KR102252855B1 - Cosmetic composition comprising nano-ized lactobacillus genus dead cells and a method for producing the same - Google Patents

Abstract

The present invention relates to a cosmetic composition containing nano-sized dead cells of genus Lactobacillus and a method for manufacturing the same. The composition may be utilized in an anti-oxidant or moisturizing cosmetic composition.

Description

Cosmetic composition containing dead cells of the genus nanonized Lactobacillus and a method for manufacturing the same {COSMETIC COMPOSITION COMPRISING NANO-IZED LACTOBACILLUS GENUS DEAD CELLS AND A METHOD FOR PRODUCING THE SAME}

It relates to a cosmetic composition and a method for producing the same comprising the nano-ized Lactobacillus sp. dead cells. The composition may be used in anti-acid ash or moisturizing cosmetic composition.

The skin is a membrane that covers the outside of the body and plays a role in protecting and regulating internal organs and other internal organs by performing various physiological functions that protect the body from environmental factors such as various external stimuli, obstacles, and dryness. do.

However, as the skin ages, the physiological function of the living body decreases, the secretion of various hormones regulating metabolism decreases, and the function of immune cells and the activity of cells decrease. In addition, the appearance or function of the skin changes due to repeated exposure to light or heat. As a result, free radicals and free radicals and free radicals increase, and excessive physical and chemical irritation and stress are caused by deterioration of the normal function of the skin and melanin deposition. It damages the skin by promoting the generation of freckles and aging of the skin.

In order to prevent such skin aging and damage to maintain healthier and more beautiful skin, by using physiologically active substances obtained from various animals, plants, microorganisms, etc. in addition to cosmetics, it maintains the skin's own function and activates skin cells to make the skin look brighter. Efforts to maintain skin health by promoting metabolism are continuing, and cosmetics and external preparations for skin are being developed accordingly.

However, when these substances are applied to the skin, the amount of use of these substances is limited due to safety issues such as irritation, erythema, and redness, or the effect is insignificant, making it difficult to substantially improve skin function. Therefore, there is a need to develop a new composition for external application for skin that is safer to the living body and has a higher effect than the existing composition for external application for skin.

Korean Patent Registration No. 10-1961149 Korean Patent Registration No. 10-1968242

It is an object of the present invention to provide a composition for antioxidant comprising a nanonized Lactobacillus (Lactobacillus) genus dead cells.

Another object of the present invention is to provide a cosmetic composition or a food composition comprising the antioxidant composition.

Another object of the present invention is to provide a method for producing nano-ized Lactobacillus genus dead cells.

One aspect of the present invention for achieving the above object relates to a composition for antioxidant comprising a nano-ized Lactobacillus (Lactobacillus) genus dead cells.

In the present invention, “dead cells”, that is, dead cells of lactic acid bacteria, are the opposite concept of live cells, and are in a form in which the growth of bacteria is prevented by heat treatment of live cells and metabolites obtained through fermentation. It is characterized by containing antimicrobial active substances such as cytoplasm, cell wall, bacteriocin, polysaccharide, organic acid, and the like. Lactobacillus dead cells are used in terms of lactic acid bacteria dead cells, lactic acid bacteria cultures, lactic acid bacteria extracts, and lactic acid bacteria cell components.

Lactobacillus dead cells have high stability compared to live cells. In particular, it has excellent heat resistance and high stability against the external environment, so it is easier to store than existing lactic acid bacteria products and has the advantage of extending the shelf life. Therefore, the dead cell product can be applied as a cosmetic raw material in addition to the fields where the existing live lactic acid bacteria such as health functional foods, food additives, pharmaceuticals, and animal feed have been applied.

In the present invention, “antioxidation” refers to an action of inhibiting oxidation, and the human body has a balance between an oxidation promoting substance and an oxidation inhibitor, but due to various factors, this balance is lost and the direction of promoting oxidation If it is tilted toward, oxidative stress is induced in the living body, leading to cell damage and pathological diseases. Reactive oxygen species (ROS), which are the direct cause of oxidative stress, are chemically unstable and highly reactive, so they can easily react with various biomaterials such as DNA, proteins, lipids, and carbohydrates. Attacks cause irreversible damage to cells and tissues, or cause mutations, cytotoxicity, and cancer, and may be a direct cause of aging. By removing or reducing these reactive oxygen species, it is possible to prevent aging and maintain health by obtaining an antioxidant effect.

