KR20230143669A - Composition for anti-oxidant comprising Lactobacillus plantarum MG989 - Google Patents

Abstract

The present invention relates to the activity of Lactobacillus plantarum MG989 deposited with deposit number (KFCC 11650P). Lactobacillus plantarum MG989 according to the present invention not only shows excellent antioxidant activity, but also reduces oxidative stress. It suppresses the production of NO and the expression of inflammation-related factors in macrophages induced by an inflammatory response in the environment, and is useful for various purposes such as food compositions for antioxidant or anti-inflammatory purposes, health supplements, cosmetic compositions, and feed additive compositions. It can be utilized effectively.

Description

Antioxidant composition comprising Lactobacillus plantarum MG989 {Composition for anti-oxidant comprising Lactobacillus plantarum MG989}

The present invention relates to Lactobacillus plantarum strains having antioxidant activity.

Inflammation is a non-specific defense action to protect the body from external physical injury or in vivo infection by microorganisms. Nitric oxide (NO), a diatomic free radical, is a representative indicator of inflammatory response. Nitric oxide is a highly reactive free radical generated from L-arginine by nitric oxide synthase (NOS) in vivo. Nitric oxide is known to function as a physiological phenomenon, blood pressure regulation, neurotransmitter, and thromboimmune function.

However, when nitric oxide is produced more than necessary, it causes an inflammatory response and causes tissue destruction and abnormalities in the immune system. Nitric oxide is a mediator for inflammatory reactions, and is known to exist as stable compounds such as nitrite (NO ₂ ) and nitrate (NO ₃ ) over time, where peroxynitrite anion (ONOO- ) is inflammatory and causes intracellular toxins, which cause tissue damage [Szabo et al., 1996].

Additionally, excessive reactive oxygen species (ROS) in the body cause oxidative stress and cause inflammatory diseases, sepsis, cancer, aging, skin aging, sarcopenia, autoimmune diseases, lung diseases (asthma, chronic bronchitis), and kidney diseases. (nephritis, chronic heart failure), joint disease (rheumatism, arthritis), brain disease (Alzheimer's disease, Parkinson's disease, memory loss, depression, stroke), eye disease (cataract, retinal disease), cardiovascular disease (arteriosclerosis, hypertension, ischemia, It is known to promote numerous diseases, including cardiomyopathy, cardiac arrest), fetal abnormalities (pre-eclampsia, intrauterine growth slowing), and neurodegenerative diseases.

Meanwhile, live microorganisms that have a beneficial effect on the host's health by improving the host's intestinal microbial environment within the gastrointestinal tract of animals, including humans, are collectively called probiotics. In order to be effective as a probiotic, when ingested orally, most of it must reach the small intestine and attach to the intestinal surface, so it must basically have excellent acid resistance, bile resistance, and the ability to adhere to intestinal epithelial cells. Lactic acid bacteria are used as probiotics because they coexist in the human digestive system and play a role in breaking down fiber and complex proteins into important nutrients. It has been reported that lactic acid bacteria exhibit effects such as maintenance of normal intestinal flora, improvement of intestinal flora, anti-diabetic and anti-hyperlipidemic effects, inhibition of carcinogenesis, inhibition of colitis, and non-specific activation of the host's immune system. Among them, Lactobacillus strains are major members of the normal microbial community living in the human intestine, and have long been known to be important in maintaining a healthy digestive system and vaginal environment. According to guidelines, all Lactobacillus strains currently deposited at the American Strain Consignment Center (ATCC) are classified as 'Bio-safety Level 1', which recognizes that there is no known potential risk of causing disease in humans or animals. there is.

As the various physiological activities of lactic acid bacteria become known, research has recently been actively conducted to develop lactic acid bacterial strains that are safe for the human body and have excellent functionality and to apply them as medicines or functional foods.

