KR101915463B1 - Novel lactobacillus pentosus, and probiotics and composition containing the same - Google Patents

Abstract

The present invention relates to a novel Lactobacillus pentosus HB-8023 strain, and a culture, a probiotic preparation, a pharmaceutical composition, a health functional food composition, and a cosmetic composition including the same. The novel Lactobacillus pentosus HB-8023 strain separated and identified from Jeju tangerine kimchi has excellent acid resistance, biliary resistance and lactic acid production ability, and thereby can be used as a probiotic preparation. Also, the novel Lactobacillus pentosus HB-8023 strain has the antimicrobial activity against various pathogenic bacteria and the expression inhibitory ability for inflammatory cytokines, and thus can be used as an active ingredient of the pharmaceutical composition and food composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel Lactobacillus pentosus strain, and a probiotic preparation or composition comprising the Lactobacillus pentosus strain.

The present invention relates to a novel Lactobacillus And a cultured product, a probiotic preparation, a pharmaceutical composition, a health functional food composition, and a cosmetic composition comprising the same.

 Kimchi, a traditional Korean fermented food made from various vegetables, is known to have various functions such as antioxidant, anti-aging, anti-obesity, antimutagenic, anti-inflammatory, antibacterial, antiallergic and immunity enhancing effect. These beneficial effects are derived from the fermentation products of lactic acid bacteria and nutrients in kimchi. Since Kimchi contains bacteria such as Lactobacillus, Leuconostoc and Pediococcus, it is involved in the fermentation and aging of kimchi. Therefore, researches using lactic acid bacteria isolated from kimchi, and probiotics and health Food and the like. Probiotics (probiotics) is a living microorganism that enters the body and gives immunity and various functions to help health. Most probiotics known to date are lactic acid bacteria and have long been used in a variety of fermented foods such as meat, vegetables, milk, and coffee. To be recognized as a probiotic, the strain must survive in the body's stomach and bile acid to reach the small intestine and attach to the epithelial cells to grow. Also, it should be non-toxic and non-pathogenic, and it is preferable to exhibit beneficial effects such as antibacterial, anti-inflammatory, anti-cancer, antioxidant, anti-diabetic and anti-obesity in the intestinal tract.

It has been known to date that lactic acid bacteria isolated from fermented foods such as soybean paste or kimchi can be used as probiotics and can be applied as pharmaceutical therapeutic applications through their various physiological functions. For example, Korean Patent No. 10-161653 discloses a probiotic composition comprising a novel Lactococcus lactis KR-WW-2 strain isolated from flounder, Korean Patent Laid-Open No. 10-2015-0106093 discloses that Bacillus methylotrophicus C14 strain isolated from soybean can be used as a antibacterial composition by using as a probiotic preparation. In addition, studies have been continuing to use various lactic acid bacteria as useful applications.

In this study, useful lactic acid bacteria were isolated and identified from Jeju citrus kimchi made with citrus as a special product of Jeju Island and its characteristics were investigated. Specifically, the acid resistance and biliary cholestasis of the isolated strains were measured and evaluated for their beneficial effects in a multifaceted manner when they were used in probiotic preparations and various compositions.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The first aspect of the present invention relates to a novel lactobacillus < RTI ID = 0.0 > A strain of Lactobacillus pentosus can be provided.

A second aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > Comprising a culture medium containing a microorganism belonging to the genus Lactobacillus , To provide a pentosus strain culture.

A third aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > The pentosus strain is cultivated in a medium containing glucose 0.2 to 10% (w / v), beef extract 0.01 to 10% (w / v), yeast extract 0.01 to 10% (w / v), magnesium sulfate 0.004 to 0.4% ), Manganese sulfate 0.004 to 0.4% (w / v), and plant pulverized material 1 to 30% (w / v).

A fourth aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > Pento suspension the strain, or a culture, concentrate or dry product may provide a probiotic preparation containing as an active ingredient.

A fifth aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > A pharmaceutical composition for antimicrobial and anti-inflammation, which comprises as an active ingredient a pentosus strain, or a culture, concentrate or dried product thereof, can be provided.

A sixth aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > There can be provided a health functional food composition for antimicrobial and anti-inflammatory use containing as an active ingredient a pentosus strain, or a culture, concentrate or dried product thereof.

A seventh aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > Pento suspension the strain, or a culture, concentrate or dry product may provide a cosmetic composition containing, as an active ingredient.

The above-described task solution is merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary implementations described above, there may be additional implementations and embodiments described in the drawings and detailed description of the invention.

The present invention relates to novel Lactobacillus strains isolated from Jeju citrus kimchi A strain of Pentosus HB-8023 can be provided. The strains have excellent acid resistance, bile resistance and lactic acid production ability and can be used as probiotic preparations. In addition, the strains have antimicrobial activity against various pathogenic bacteria and inhibit the expression of inflammatory cytokines, and thus can be used as an active ingredient of pharmaceutical composition and food composition And has an antioxidant effect, a whitening effect and an exfoliation improving effect, a skin moisturizing effect and soothing effect, and therefore can be used as an effective ingredient of a cosmetic composition.