Meanwhile, in the present invention, microorganisms having excellent antioxidant and skin moisturizing effects were selected by separating lactic acid bacteria from kimchi in Jeju Island, and as a result of identification, Lactobacillus paraplantarum AMI-1101, Lactobacillus plantarum AMI-1101 Room AMI-1103 ( Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 ( Lactobacillus brevis AMI-1109).

Thus, the Lactobacillus genus strain was used to prepare dead cells, and it was found that the antioxidant effect can be remarkably improved in the process of nanosizing the size of the dead cells using a microfludizer. To provide the genus nano dead cells.

Specifically, the Lactobacillus strains of the genus Lactobacillus paraplantarum AMI-1101 (Lactobacillus paraplantarum AMI-1101), Lactobacillus plantarum AMI-1103 (Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 ( Lactobacillus brevis AMI-1109).

Specifically, the dead cells are Lactobacillus paraplantarum AMI-1101 (Lactobacillus paraplantarum AMI-1101), Lactobacillus plantarum AMI-1103 (Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 ( Lactobacillus brevis AMI). -1109) may be a nano-dead cell of the genus Lactobacillus including any one or more selected from the group consisting of.

More specifically, the dead cells are Lactobacillus paraplantarum AMI-1101 (Lactobacillus paraplantarum AMI-1101), Lactobacillus plantarum AMI-1103 (Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 ( Lactobacillus brevis AMI-1109).

"Lactobacillus paraplantarum AMI-1101 (accession number KCCM12383P)" of the present invention is a species of lactic acid bacteria of Lactobacillus paraplantarum isolated and identified from kimchi in Jeju Island. For the Lactobacillus paraplantarum AMI-1101 strain, reference may be made to Korean Patent Registration No. 10-1961149.

"Lactobacillus plantarum AMI-1103 (KCCM12384P)" of the present invention is a novel lactic acid bacteria of Lactobacillus plantarum isolated and identified from kimchi in Jeju Island.

"Lactobacillus brevis AMI-1109 (KCCM12385P)" of the present invention is a novel strain of Lactobacillus brevis isolated and identified from kimchi in Jeju Island. For the Lactobacillus brevis AMI-1109 strain, reference may be made to Korean Patent No. 10-1968242.

The nano dead cells of the Lactobacillus genus may be nano-ized at a specific pressure using a microfluidizer. Specifically, the pressure may be 10,000 psi to 20,000 psi.

In addition, specifically, the size of the nano dead cells may be 700 nm or more and 1,000 nm or less. More specifically, it may be 750 nm or more and 990 nm or less. More specifically, it may be 790 nm or more and 990 nm or less.

As a result of measuring the size of the nano dead cells in an embodiment of the present invention, it was confirmed that the size of the normal dead cells exceeded 1,000 nm, while the size of the complex dead cells in Lactobacillus genus was 1,000 nm or less (Table 1). .

In one embodiment of the present invention, as a result of confirming the antioxidant effect by measuring the free radical scavenging activity, it was confirmed that the antioxidant effect of the nano dead cells was remarkably improved compared to that of ordinary dead cells (FIG. 3).

The antioxidant composition may further include a skin moisturizing application.

Specifically, the composition may increase the expression levels of hyaluronic acid and filaggrin.

In one embodiment of the present invention, as a result of measuring the expression levels of hyaluronic acid and filaggrin by inoculating nano dead cells, it was confirmed that the expression of the moisturizing factor was promoted, and in particular, the increase in the expression of hyaluronic acid was superior compared to the normal dead cells. (Fig. 4 to Fig. 5).

Another aspect of the present invention relates to a cosmetic composition or a food composition comprising the antioxidant composition.

The cosmetic composition may be used for antioxidant and/or skin moisturizing.