The present inventors have found that among Lactobacillus strains isolated from the vagina of Korean women, certain strains exhibit excellent antioxidant activity and are effective in suppressing NO production and expression of inflammation-related factors in macrophages induced by an inflammatory response in an oxidative stress environment. After confirmation, the present invention was completed.

Therefore, the purpose of the present invention is to provide a food composition for antioxidant use.

Another object of the present invention is to provide an antioxidant health supplement.

Another object of the present invention is to provide a health functional food composition for antioxidant purposes.

Another object of the present invention is to provide an antioxidant cosmetic composition.

Another object of the present invention is to provide a composition for adding feed to increase antioxidant activity.

Another object of the present invention is to provide a food composition for preventing or improving inflammatory diseases.

Another object of the present invention is to provide a cosmetic composition for preventing or improving inflammatory diseases.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases.

In order to achieve the above object, the present invention provides an antioxidant agent comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. A food composition is provided.

In order to achieve the above other objects, the present invention is an antioxidant containing at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. Provides health supplements for use.

In order to achieve the above other objects, the present invention is an antioxidant containing at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. Provides a health functional food composition for use.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. , provides an antioxidant cosmetic composition.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. , provides a composition for adding feed to increase antioxidant activity.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. , provides a composition for adding feed to increase antioxidant activity.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. , providing a food composition for preventing or improving inflammatory diseases.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. Provides a cosmetic composition for preventing or improving inflammatory diseases.

In order to achieve the above further object, the present invention includes at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P. , and provides a pharmaceutical composition for preventing or treating inflammatory diseases.

Lactobacillus plantarum MG989, deposited under the deposit number (KFCC 11650P) according to the present invention, not only shows excellent antioxidant activity, but also NO Since it suppresses the production and expression of inflammation-related factors, it can be usefully used for various purposes for antioxidant or anti-inflammatory purposes.

Figure 1 shows the results of confirming the production of nitric oxide (NO) after treating macrophages in which an inflammatory response in an oxidative stress environment was induced with the strain according to the present invention (*** P <0.001, compared to the LPS experimental group) .
Figure 2 shows the results of confirming the cytotoxicity of the strain according to the present invention.
Figure 3 shows the results of confirming the expression of inflammation-related factors after treating macrophages in which an inflammatory response in an oxidative stress environment was induced with the strain according to the present invention. In Figure 3, A indicates tumor necrosis factor-α (TNF-α). This is the result of confirming the expression of , B in Figure 3 is the result of confirming the expression of iNOS (inducible nitric oxide synthase), and C in Figure 3 is the result of confirming the mRNA expression of COX-2 (cyclooxygenase 2) (* P <0.05 ; ** P <0.01; *** P < 0.001, compared to LPS experimental group).

Hereinafter, the present invention will be described in detail.

The present invention is an antioxidant food composition containing at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, with accession number KFCC11650P, and antioxidant health supplement. Provides food and antioxidant health functional food compositions.

The Lactobacillus plantarum MG989 strain deposited under the accession number KFCC11650P of the present invention is a Lactobacillus genus strain isolated from the vagina of a Korean woman.

According to one embodiment of the present invention, the Lactobacillus plantarum MG989 is DPPH (2,2-diphenyl-1-picrylhydrazyl) or ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) ), the composition according to the present invention is characterized by having DPPH or ABTS radical scavenging activity.

According to another embodiment of the present invention, the Lactobacillus plantarum MG989 showed an effect of suppressing the increased production of nitric oxide when treated with macrophages in which an inflammatory response was induced in an oxidative stress environment.

In addition, according to another embodiment of the present invention, when the Lactobacillus plantarum MG989 is treated with macrophages in which an inflammatory response in an oxidative stress environment is induced, inflammation-related factors such as tumor necrosis factor-α (TNF-α) and iNOS ( It was confirmed that the expression of inducible nitric oxide synthase) and COX-2 (cyclooxygenase 2) was significantly suppressed.

Therefore, Lactobacillus plantarum MG989 according to the present invention can suppress NO production through down-regulation of iNOS and COX-2.