FIG. 1 is a graph showing the results of an experiment in which a lactobacillus strain derived from Jeju citrus kimchi was cultured according to an embodiment of the present invention and its organic acid production ability was confirmed.
2 is a graph showing the growth curves of Lactobacillus strains derived from Jeju citrus kimchi according to one embodiment of the present invention.
FIG. 3 is a flow diagram of genetically classifying Lactobacillus strains derived from Jeju citrus kimchi according to one embodiment of the present invention.
FIG. 4 is a graph showing the activity of Lactobacillus < RTI ID = 0.0 > This is the result of the protease secretion experiment of Pentosus HB-8023 strain.
FIG. 5 is a graph showing the activity of Lactobacillus < RTI ID = 0.0 > This is the result of lipase secretion experiment of Pentosus HB-8023 strain.
FIG. 6 is a graph illustrating the activity of Lactobacillus < RTI ID = 0.0 > The result of measurement of lactic acid secretion amount according to mixed culture of natural product of Pentosus HB-8023 strain.
FIG. 7 is a graph showing the activity of Lactobacillus < RTI ID = 0.0 > This is the cytotoxicity test result of Pentosus HB-8023 strain.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly explain the present invention in the drawings, portions not related to the description are omitted.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The terms "about "," substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) "or" step "used to the extent that it is used throughout the specification does not mean" step for.

Throughout this specification, the term "combination thereof" included in the expression of the machine form means one or more combinations or combinations selected from the group consisting of the constituents described in the expression of the machine form, And the like.

Throughout this specification, the description of "A and / or B" means "A, B, or A and B".

Throughout this specification, " prevention " means any action that inhibits or delays disease by administration of a composition according to the invention.

Throughout this specification, "improvement" means any action that at least reduces the degree of symptom associated with the condition being treated.

Throughout this specification, " treatment " means any action that improves or alters the condition of the disease by administration of the composition according to the invention.

Throughout the present specification, " comprising as an active ingredient " means that the lactobacilli Quot; is meant to include an amount sufficient to achieve efficacy or activity of a Pentosus strain. The Lactobacillus < RTI ID = 0.0 > Since the pentosus strain is a microorganism isolated from Jeju citrus kimchi which is food, there is no significant side effect on the human body even when administered in an excessive amount. Therefore, the quantitative upper limit of the strain contained in the composition of the present invention can be selected by a person skilled in the art have.

Hereinafter, the Lactobacillus The pentosus strain, the culture including the same, the probiotic preparation and the composition will be specifically described with reference to the embodiments, examples and drawings. However, the present invention is not limited to these embodiments and examples and drawings.

The first aspect of the present invention relates to a method for producing lactobacillus Lt; / RTI > strain.

The Jeju citrus kimchi is prepared by adding raw materials of Jeju citrus fruit such as Jeju citrus fruit, cut pieces, crushed water or extract, etc. in the production of kimchi, and has a characteristic of citrus flavor in kimchi.

According to one embodiment of the invention, the strain may be, but is not limited to, deposited with accession number KCTC 13322BP.

According to one embodiment of the present invention, the strain has acid resistance and biliary cholesterol, so that it may be suitable for use as an active ingredient of various compositions including probiotic agents.

For example, the acid tolerance may be a survival rate of about 70% or more even after culturing for a certain period of time in a medium having a pH of 2 to 3, and the biliary properties may show a survival rate of about 50% or more in an environment similar to human bile . When the Lactobacillus pentosus strain of the present invention is used as a probiotic preparation, the effect can be lowered or promoted by maintaining the viability of the strain when administered orally in the body, so that the acid tolerance and bile salt It is possible to show a significant effect in having the property.

According to one embodiment of the present invention, the strain may secrete at least one selected from lactic acid, lipase, and protease, but may not be limited thereto.

Lactobacillus cells secrete various enzymes such as protease, amylase, cellulase, and lipase during the fermentation process. As a result, secondary products such as amino acids, free sugars, isoflavones, aglycons and fatty acids are produced during fermentation, It is known to provide functionality. A double protease is a generic term for enzymes that hydrolyze peptide bonds of proteins and various proteolytic degradation products to produce small peptides and amino acids as final products. This enzyme plays a role in the digestion of food in the body, activation of enzyme precursor, conversion of proteins other than enzymes into functional form, release of physiologically active peptides, decomposition of unwanted proteins, and in cosmetics, EWG . In addition, lipase has a characteristic of decomposing triglycerides, which can decompose neutral fat in the blood to prevent adult diseases related to blood vessel diseases and prevent obesity.