In one embodiment of the present invention, it was confirmed that there is no cytotoxicity in the skin keratinocytes (HaCaT cells) of the nano dead cells of the Lactobacillus genus (FIG. 3), and the nano dead cells are used as cosmetics for antioxidant and/or skin moisturizing. It was confirmed that it can be used as a composition or as an external application for skin.

The cosmetic composition of the present invention is a solution, external ointment, cream, foam, nutritional lotion, flexible lotion, pack, softening water, emulsion, makeup base, essence, soap, liquid detergent, bathing agent, sunscreen cream, sun oil, suspension, emulsion It can be prepared in a formulation selected from the group consisting of liquid, paste, gel, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, patch, and spray, but is limited thereto. no.

The cosmetic composition of the present invention may additionally contain one or more cosmetically acceptable carriers blended in general skin cosmetics, and as common ingredients, for example, oil, water, surfactants, moisturizers, lower alcohols, thickeners , A chelating agent, a colorant, a preservative, a fragrance, and the like may be appropriately blended, but are 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, tracant, 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 spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component. It may include a propellant such as locarbon, propane/butane or dimethyl ether, 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 solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, Propylene glycol, 1,3-butyl glycol oil, and the like may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of 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, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used, but is not limited thereto. These may be used alone or in combination of two or more.

Specifically, in the cosmetic composition, the dead cells, cultures thereof, lysates or extracts thereof may be included in an amount of 0.005% to 30.0% by weight based on the total weight of the cosmetic composition. More specifically, it may be included in an amount of 0.01% to 20% by weight. There is an advantage of showing an excellent skin whitening effect within the above range, and there is an advantage of stabilizing the formulation of the composition.

In addition, the composition of the present invention may be used as a transdermal administration method such as direct application or spraying on the skin, and the administration route of the composition of the present invention may be administered through any general route as long as it can reach the target tissue.

The amount of use of the composition of the present invention may be appropriately adjusted according to individual differences such as age and degree of lesions or formulation, and may be used for 1 week to several months by applying the appropriate ?韜? to the skin once to several times a day.

The food composition may be used for antioxidant and/or skin moisturizing.

The food composition includes all uses for humans and companion animals. For example, companion animals include dogs, cats, hamsters, rabbits, chickens, iguanas, guinea pigs, gerbils, snails, raccoons, parrots, lizards, fish including tropical fish, etc. Animals of any kind are included.

Foods to which the Lactobacillus genus nano dead cells of the present invention can be added include meat, bread, sausage, chocolate, snacks, candy, confectionery, ramen, pizza, other noodles, gums, dairy products including ice cream, various soups, There are beverages, teas, vitamin complexes, and health supplements.

The type of food may specifically be a health functional food. The health functional foods include various nutrients, vitamins, minerals (electrolytes), synthetic flavors and flavoring agents such as natural flavors, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, organic acids, protective colloidal growth. The health functional food, which will contain an agent, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, and a carbonation agent used in carbonated beverages, is a food that emphasizes the biological control function of food, and uses physical, biochemical, and bioengineering methods. It is a food that has added value to act and manifest itself for a specific purpose. The ingredients of these health functional foods are designed and processed to sufficiently exert the body's control functions related to the body defense, regulation of body rhythm, prevention and recovery of diseases to the living body, and food supplementary additives, sweeteners, or functional foods that are acceptable as foods. It may contain raw materials.

The health functional food may be any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills, but is not limited thereto. Specifically, for the health functional food in the form of a tablet, a mixture of lava seawater, mineral water, excipients, binders, disintegrants and other additives is granulated in a conventional manner, and then compression molded by putting a lubricant or the like, or directly compression molding the mixture. It can be manufactured by doing. In addition, the health functional food in the form of a tablet may contain a bitter agent or the like if necessary, and may be coated with a suitable skin-forming agent if necessary.

Among the capsule-type health functional foods, the hard capsule may be prepared by filling a conventional hard capsule with a mixture of lava seawater, mineral water and additives such as excipients, or granular or skinned granules thereof. Soft capsules can be prepared by filling a mixture of lava seawater mineral water and additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The ring-shaped health functional food may be prepared by molding a mixture of lava seawater, mineral water, excipients, binders, disintegrants, etc. by appropriate methods. You can also create a ring with a suitable substance.

