β-scintillation counter (Beckman LS 6000 TA, USA). The amplification degree (dpm, disintegrations per minute) of the cells was measured using. The results are shown in Table 7 below.

sample Lymph node cells (10 ⁷ cells / ml) cpm (average) Amplification Acetone: Olive Oil (4: 1) 1.70 2870 Transportation Fermentation metabolite at room temperature (25 ℃) 1.88 3852 2.5 Low temperature condition (4 ℃) fermented metabolite 1.71 2296 0.8 Low temperature condition (10 ℃) fermented metabolite 1.72 2538 0.9

Referring to Table 7, the composition containing the low temperature microbial fermentation according to the present invention was shown to have an amplification degree (SI) of 2.0 or less, which is unlikely to cause allergy.

Claims (5)

Cosmetic composition comprising a fermentation product of a low temperature microorganism producing a fermentation product at a temperature of 3 ℃ to 15 ℃.
The method of claim 1,
The cosmetic composition further comprises a fermentation product obtained by fermenting a natural medicinal substance, fruits, vegetables or herbal medicines at low temperature using the low temperature microorganisms.
The method of claim 1,
The low temperature microorganisms include Arthrobacter agilis, Arthrobacter globiformis, Arthrobacter psychrolactophilus, Arthrobacter rhombi, Art Arthrobacter sulfonivorans, Bacillus macroides, Bacillus psychrodurans, Colwellia aestuarii, Colwellia piezophila, Flawell Flabacacterium frigidarium, Flavobacterium segetis, Lacinutrix mariniflava, Pseudoalteromonas arctica, Pseudoalteria thymos (Pseudoalteromonas citrea), Pseudoalteromonas elyakovii, Pseudoaltermonas haloplanktis (Pseudoalt) eromonas haloplanktis, Pseudomonas veronii, Pseudomonas viridiflava, Psychrobacter aquimaris, Psychrobacter fozii, Psychrobactar arsyomas, Arcticachroma Group consisting of Psychromonas profunda, Stenotrophomonas maltophilia, Thalassomonas sediminis, Vibrio logei, and Zogloa ramigera Cosmetic composition, which is at least one selected from.
The method of claim 1,
The low temperature microorganisms include Acinetobacter johnsonii, Algibacter lectus, Alteromonas hispanica, Alteromonas macleodii, and Altermonas tagae. Alteromonas tagae, Arthrobacter globiformis, Arthrobacter nicotianae, Arthrobacter protophormiae, Arthrobacter sulfous, Bacillus Bacillus aquimaris, Bacillus baekryungensis, Bacillus cereus, Bacillus firmus, Bacillus fusiformis, Bacillus coguryoa (Bacillus koguryoa) ), Bacillus megaterium, Cobetia marina, Exiguobacterium oxidotolerum (Exiguobacteriu) m oxidotolerum, Gillisia illustrilutea, Glaciecola mesophila, Halomonas marina, Maribacter forsetii, Paracoccus homiensis ), Photobacterium leiognathi, Photobacterium phosphoreum, Photobacterium profundum, Planococcus donghaensis, Planococcus marittis Planococcus maritimus, Pseudoalteromonas denitrificans, Pseudoalteromonas ganghwensis, Pseudoaltermonas nigrifacius, Pudoudoteroteromonas nidofamonas , Pseudomonas syringae, Pseudomonas tolaasii, Pseudomonas denite Pecans (Pseudovibrio denitrificans), Psychrobacter marincola, Psychrobacter okhotskensis, Psychrobacter urativorans, Sheanella prigidi marina Shewanella gelidimarina, Staphylococcus saprophyticus, Sulphitobacter dubius, Vibrio cyclitrophicus, Vibrio gontis , Vibrio pomeroyi, Vibrio splendidus, Vibrio tasmaniensis, at least one microorganism selected from the group consisting of, cosmetic composition.
The method of claim 1,
The cosmetic composition is at least one member selected from the group consisting of skin external ointment, softening lotion, nourishing lotion, cream, gel, essence, pack, and emulsion.