A second aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > Comprising a culture medium containing a microorganism belonging to the genus Lactobacillus , Lt; RTI ID = 0.0 > Pentosus < / RTI >

According to one embodiment of the present invention, the plant pulverized material may be selected from fruit pulverized material, pulverized plant material and vegetable pulverized material, but the present invention is not limited thereto. The fruits, medicines, and vegetables may be selected from a variety of fruits, medicines, and vegetables known in the art without particular limitation, and the pulverized product may be produced by using the pulverizing method generally performed in the art. The pulverized product may contain both the solid mulch and the juice of the plant obtained by pulverizing the plant. For example, the fruit may be selected from the group consisting of lemon, pomegranate, banana, apple, grape, plum, mango, kiwi, cranberry, blueberry, apricot, fig, persimmon, jujube, pineapple, papaya, And may be selected from the group consisting of watermelon, melon, persimmon, horse mackerel, cherry, oak, jujube, plum, and combinations thereof. The medicament may be selected from the group consisting of green tea, burdock, Digs. And subtract. From the group consisting of Rhizoctonia sp., Aristocratis spp., Cedarwood, Prunus persimilis, Pinus bean, Chrysanthemum, Leaf, Taran, Taran leaf, Mint, Cattle, Lemon Myrtle, Mulberry leaf, The vegetables may be selected from the group consisting of avocados, zucchini, carrots, tomatoes, zucchini, onions, garlic, turmeric, beets, potatoes, peppers, spinach, corn, artichoke, eggplant, cucumbers, radishes, yams, cauliflowers, Broccoli, red cabbage, white cabbage, peas, beans, fennel, ginger, cola bean, parsnip, rhubarb, sprouts cabbage, celery, cabbage, modern chicory, kale, lettuce, mushroom, pepper, olive and combinations thereof But are not limited thereto.

According to one embodiment of the invention, the culture medium comprises about 0.2-10% w / v glucose, about 0.01-10% w / v beef extract, about 0.01-10% w / v yeast extract, But are not limited to, about 0.004 to 0.4% (w / v) magnesium sulfate, about 0.004 to 0.4% (w / v) manganese sulfate, and about 1 to 30% (w / v) And may further include, in addition to the above components, additional nutrients, growth factors, trace elements or residual water used for culturing Lactobacillus. For example, the plant pulverized material may be present in the culture medium in an amount of about 1 to 20% (w / v), about 1 to 10% (w / v), about 5 to 30% (w / v), about 10 to 20% (w / v), about 5 to 15% (w / v), or about 10% (w / v).

A third aspect of the present invention is a method for producing Lactobacillus < RTI ID = 0.0 > The pentosus strain is cultivated in a medium containing about 0.2-10% w / v glucose, about 0.01-10% w / v beef extract, about 0.01-10% w / v yeast extract, about 0.004-0.4% magnesium sulfate, (w / v), about 0.004 to 0.4% (w / v) manganese sulfate, and about 1 to 30% (w / v) of a plant pulverized product. When using media compositions as described above, Lactobacillus The amount of lactic acid produced from the pentosus strain is maximized to exhibit more excellent antibacterial activity.

A fourth aspect of the present invention is a method for producing Lactobacillus < RTI ID = 0.0 > Pento suspension directed to the strain, or a culture, concentrate or dry product the probiotic preparation containing as an active ingredient.

For example, the probiotic agent can be prepared according to a conventional method for producing a probiotic composition, and may generally be in the form of a culture suspension or a dry powder.

A fifth aspect of the invention is a pharmaceutical composition comprising lactobacillus < RTI ID = 0.0 > The present invention also relates to a pharmaceutical composition for antibacterial and anti-inflammation containing a pentosus strain or a culture, concentrate or dried product thereof as an active ingredient. The Lactobacillus < RTI ID = 0.0 > Since the pantothecus strains exhibit antibacterial activity against various pathogenic microorganisms, the pantothecia strains can exhibit therapeutic, preventive or ameliorative effects for diseases caused by pathogenic microorganisms without any particular limitation. The Lactobacillus < RTI ID = 0.0 > Since the pentose strain exhibits an inflammatory cytokine inhibitory effect, it can exhibit therapeutic, preventive or ameliorative effects on inflammation-related diseases.

According to one embodiment of the invention, the pharmaceutical composition is Staphylococcus genus (Staphylococcus spp.), Escherichia coli (Escherichia coli), Candida genus (Candida spp.), Pseudomonas species (Pseudomonas spp.), propionic sludge tumefaciens in (Propionibacterium spp.), dry aged Jia in (Malassezia spp.), Tricot piton in (Trichophyton spp.), and their But are not limited to, for preventing or treating infection of a microorganism selected from combinations of < RTI ID = 0.0 >

Alternatively, according to one embodiment of the present invention, the pharmaceutical composition may be one for preventing or treating an allergic disease by inhibiting the secretion of an inflammation-related cytokine. The allergic disease specifically includes food allergy, bronchial asthma, allergic Allergic conjunctivitis, allergic otitis media, urticaria and anaphylactic shock, contact hypersensitivity, allergic contact dermatitis, bacterial allergy, fungal allergy, viral allergy, drug allergy , Thyroid gland and allergic encephalitis, but the present invention is not limited thereto.

The probiotic preparation or the pharmaceutical composition may contain the lactobacillus Pento strain suspension, or select an effective amount alone or in combination of two or more possible pharmaceutically acceptable conventional carriers or of one or more additives to the culture medium thereof, to the composition may be prepared by conventional formulation.