The granular health functional food may be prepared in a granular form of a mixture of lava seawater, mineral water, excipients, binders, disintegrants, etc., and may contain flavoring agents, bittering agents, and the like, if necessary.

The definition of terms for the excipients, binders, disintegrants, lubricants, bitters, flavoring agents, and the like are known in the art, and may include the same or similar functions such as the function thereof.

In addition, the type of food may be a food additive, and the food additive refers to a substance used by a method such as adding, mixing, infiltrating or adding to food for the preparation, processing, or preservation of food. The food additives include natural products and synthetic products, and can be classified according to functions and uses. Currently, 370 kinds of chemical synthetic products and 50 kinds of natural additives are approved as food additives in Korea. Depending on the purpose, preservatives, fungicides, antioxidants, coloring agents, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, reinforcing agents, and improving agents , Emulsifiers, thickeners and stabilizers, coating agents, gum base agents, defoaming agents, solvents, release agents, insect repellents, quality improvers and other additives for food manufacturing.

The form of the food additive may include a powder, granule, tablet, capsule or liquid form, and specifically may be a capsule form, but is not limited to the form.

Another aspect of the present invention is the step of killing the fermented cells in the a) Lactobacillus genus; And b) It relates to a method for producing nano-dead cells of the genus Lactobacillus comprising the step of nano-ization to 10,000 to 20,000 psi with a microfluidizer.

The nano dead cells were obtained by inoculating a complex strain of the genus Lactobacillus in green barley to obtain a fermented strain, followed by heat sterilization to prepare dead cells. It can be prepared by pulverizing the dead cells at a pressure of 10,000 psi to 20,000 psi using a microfluidizer.

Specifically, step a) may be a step of killing the fermented cells in Lactobacillus by heating them at 100 to 160° C. for 10 to 60 minutes.

In an embodiment of the present invention, it was confirmed that the antioxidant effect of the complex cells of the Lactobacillus genus can be remarkably improved by preparing nano dead cells by the microfluidizer method (FIG. 2).

In addition, in an embodiment of the present invention, when the microfluidizer is controlled at a pressure of 10,000 psi or 20,000 psi to make the dead cells nano, the free radical scavenging ability and the ability to promote the expression of skin moisturizing factors are remarkably improved. It shows the possibility of application of various uses of the method for manufacturing nano dead cells using the method.

The nano dead cells of the Lactobacillus genus of the present invention are excellent in antioxidant and skin moisturizing effects.

In addition, the nano-dead cells are not toxic and do not cause side effects even when applied to the human body, and thus the use of the nano-dead cells is high.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 shows the result of confirming the size of the nano-dead cells in Lactobacillus genus.
Figure 2 shows the results of confirming the antioxidant efficacy of Lactobacillus genus nano dead cells.
Figure 3 shows the results of confirming the cytotoxicity of the nano-dead cells in Lactobacillus genus.
Figure 4 shows the results of measuring the amount of hyaluronic acid produced by nano-dead cells of the genus Lactobacillus.
Figure 5 shows the results of measuring the amount of pillar green production of nano dead cells in Lactobacillus genus.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, but it is obvious that the present invention is not limited by the following examples.

Example 1. Preparation of complex nano dead cells in Lactobacillus genus

1-1. Production of dead cells of Lactobacillus sp. using fermented green barley

The green barley outpost washed with purified water and dried was coarsely pulverized and extracted with high pressure hot water at 121°C for 15 minutes with 20 times the extraction solvent (purified water). Inoculum for the fermentation of lactic acid bacteria Lactobacillus Planta room AMI-1101 cultured on sterilized lactic acid bacteria culture medium (Lactobacillus MRS Broth, deep nose, the United States of America) (Lactobacillus paraplantarum AMI-1101) , Lactobacillus Planta room AMI-1103 (Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 were used, and after the culture was completed, only the cells were recovered and inoculated into the green barley extract at a concentration of 1 ^{×10 7 cfu/ml.} Fermentation was carried out while maintaining at 25 to 35 °C for 1 to 3 days using a fermentation tank. The fermented green barley fermented product was fermented using 0.2 μm filter paper to separate the cells remaining on the filter. After the cells left on the filter surface were washed with purified water and recovered, only the precipitated cells were recovered using a centrifugal separator. The fermented cells were killed by heating at 100 to 160° C. for 10 to 60 minutes, then sterilized and lyophilized to powder.