Therefore, the present invention is for the prevention or improvement of inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant. A food composition is provided.

In the present invention, the strain may be included in the form of dead cells as well as live cells. The term “dead cells” is the opposite of live cells and refers to a form in which live cells and metabolites obtained through fermentation are prevented from growing by heat treatment, etc. Dead cells may include cytoplasm, cell walls, antibacterial substances such as bacteriocin, polysaccharides, and/or organic acids. In one example of the present invention, dead cells were obtained through heat treatment and their anti-inflammatory effect was confirmed.

In the present invention, the "culture medium" refers to the strain, its metabolites, and excess fluid obtained by culturing the strain for a certain period of time in a medium that can supply nutrients so that the Lactobacillus Plantarum MG989 strain can grow and survive in vitro. It refers to the entire medium containing nutrients, etc., but it also includes the supernatant from which the strain is removed after culturing the strain, as well as their extracts and fractions. The liquid from which the bacterial cells are removed from the culture medium is also called “supernatant” or “cell-free supernatant.” The culture medium is left alone for a certain period of time and only the liquid in the upper layer excluding the part that has settled in the lower layer is taken, or the bacterial cells are removed through filtration, or the culture liquid is removed. It can be obtained by centrifuging to remove the sediment at the bottom and taking only the liquid at the top. Additionally, the culture medium may include fermented product. In one example, the culture may be a fermented product obtained by culturing the strain in a medium. The “fermented product” refers to the result of enzymatic or metabolic decomposition of organic substances using microorganisms. In the present invention, “fermentation” may mean any activity or process including enzymatic or metabolic decomposition of organic substances using microorganisms that is not a putrefactive reaction. The culture medium may be used as such, or may be obtained by performing known filtration, concentration, and drying processes. It can be used in the form of a powder in which moisture has been completely removed through a drying process as described above, or the powder can be used by dissolving it in distilled water or a common solvent, but is not limited thereto.

The above-mentioned known filtration method is not limited thereto, but may be, for example, filtration using a filter net, microfilter, centrifugation, or filtration using a separatory funnel.

The known concentration method is not limited thereto, but may include, for example, precipitation concentration, evaporation concentration, reduced pressure concentration, ultrafiltration, reverse osmosis, or centrifugation.

The above-mentioned known drying method is not limited thereto, but may be, for example, freeze drying, spray drying, or hot air drying methods.

Food compositions according to the present invention include all types of functional foods, nutritional supplements, health foods, and food additives. The food composition of the above type may be formulated in any form selected from the group consisting of powder, tablet, capsule, pill, and liquid according to a conventional method known in the art, but is not limited thereto. It can be manufactured in various forms using methods known in the art.

For example, as a health food, one or more selected from the group consisting of the Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, which is the accession number KFCC11650P of the present invention, is granulated. , it can be consumed by encapsulating and powdering, or it can be prepared and consumed in the form of tea, juice, and drinks. In addition, at least one selected from the group consisting of the Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, which is the accession number KFCC11650P of the present invention, is a known substance known to have antioxidant activity or It can be prepared in the form of a composition by mixing it with the active ingredient.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, marmalades, etc.), fish, meat and their processed foods (e.g. ham, sausage corn beef, etc.) , bread and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort. Food, frozen food, various seasonings (e.g. soybean paste, soy sauce, sauce, etc.), etc., consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P of the present invention, its culture medium, its extract, and its cell-free supernatant. It can be prepared by adding one or more selected from the group.

In addition, the food composition of the present invention may contain common food additives, and its suitability as a "food additive" is determined in accordance with the general provisions of the food additive code and general test methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is determined based on the specifications and standards for the relevant item. Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; natural additives such as subchromic pigment, licorice extract, crystalline cellulose, high-liquid pigment, and guar gum; Examples include mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

In the antioxidant food composition of the present invention, any one or more selected from the group consisting of the Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with the accession number KFCC11650P, its culture, its extract, and its cell-free supernatant is preferably used as a food. It may be included in 0.00001 to 50% by weight of the composition. If the content is less than 0.00001% by weight, the effect is insignificant, and if it exceeds 50% by weight, the increase in effect compared to the amount used is minimal, making it uneconomical.