For example, the carrier may be selected from one or more of diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives, and one or two kinds of additives such as flavorings, vitamins and antioxidants More than one species can be selected and used. Examples of the diluent include lactose monohydrate, trehalose, cornstarch, soybean oil, microcrystalline cellulose (microcrystalline cellulose), and the like. magnesium stearate or talc is preferable as a binder and polyvinyipyrolidone (PVP) or hydroxypropylcellulose (D-mannitol) is used as a binding agent. HPC: hydroxypropylcellulose), but the present invention is not limited thereto. The disintegrant may be selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium or cross-linked polyvinylpyrrolidone, The sweetener may be selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta-cyclodextrin) white bee's wax or xanthan gum. Preservatives include methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate, but may not be limited thereto.

Alternatively, the pharmaceutically acceptable carrier may include conventionally acceptable saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, or liposome. The pharmaceutical composition of the present invention may be formulated into injections, inhalants, external skin preparations and the like, though there is no particular limitation on the formulations.

The pharmaceutical composition of the present invention may be orally administered or parenterally administered (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method, but is preferably orally administered, Depends on the condition and the weight of the patient, the degree of the disease, the type of the drug, the administration route and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, " pharmaceutically effective amount " means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable for medical treatment or diagnosis, and the effective dose level will depend on the disease type, severity, Activity, sensitivity to the drug, time of administration, route and rate of excretion, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The effective amount of the pharmaceutical composition of the present invention may be varied depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, type of disease, 0.001 to 150 mg per kg, preferably 0.01 to 100 mg per day, or one to three times per day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of obesity, sex, weight, age and the like.

A sixth aspect of the present invention is a method for producing lactobacillus < RTI ID = 0.0 > The present invention relates to a health functional food composition for antimicrobial and anti-inflammatory use, which comprises as an active ingredient a pentosus strain, or a culture, concentrate or dried product thereof.

When the composition of the present invention is made from a health functional food composition, the composition may be in the form of tablets, pills, powders, granules, powders, capsules, and liquid formulations, including one or more of carriers, diluents, excipients, , But may not be limited thereto. Examples of foods that can be added to the composition of the present invention include various foods, powders, granules, tablets, capsules, syrups, drinks, gums, tea, vitamin complexes, and health functional foods. Examples of the additive that can be further included in the present invention include natural carbohydrates, flavors, nutrients, vitamins, minerals (electrolytes), flavors (synthetic flavors, natural flavors and the like), colorants, fillers, But are not limited to, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, antioxidants, glycerin, alcohols, carbonating agents and fats have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As the above-mentioned flavors, natural flavors (tautatin, stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (saccharine, aspartame, etc.) can be advantageously used. The composition according to the present invention can be used in various forms such as flavorings such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies, factic acid and its salts, alginic acid and its salts, , pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components may be used independently or in combination. Specific examples of the carrier, excipient, diluent, and additive include, but are not limited to, lactose, dextrose But are not limited to, sucrose, sorbitol, mannitol, erythritol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium phosphate, calcium silicate, microcrystalline cellulose, polyvinylquilolidone, cellulose, polyvinylpyrrolidone, methylcellulose, water , Sugar syrup, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil may be used, but the present invention is not limited thereto.

The health functional food composition according to the present invention can be prepared in various forms according to a conventional method known in the art. Common foods include but are not limited to beverages (including alcoholic beverages), fruits and processed foods (eg, canned fruits, jam, maamalade, etc.), fish, meat and processed foods (Eg butter, chewing), edible plant oil, margarine, soybean oil, etc., etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, plant proteins, retort foods, frozen foods, various seasonings can be prepared by adding Lactobacillus pento gyunjueul suspension of the present invention or the like (for example, soybean paste, soy sauce, sauce, etc.). In addition, nutritional supplement includes, but is not limited to, lactose bacillus of the present invention in capsules, tablets, Pento gyunjueul suspension can be produced by the addition. In addition, the health functional foods include, but are not limited to, for example, lactobacillus The pentosus strains themselves can be prepared in the form of tea, juice and drink and liquefied, granulated, encapsulated and powdered for drinking (health drinks). The Lactobacillus < RTI ID = 0.0 > In order to use the pentosus strain in the form of a food additive, it may be prepared in the form of powder or concentrate. The Lactobacillus < RTI ID = 0.0 > With pento suspension strains and, of active ingredient that may assist the desired effect known it can be prepared by mixing in the form of a composition.

A seventh aspect of the invention is a method for producing Lactobacillus < RTI ID = 0.0 > Pento relates to a suspension strain, or a culture, concentrate or dry product in the cosmetic composition containing, as an active ingredient. Lactobacillus according to the invention Since the Pantosus strain has antioxidative effect, exfoliation effect, whitening effect, and moisturizing effect, the cosmetic composition can exhibit excellent skin whitening and moisturizing effect, and has anti-bacterial effect against acne bacteria, .