1-2. Nanoization of complex dead cells in Lactobacillus genus

Samples prepared by mixing freeze-dried dead cell powder in 70% EtOH at 0.01% were collected after homogenization by repeating 3 times each at pressures of 10,000, 15,000, and 20,000 psi using a microfludizer. The recovered nano dead cells were concentrated in vacuum at a low temperature of 40 °C, and then lyophilized to powder.

Comparative Example 1. Preparation of general Lactobacillus genus complex dead cells

The green barley outpost washed with purified water and dried was coarsely pulverized and extracted with high pressure hot water at 121°C for 15 minutes with 20 times the extraction solvent (purified water). The seeds for fermentation are lactic acid bacteria Lactobacillus plantarum AMI-1101 (Lactobacillus paraplantarum AMI-1101), Lactobacillus plantarum AMI-1103 (Lactobacillus plantarum AMI-1103) grown in sterilized lactic acid bacteria culture medium (Lactobacillus MRS Broth, Difco, United States of America) plantarum AMI-1103) and Lactobacillus brevis AMI-1109 were used, and after the culture was completed, only the cells were recovered and inoculated into the green barley extract at a concentration of 1 ^{×10 7 cfu/ml.} Fermentation was carried out while maintaining at 25 to 35 °C for 1 to 3 days using a fermentation tank. The fermented green barley fermented product was fermented using 0.2 μm filter paper to separate the cells remaining on the filter. After the cells left on the filter surface were washed with purified water and recovered, only the precipitated cells were recovered using a centrifugal separator, mixed again by 1/10 of the extract supernatant removed with purified water, sterilized and lyophilized to powder.

Experimental Example 1. Confirmation of the size of nano dead cells in Lactobacillus genus

The dead cells of Examples and Comparative Examples were observed with a scanning electron microscope (SEM).

The size of each dead cell is shown in Table 1 below.

division Common dead cells
(Comparative Example 1) Nano dead cells
(10,000 psi) Nano dead cells
(15,000 psi) Nano dead cells
(20,000 psi) size 1455.9 nm 971.9 nm 985.3 nm 799.0 nm

As shown in Table 1, the size of the dead cells were all 10,000 nm or less, which was smaller than that of the normal dead cells, and specifically, the size of the dead cells was 971.9 nm at a pressure of 10,000 psi, and at a pressure of 15,000 psi. It was confirmed that it was 985.3 nm, and that it was 799.0 nm at 20,000 psi (FIG. 1).

Experimental Example 2. Confirmation of cytotoxicity of complex nano dead cells in Lactobacillus genus

HaCaT cells, which are skin keratinocytes, are distributed from Cell Line Service GmbH.(Germany) and contain 100 units/ml penicillin-streptomycin and Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS). It was cultured in a 37°C, 5% CO ₂ incubator using a medium, and subculture was performed at intervals of 3 to 4 days.

To confirm the skin safety of the examples, cytotoxicity evaluation was performed using the EZ-cytox assay method. EZ-cytox assay is a representative method of measuring cell viability using the principle of producing orange water-soluble formazan by reacting with dehydrogenase of living cells.

Specifically, in order to confirm the toxicity in skin keratinocytes, HaCaT cells were dispensed into a 24-well plate at ^{1×10 5} _{cells/mL using DMEM medium added with 10% FBS, and 37°C, 5% CO 2} At 24 hours, the cells were attached, and the cells were exchanged with serum-free DMEM, and the nano-dead cells of Example 1 were treated for 24 hours. EZ-Cytox was treated with the cell culture solution to react, and absorbance was measured at 450 nm using a microplate reader. The average absorbance value for each sample group was obtained, and the cell viability was evaluated by comparing it with the absorbance value of the control group. The results are shown in Table 2.