In addition, in order to use at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, which is accession number KFCC11650P of the present invention, in the form of a food additive, it must be purified. It can be manufactured and used in the form of capsules, powders, granules, liquid, pills, etc.

When the composition of the present invention is manufactured into a beverage, it may contain various flavoring agents or natural carbohydrates as additional ingredients, like conventional beverages. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. The proportion of natural carbohydrates is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g, per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may include carbonating agents used in carbonated drinks. Additionally, the composition of the present invention may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, “health supplement” or “health functional food” refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Act on Health Functional Food, and “functional” refers to food. This means taking it for the purpose of controlling nutrients for the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects.

In addition, the present invention provides an antioxidant cosmetic composition comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant. .

In addition, the present invention is for the prevention or improvement of inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant. A cosmetic composition is provided.

The strain according to the present invention has excellent DPPH and ABTS radical scavenging ability, and can exhibit an anti-aging effect on the skin by reducing cell damage. In addition, it can exhibit an anti-inflammatory effect by inhibiting the production of NO and the expression of inflammation-related factors increased by the inflammatory response.

The ingredients included in the cosmetic composition of the present invention are cosmetics in addition to one or more selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P as an active ingredient, its culture medium, its extract, and its cell-free supernatant. It may contain ingredients commonly used in the composition, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments and flavorings.

The cosmetic composition of the present invention can be manufactured in any formulation commonly manufactured in the art, and examples include emulsions, creams, lotions, packs, foundations, lotions, beauty essences, hair cosmetics, etc.

Specifically, the cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence, It includes formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions, and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier ingredient. You can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is 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, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include 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, and microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

In addition to the above active ingredients, the cosmetic composition of the present invention may further contain one or more ingredients that help improve skin condition, including ingredients that exhibit the same or similar antioxidant activity or anti-inflammatory activity. The ingredients include hyaluronic acid, butylene glycol, glycerin, amino acid, trehalose, kojic acid and its derivatives, arbutin, and ascorbic acid. acid) and its derivatives, hydroquinone and its derivatives, resorcinol, 2,7-dinitroindazole, adenosine, retinol, retinyl Retinyl palmitate, polyethoxylated retinamide, yeast, dipeptide, Palmitoyl oligopeptide & Palmitoyl Tripeptide-7, Acetyl Examples include, but are not limited to, acetyl hexapeptide, EGF (epidermal growth factor), or plant extracts such as tangerine, rice, licorice, shea butter, aloe vera, coconut, olive, and avocado.

Additionally, the cosmetic composition of the present invention may be provided included in a pack. The pack is preferably manufactured with any one formulation selected from a mask pack, sleeping pack, cleansing pack, wash off pack, and peel off pack, but is not limited thereto.

In addition, the present invention provides a feed for increasing antioxidant activity, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant. A composition for addition is provided.

The term "feed" of the present invention refers to any natural or artificial diet, meal, etc., or an ingredient of the meal eaten by animals, and the composition for adding feed to increase antioxidant activity according to the present invention is used as an active ingredient. The feed included can be manufactured from various types of feed known in the art, and may preferably include concentrated feed, roughage and/or special feed, but is not limited thereto.

In the present invention, the term “feed additive composition” may mean “feed additive”, which can alleviate disease symptoms in animals, supplement nutrients and prevent weight loss, improve digestibility of fiber in feed, improve oil quality, It contains substances added to feed for various effects, such as preventing reproductive disorders, improving conception rates, and preventing high temperature stress in the summer.