The Lactobacillus < RTI ID = 0.0 > When the pantothecia strain is produced from a cosmetic composition, it is preferable that the lactobacillus May contain ingredients commonly used in cosmetic compositions other than pentoses , and may include, but are not limited to, conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art, and may be prepared, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, , Oil, powder foundation, emulsion foundation, wax foundation, and spray, but the present invention is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide Can be used. Lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as the carrier component when the formulation of the cosmetic composition of the present invention is a powder or a spray, Propellants such as hydrocarbons, propane / butane or dimethyl ether. When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , Propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters. When the formulation of the cosmetic composition of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

1. From Jeju Citrus Kimchi Lactobacillus  Isolation culture and identification

10 g of Jeju tangerine kimchi was collected and placed in sterilized water (100 ml) at 37 ℃ for 1 hour. The stationary sterilized water was diluted with saline, and 100 μl of each dilution step was dispensed into a lactobacillus MRS broth (difco) solid medium. Plate-size solid medium was cultured at 37 ° C for 2 days. The colonies produced after cultivation were each aliquoted and the cells were also subjected to a linear culture on a BCP plate count agar (EIKEN) solid medium to confirm the presence or absence of organic acid secretion. Since this medium contains Bromocresol Purple reagent, the pH was lowered by the organic acid secreted from the strain, and thus a yellow rim was formed around the colonies. As a result, it was confirmed that the strain secreted the organic acid (FIG. 1).

The isolated strains were cultured in the MRS liquid medium at 37 ° C. and were scanned for each hourly (0, 4, 7, 12, 24 hours (1 day), 36 hours and 48 hours (2 days) ) At 520 nm using a UV spectrophotometer, and the growth curve thereof is shown in Fig. As a result, the highest growth rate was observed at 1 day.

In addition, the strain was cultured in MRS broth for one day, and the culture was incubated in a solid medium, followed by homogenization. The PCR conditions were: 60 seconds at 94 ° C, 30 cycles at 94 ° C, 30 seconds at 55 ° C, 90 seconds at 72 ° C, 50 minutes at 72 ° C and 15 ° C (ever) 8 primers having the same primer sequence were used.

[Table 1]

16S ribosomal RNA sequence was aligned with ClustalW (San Diego Supercomputer Center, University of California, San Diego) by BLAST (National Center for Biotechnology Information, US National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA) Then, a schematic diagram of the MEGA 6 program is shown in FIG.

In the case of the strain isolated in the present embodiment is a genetically Lactobacillus Because it was similar to the Lactobacillus plantarum , the final classification was carried out through biochemical experiments (Table 2).

[Table 2]

As a result, it was found that the strains isolated in this Example were Lactobacillus When the glycerol and D- xylose as a carbon source, such as pento suspension (Lactobacillus pentosus) been tested and found to produce an organic acid, the Lactobacillus strains according to the present embodiment Bacillus Pentosus . Thus, this strain was transformed into Lactobacillus Pentosus HB-8023, and deposited with Accession No. KCTC 13322BP at the BioResource Center, Sep. 19, 2013.

2. From Jeju Citrus Kimchi Derived Lactobacillus  Medium composition for culture of strain

Lactobacillus In order to increase the content of lactic acid produced from Pentosus HB-8023, culture medium was regulated for 2 days and the content of lactic acid produced was confirmed by HPLC analysis.

를 이용하여 20 분간 등용매(isocratic)로 시료를 분석하였다. 표준(standard)으로는 L-(+)-락트산 (sigma)을 이용하였다.The HPLC instrument used was a Waters 2695 Alliance separation module (waters, Milford, MA) and a Photo Diode Array 2998 (Waters, Milford, Mass.). As the column, YMC ODS-A 150X 4.6 mm, 5 m, 12

The samples were analyzed with isocratic for 20 minutes. L - (+) - lactic acid (sigma) was used as the standard.

Experimental results, Lactobacillus bacteria in a culture medium composition containing glucose 2%, as shown in the following Table 3, 0.1% beef extract, 0.1% yeast extract, 0.04% magnesium sulfate and manganese sulfate, 0.04% Pento confirmed that the suspension of lactic acid producing HB-8023 is particularly maximized.

[Table 3]

3. From Jeju Citrus Kimchi Derived Lactobacillus  Characterization of strains

3-1. Antimicrobial activity

To determine the antimicrobial activity of Lactobacillus pentosus HB-8023, Staphylococcus aureus (ATCC 6538P), Pseudomonas aeroginosa (ATCC 9027), Escherichia coli (ATCC 8739) And fungi (Candida albicans, ATCC 10231) were used as pathogenic strains. The fungi were cultured in PDB and the other three strains were cultured in TSB at 32 ° C and 150 rpm in a water bath. Malassezia restricta, KCTC 27539, Trichophyton rubrum, KCTC 6375, and Propionibacterium acnes subsp. Acnes , KCTC 3314 were cultivated on a special medium, respectively.

To measure the antimicrobial activity, all pathogenic strains were plated on a medium of 10 ⁶ cfu / ml, and a paper disk method was used to measure the inhibitory rings by placing filter paper impregnated with the sample. 100 μl of each sample was added to each medium to which the pathogenic strain had been streaked, and the filter paper was placed thereon for 24 hours at 32 ° C. Specially, acne, dandruid and athlete 's disease were inhibited after incubation for 5 days. At this time, the sample used was Lactobacillus The culture of the pentosus HB-8023 strain itself was used as a filter, and the control was performed in a conventional MRS broth medium.