Cytotoxicity (%) Concentration (μg/ml) 2.5 5 10 20 Nano dead cells
(10,000 psi) 93.94 91.81 98.32 98.95 Nano dead cells (15,000 psi) 95.08 96.68 93.24 104.36 Nano dead cells (20,000 psi) 90.54 100.14 93.11 98.25

As shown in Table 2, the nano dead cells showed excellent cell viability regardless of the concentration. These results suggest that not only the nano-dead cells are harmless to the human body, but also excellent human safety (FIG. 2).

Experimental Example 3. Confirmation of antioxidant effect of complex nano dead cells in Lactobacillus genus

ABTS ⁺ [2,2'- ^{Azino-Beads (3-ethyl benzothiazoline-6-sulfonic acid (ABTS +} [2,2'-Azino-bis(3-ethyl benzothiazoline-6-sulfonic acid))]) radical scavenging activity Is capable of measuring both hydrogen-donating antioxidants and chainbreaking antioxidants, and is a method applicable to both the aqueous and organic phases.

The 7 mM ABTS solution was mixed so that potassium persulfate became 2.45 mM, reacted in the dark for about 24 hours, and the ABTS ⁺ solution was mixed with the sample and reacted for 6 minutes in the dark, and the absorbance was measured at 734 nm. The resulting value was expressed as a percentage (%) of radical scavenging activity through calculation using the following equation 1 by comparing the extract sample-treated group and the untreated group, and BHT was used as a positive control group.

[Equation 1]

ABTS scavenging activity (%) = [1-(absorbance of sample treatment group in Example or Comparative Example / absorbance of sample untreated group)] X 100

As a result, the antioxidant efficacy of Lactobacillus nano dead cells nano-ized with a microfluidizer was remarkably improved compared to general nano dead cells, and a microfluidizer was used to nanonize dead cells in Lactobacillus with a size of 10,000 psi or 20,000 psi. When it was confirmed that the antioxidant effect was particularly excellent (Fig. 3).

Experimental Example 4. Confirmation of moisturizing effect of complex nano dead cells in Lactobacillus genus

4-1. Confirmation of the effect of increasing the amount of hyaluronic acid produced

In order to confirm the moisturizing effect of Examples and Comparative Examples, an experiment was performed to confirm the effect of increasing the amount of hyaluronic acid produced.

Specifically, HaCaT cells were dispensed into a 24-well plate at 1.0 × 10 ⁵ cells/mL and cultured for 18 hours at 37° C. and 5% CO _2. It was exchanged for serum-free DMEM, treated with Example 1 and Comparative Example 1, and cultured for 24 hours. Thereafter, the culture medium was removed, centrifuged at 15,000 rpm for 5 minutes, and the supernatant was removed and stored frozen (-20°C) until quantification. The enzyme immunoassay (ELISA: Enzyme-Linked Immunosorbent Assay) was performed using a hyaluronic acid ELISA kit (Elabscience Biotechnology Co., Ltd.) and was carried out by the method provided by the manufacturer. The results are shown in Table 3.

Hyaluronic acid production increase (%) - Concentration (μg/ml) % - - 100 Common dead cells
(Comparative Example 1) 2.5 108.67 5 117.99 10 124.13 20 132.04 Nano dead cells
(10,000 psi) 2.5 122.92 5 124.60 10 147.07 20 147.87 Nano dead cells
(15,000 psi) 2.5 112.19 5 118.44 10 128.44 20 128.58 Nano dead cells
(20,000 psi) 2.5 127.41 5 122.22 10 146.59 20 153.08 Retinoic acid 10 μM 130.06

As shown in Table 3, it was confirmed that the amount of hyaluronic acid produced was remarkably increased compared to the normal dead cells when the Lactobacillus genus complex nano dead cells were treated (FIG. 4).

4-2. Confirmation of the effect of increasing the production of Filaggrin

HaCaT cells were dispensed into a 24-well plate at 1.0 × 10 ⁵ cells/mL, and cultured for 18 hours at 37° C. and 5% CO _2. It was exchanged for serum-free DMEM, and the sample was treated and incubated for 24 hours. After removing the culture medium for each group and washing with PBS, PBS containing no drug that may affect protein quantification was treated. After the cells were lysed by repeating incubation at low temperature and room temperature, proteins were collected. Quantification was performed using a Filaggrin-ELISA kit (Elabscience Biotechnology Co., Ltd.) and was performed by a method provided by the manufacturer. The results are shown in Table 4.

Pillar green (FLG) production increase (%) - Concentration (μg/ml) % - - 100 Common dead cells
(Comparative Example 1) 2.5 112.17 5 105.40 10 138.70 20 160.52 Nano dead cells
(10,000 psi) 2.5 114.86 5 102.28 10 137.46 20 160.66 Nano dead cells
(15,000 psi) 2.5 86.99 5 94.11 10 152.52 20 169.49 Nano dead cells
(20,000 psi) 2.5 109.63 5 114.18 10 114.47 20 140.76 Retinoic acid 10 μM 122.89

As shown in Table 4, it was confirmed that the Lactobacillus complex nano-dead cells promote the production of filaggrin, and the amount of filaggrin production increases depending on the concentration of the dead cells (FIG. 5).

The above results show that the complex nano dead cells of the Lactobacillus genus of the present invention generally promote the generation of skin moisturizing factors, and in particular, have excellent effects of promoting the production of hyaluronic acid and filaggrin, and thus can be used in various ways for skin moisturizing Show that there is.

Preparation Example 1. Preparation of essence containing Lactobacillus complex nano dead cells

As shown in Table 5 below, a cosmetic composition of an essence formulation was prepared (unit: wt%).

Raw material name Content (% by weight) Cetearyl alcohol 2 Cetearyl glucoside One Jojoba seed oil 5 Silica 0.5 glycerin 10 Complex nano dead cells of the genus Lactobacillus (Example) 0.01 Purified water to 100

Preparation Example 2. Preparation of a cream containing complex nano dead cells in Lactobacillus genus

As shown in Table 6 below, a cream-formed cosmetic composition was prepared (unit: wt%).

Composition Content (% by weight) Lactobacillus complex nano dead cells
(Example) 0.01 Stearic acid 15 ethanol One Potassium hydroxide 0.7 glycerin 5 Propylene glycol 3 antiseptic Appropriate amount incense Appropriate amount Purified water to 100

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the technical field to which the present invention pertains will be able to understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and the concept of equivalents thereof should be construed as being included in the scope of the present invention.

Name of Depository Organization: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12383P

Consignment Date: 20181112

Name of deposit institution: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12384P

Consignment Date: 20181112

Name of Depository Organization: Korea Microorganism Conservation Center (overseas)

Accession number: KCCM12385P

Consignment Date: 20181112

Claims (10)

Contains dead cells of the genus nanonized Lactobacillus,
The dead cells of the genus Lactobacillus are Lactobacillus paraplantarum AMI-1101 (Lactobacillus paraplantarum AMI-1101), Lactobacillus plantarum AMI-1103 (Lactobacillus plantarum AMI-1103) and Lactobacillus brevis AMI-1109 ( Lactobacillus brevis AMI). -1109) complex fermentation dead cells, antioxidant and skin moisturizing composition. delete The method of claim 1,
The size of the dead cells is 700 nm or more and 1000 nm or less, antioxidant and skin moisturizing composition. The method of claim 1,
The composition is to increase the expression level of hyaluronic acid and filaggrin, antioxidant and skin moisturizing composition. A cosmetic composition comprising the composition of claim 1. A food composition comprising the composition of claim 1. The method of claim 6,
The food composition is for companion animals, food composition. a) killing the fermented bacteria in Lactobacillus genus; And
b) nanofluidizing with a microfludizer to 10,000 to 20,000 psi,
The Lactobacillus bacteria in the fermentation cells are Lactobacillus para Planta room AMI-1101 (Lactobacillus paraplantarum AMI- 1101), Lactobacillus Planta room AMI-1103 (Lactobacillus plantarum AMI- 1103) and Lactobacillus brevis AMI-1109 (Lactobacillus brevis AMI -1109) of the complex fermentation bacteria, a method for producing nano-ized Lactobacillus genus dead cells. delete The method of claim 8,
The step a) is a step of killing the fermented cells of the genus Lactobacillus by heating them at 100 to 160° C. for 10 to 60 minutes.

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