In the case of anthracnose, the plug is cut into a solid state, so that a plug of the same size is placed on a fungal athlete's culture medium, and the filter paper on which the sample is dropped is placed thereon for 5 days at 25 ° C Post - inhibition rings.

As a result of the experiment, it was found that lactobacillus cultured in a culture solution having increased lactic acid production according to Example 2, It was confirmed that the antibacterial activity of the pentosus HB-8023 strain was increased, and more than 50% of the antibacterial activity was confirmed in acne bacteria and dandruff (Table 4, unit of inhibition rate:%).

[Table 4]

3-2. Acid resistance  And My bile

Experiments were performed to determine whether the Lactobacillus pentosus HB-8023 isolate identified in this Example had acid resistance. The strains were incubated at 35 ~ 37 ℃ for 2 days and pH was adjusted to pH 2 using citric acid or HCl, respectively. The colonies of yellow color after culturing under the above conditions were measured by colonization of Lactobacillus pentosus HB-8023, and the results are shown in Table 5 below. Myocardial measurements were performed in MRS (Lactobacilli, Difco) liquid medium for 2 days at 37 ° C and strains were plated on MRS agar plates supplemented with oxgall (0, 0.1, 0.5, 1 and 5% And then cultured at 37 DEG C for 2 days. The results are shown in Table 5 below. The survival rate was 79.2% in case of acid tolerance and 50% in case of bovine intestine.

[Table 5]

3-3. Protease secretion

Agar supplemented with 2% skim milk was prepared, and the strain was linearly cultured at 37 占 폚 for 2 days to confirm the presence or absence of protease secretion. When the medium containing skim milk was used, decomposition of the medium during the growth of the strain resulted in a transparent border around the colonies, and the presence of protease secretion could be confirmed by the sharpness of the border (FIG. 4).

3-4. Secretion of lipase

To confirm the presence or absence of lipase, a spirit blue agar (Sigma) was used, and as a control group, bacteria that did not secrete lipase were used. When the strain cultured in Spirit Blue Agar secretes lipase, the colony surrounds blue color, and when lipase is not secreted, the color does not change. In this embodiment, Lactobacillus Since pento suspension during incubation colonies of HB-8023 is a blue color, Lactobacillus It was confirmed that lipase was secreted from Pentosus HB-8023 (FIG. 5).

4. From Jeju Citrus Kimchi Derived Lactobacillus  Characterization of strains according to culture

4-1. Preparation of mixed culture using natural materials and analysis of lactic acid content according to sample condition

Table 6 below shows the composition of the mixed culture of natural products for the cultivation of Lactobacillus pentosus HB-8023 according to this example. Pomegranate and lemon were used as natural products, and cultured at 37 ° C and 150 rpm for 2 days using a culture solution containing crushed natural products. As the pulverized product of the natural product, pomegranate or lemon was pulverized and then pulverized solid and juice were used together. The pulverized product was used in an amount of 10% in the medium composition in which the lactic acid content was increased according to Example 2, Was made to be 1 x 10 < ⁶ > cfu / ml or more.

[Table 6]

Specifically, Lactobacillus The production of lactic acid from the strain was analyzed using lyophilized, concentrated, and filtrate of the culture broth of Pentosus HB-8023 or a mixed culture using at least one natural product. As a result, Of lactic acid was confirmed in the mixed culture medium. In particular, lactic acid of 6000 ppm or more was confirmed in the cultured medium supplemented with the pomegranate powder (FIG. 6).

4-2. Analysis of effects on human cells MTT  Cells by analysis Survival rate  Measure)

Human dermal fibroblast (HDF), isolated from epidermal tissue of newborn infants, was purchased from Modern Tissue Technology (MTT, Korea). The purchased HDF was cultured in DMEM / F12 (3: 1) medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin at 37 ° C under 5% CO ₂ , Cells were cultured at 4-day intervals and 5-10 generation cells were used for the experiment.

To determine cell viability, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5, -diphenyltetrazolium bromide] was quantified by modification of Mosmann's method. Wells of a 96-well plate containing HDF at a concentration of 2 X 10 ⁴ cells / well were each cultured in a CO ₂ incubator for 24 hours in a sample of Lactobacillus pentosus HB-8023 culture medium. The media for each well was removed and MTT solution (5 mg / ml in PBS) was added. After reacting at 37 ° C for 2 hours, MTT solution was removed and 100 μl of dimethylsulfoxide (DMSO) was added to each well. The plate was shaken for 15-20 minutes with a plate shaker, and then immersed in a microplate reader (Model ELX 800, BIO-TEK Instruments Inc, USA).

On both cell viability experiments were cultured using the basic minimal medium (primary culture medium) and mixed-culture solution was added to a lemon and pomegranate substrate culture, cell viability, in use, because it was 10% of the sample is more than 100%, Lactobacillus Pento suspension cultures of HB-8023 was considered to be a safe raw material (Fig. 7).

4-3. Antimicrobial activity

Lactobacillus In a similar manner to that described in Example 3-1 Bacillus By measuring the pento suspension antimicrobial activity of HB-8023 it is shown in Table 7 below.

[Table 7]

As a result of the experiment, excellent antimicrobial activity was confirmed in both of the basic culture medium and the mixed culture medium containing the natural product. Especially, the highest antimicrobial activity against acne bacteria was obtained.

4-4. Inhibitory Effect of Inflammatory Cytokine Expression upon UV Irradiation

Lactobacillus In order to confirm whether a culture solution containing Pentosus HB-8023 or a mixed culture solution containing natural substances can inhibit the expression of inflammatory cytokines expressed by ultraviolet irradiation, fibroblasts isolated from human epidermal tissue were cultured in 24- ⁵ x 10 ⁴ cells were added to the test plate and allowed to adhere for 24 hours. Each well was then washed once with PBS and 500 [mu] l PBS was added to each well. The fibroblasts were irradiated with ultraviolet rays at 10 mJ / cm 2 using an ultraviolet B (UVB) lamp (F15T8, UVB 15W, Sankyo Dennki Co., Japan), and PBS was removed. Cell culture medium (DMEM supplemented with no FBS ) Was added. Here, the Lactobacillus Pentosus HB-8023 medium was treated and cultured for 5 hours. After that, 150 占 퐇 of the culture supernatant was taken to quantify IL-1.alpha., And the inhibitory effect of the culture solution on inflammatory cytokine expression was evaluated. The amount of IL-1α was quantitated using Enzyme-linked Immunosorbent Assay, and the production rate of IL-1α was calculated by the following equation.

Bo: IL-1? Production in wells without UV irradiation

Bt: IL-1? Production in wells irradiated with ultraviolet light and not treated with the sample

St: IL-1α production in wells irradiated with ultraviolet light and treated with the sample

As a result, it was found that Lactobacillus Pento suspension can be seen that with the fermented product was produced in the IL-1α-inflammatory cytokines in the 0.25% concentration of HB-8023 inhibited more than 50%, effectively defends the inflammation due to ultraviolet radiation at low concentrations (Table 8).

[Table 8]

4-5. Water hygroscopicity experiment

Lactobacillus In order to test the water hygroscopicity of the Pentosus HB-8023 culture, three types of cultures of the above Table 6 and distilled water as the control group were saturated and absorbed, respectively, on dry epidermal cells of a person directly taken from a human or purchased commercially, After the amount was measured, the mass change after drying for 48 hours was measured. The amount of water absorbed per skin cell unit mass was compared from each measured mass change. Such an experiment is a method for comparing the amount of water absorbed in the epidermal cells, and is used as one of methods for predicting the moisturizing effect when actually applied to human skin.

Specifically, human epidermal cells were dried to make the amount of water contained per unit mass constant, and then the weight of each epidermal cell sample and the amount of water contained were measured using the Kaiser method. The epidermal cells in which water content was measured were carried on each of three kinds of culture liquids according to Table 6 and purified water for 24 hours to saturate and absorb them. Then, the epidermal cells in which the water was saturated and absorbed were taken out from the cells, weighed, The amount of water per unit mass contained in the cells was measured with a measuring device. Thereafter, the resultant was further dried under reduced pressure for 48 hours, the weight was measured, and a part of the weight was taken. The water content was measured by the Kaiser method to measure the amount of water contained per unit mass.

As it is shown in Table 9 below, Lactobacillus It was confirmed that the epidermal cells saturated with 3 kinds of Pentosus HB-8023 culture medium contained a much larger amount of moisture in the re-drying process than the saturated and desquamated epidermal cells using purified water. From this, Lactobacillus It was confirmed that Pentosus HB-8023 culture medium had excellent skin hygroscopicity and excellent skin moisturizing effect.

[Table 9]

4-6. Skin keratinization experiment

Lactobacillus To pento suspension HB-8023 to test the cultured skin keratin water improvement was carried out a clinical trial. The cosmetic compositions containing the pomegranate cultures obtained in Table 6 were prepared with the compositions shown in Table 10 below and evaluated by human skin for improving the skin horny. Twenty two patients (30-55years old) were applied to the facial area twice a day for 4 weeks. The keratin sticks and iScope were used to measure the skin keratin of the ball immediately after application, 2 weeks after application, and 4 weeks after application.

[Table 10]

As it is shown in Table 11 below, Lactobacillus The skin moisturized with the serum containing Pentosus HB-8023 pomegranate culture decreased to a statistically significant level (p <0.001) immediately after application. In addition, it was confirmed that after 2 weeks of application and after 4 weeks of application, the skin keratin value decreased to a statistically significant level (p <0.001) From this, Lactobacillus It was confirmed that the serum containing Pentosus HB-8023 pomegranate culture showed excellent skin erasing effect.

[Table 11]

4-7. Comparison of polyphenol content and flavonoid content

In order to compare the polyphenol content and the flavonoid content of the three cultures according to Table 6, the polyphenol content in the culture broth was measured by the Folin-Denis method, and the total flavonoid content was measured by modifying the method of Nieva Moreno et al. (2000) Respectively.

First, the polyphenol content was measured by dissolving the sample in distilled water at a concentration of 10 mg / ml, and then taking 0.2 ml of the sample in a test tube, adding distilled water to make 2 ml, adding 0.2 ml of Foiln-ciocalteu's phenol reagent, Lt; / RTI &gt; 0.4 ml of a saturated solution of Na ₂ CO ₃ was added thereto, and 1.4 ml of distilled water was added thereto. After reacting at room temperature for one hour, the absorbance was measured at 725 nm using a UV / VIS spectrophotometer (Shimadzu U-1201, Japan) . The total polyphenol compound was dissolved in distilled water at a concentration of 10 mg / ml and galactic acid (Sigma Co, USA) was taken to be a final concentration of 0, 37.5, 75, 150 and 300 ㎍ / And the content of polyphenol compounds in the extract was calculated. The flavonoid content was determined by modifying Nieva Moreno's method (2000), diluting 80% ethanol with a solvent to a concentration of 10 mg / ml, adding 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1 M potassium acetate, and 80% Ethanol was added and reacted at 25 ° C for 40 minutes. Then, the absorbance was measured at 415 nm using a UV / VIS spectrophotometer. The total amount of flavonoids was determined from quercetin (Sigma Chemical Co.) at the final concentrations of 0, 10, 25, 50, 100, 250 and 500 ㎍ / The contents of the flavonoid compounds were determined and the contents thereof are shown in Table 12 below.

The results of analysis of the total polyphenol content and flavonoid content of each of the three cultivars show that the content of polyphenol and flavonoid in the medium using the substrate is higher than that of the substrate-free culture.

[Table 12]

4-8. Experiment to measure free radical scavenging activity

In order to measure the free radical scavenging activity of the three cultures according to Table 6, free radical scavenging activity was measured using a DPPH method using an extract having an excellent antioxidative activity such as green tea extract under laboratory conditions as a comparative sample. Free radical scavenging activity was measured by reducing power using a free radical called DPPH (2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical). Free radical scavenging was measured at a wavelength of 560 nm by comparing the degree of reduction of the absorbance by DPPH reduction by the test substance with the absorbance of the blank test solution. The DPPH free radical scavenging activity was determined by concentration. The extracts of the above concentrations were placed in a 96-well plate, and DPPH prepared from 100 μM methanol solution was added thereto to make the total volume of the solution 200 μl. After incubation at 37 ° C for 30 minutes, the absorbance was measured at 560 nm. The free radical scavenging activity (%) was calculated by the following equation.

 A: Absorbance of the control well without treatment of the extract of the present invention

 B: Absorbance of the experimental group well treated with the extract of the present invention

As shown in Table 13 below, the free radical scavenging activity of each of the three culture media was confirmed. As a result, two kinds of culture media using the substrate exhibited excellent free radical scavenging activity as compared with the culture medium without the substrate, Which is superior to the green tea extract.

[Table 13]

4-9. Experiment to measure inhibition effect of tyrosinase

 In order to confirm the whitening effect of the three cultures according to Table 6, the degree of inhibition of the enzyme function of tyrosinase was confirmed and the whitening effect was evaluated. Tyrosinase is an enzyme that helps the production of melanin by stimulating the oxidation process of tyrosine in vivo. (Pomerantz SH: J. Biochem., 24: 161-168, 1996) which measures the degree of inhibition of the function of the enzyme to inhibit tyrosine oxidation and formation of a melanin polymer called melanin Respectively.

The inhibitory activity of each sample on tyrosinase was determined by adding 15 μl of a sample to a 96-well plate, adding 150 μl of 50 mM phosphate buffer (pH 6.5) and 25 μl of a 1.5 mM L-tyrosine solution, and then adding 1.5 μl of mushroom tyrosinase , Sigma) was added and reacted at 37 ° C for 20 minutes. Then, the inhibition rate against tyrosinase was measured by measuring the absorbance at 490 nm using a microplate reader (ELx800, USA). The inhibition rate (%) for tyrosinase was calculated by the following equation, and the IC50 value is the concentration of the substance that inhibits tyrosinase enzyme activity by 50%.

A: absorbance before reaction of the well containing the sample

B: absorbance after reaction of the well containing the sample

C: Absorbance before reaction of well without sample

D: absorbance after reaction of well without sample

As a result of measuring the inhibitory effect of tyrosinase activity of each of the three cultures, as shown in Table 14 below, the IC50 value of the culture solution without the substrate was 0.1%. Therefore, the culture solution using the substrate showed a superior tyrosinase activity inhibitory effect .

[Table 14]

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention.

BRC KCTC13322BP 20170911

Claims (1)

Jeju is derived from a citrus Kimchi Lactobacillus pento suspension (Lactobacillus pentosus) deposited as accession number of KCTC 13322BP strain; (W / v), yeast extract 0.1% (w / v), magnesium sulfate 0.04% (w / v), manganese sulfate 0.04% (w / v) And 10% (w / v) pomegranate ground product,
Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeroginosa), E. coli (Escherichia coli), Candida albicans (Candida albicans), dry three Jia less trick other (Malassezia restricta), red baekseongyun (Trichophyton rubrum) and propionyl sludge tumefaciens Exhibit antibacterial activity against Propionibacterium acnes ,
Wherein said Lactobacillus pentosus strain is capable of producing 6,000 ppm or more of lactic acid in the presence of 1 x 10 &lt; ⁶ &gt; cfu / ml.

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