The composition for adding feed of the present invention corresponds to supplementary feed under the Feed Management Act, and includes mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex mineral substances, and trace mineral preparations such as zinc, copper, cobalt, and selenium. , vitamins such as kerotene, vitamins A D, E, nicotinic acid, and vitamin B complex, protective amino acids such as methionine and lysine, protective fatty acids such as fatty acid calcium salts, probiotics (lactic acid bacteria), yeast cultures, fungal fermentation products, etc. Probiotics, yeast, etc. may be additionally included.

Among the above feeds, concentrated feeds include seeds and fruits containing grains such as wheat, oats, and corn, bran containing rice bran, bran, and barley bran as by-products obtained by refining grains, soybeans, milkweed, sesame seeds, linseed, and coco. Sesame seeds, which are by-products obtained from oil extraction, etc., residues such as residual starch, which is the main component of starch residue that is left over after removing the starch from sweet potatoes, potatoes, etc., fishmeal, fish residue, and fresh liquid obtained from fish. Animal substances such as concentrated fish soluble, meat meal, blood meal, feather meal, skim milk powder, dried whey that is the remaining whey when producing cheese from milk, and casein from skim milk. Includes, but is not limited to, feed, yeast, chlorella, and seaweed.

Among the above feeds, roughage includes raw grass feeds such as wild grasses, grasses, and green cuttings, turnips for feed, beets for feed, root vegetables such as Luther Bearger, a type of turnip, raw grass, green cuttings, and grains. These include, but are not limited to, silage, which is stored feed that is filled in a silo and fermented with lactic acid, field grass, hay made by cutting and drying grass, straw from crops for breeding stock, and leaves from legumes. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea and its derivative diureide isobutane, and mixed feeds to supplement ingredients that are likely to be lacking when mixing only natural feed ingredients, or to increase the storability of the feed. Substances added in trace amounts include feed additives and dietary supplements, but are not limited to these.

The composition for feed addition according to the present invention is Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, with accession number KFCC11650P according to the present invention, in an appropriate effective concentration range according to various feed production methods known in the art. It can be prepared by adding any one or more selected from the group consisting of its extract and its cell-free supernatant.

Additionally, the composition for adding feed according to the present invention can be applied without limitation to any entity aiming to increase antioxidant activity. For example, it can be applied to any entity, including non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds, and fish.

At least one selected from the group consisting of the Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, its extract, and its cell-free supernatant, which has accession number KFCC11650P according to the present invention, has excellent antioxidant activity and increases by inflammatory response. It has the activity of suppressing the production of NO and the expression of inflammation-related factors.

Therefore, the present invention is for the prevention or treatment of inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant. Pharmaceutical compositions are provided.

The inflammatory diseases include allergic asthma, allergic rhinitis, allergic mucositis, allergic diseases including urticaria and anaphylax, systemic sclerosis, dermatomyositis, and inclusion body myositis. ), including myopathies, arthritis, atopic dermatitis, psoriasis, asthma, multiple sclerosis, sepsis, polychondritis, scleroderma, eczema, gout, periodontal disease, Behcet syndrome, edema, vasculitis, Kawasaki disease, diabetic retinitis, autoimmune It may be one or more types selected from the group consisting of pancreatitis, vasculitis, glomerulonephritis, acute and chronic bronchitis, and influenza infection, but is not limited thereto.

The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. . Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture medium, and its accession number KFCC11650P of the present invention. It is prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, and gelatin, with one or more selected from the group consisting of extracts and cell-free supernatants thereof. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, gender, and weight of the subject to be treated, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of one skilled in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 1000 mg/kg/day. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, intrathecal or intracerebrovascular injection.

In the present invention, the pharmaceutical composition for preventing or treating inflammatory diseases is a group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P according to the present invention, its culture medium, its extract, and its cell-free supernatant. In addition to any one or more selected from the above, any compound or natural extract that has already been proven to be safe and is known to have a therapeutic effect for inflammatory diseases may be additionally included to increase or reinforce the anti-inflammatory effect.

In addition, the pharmaceutical composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemical therapy, and methods using biological response regulators to treat inflammatory diseases.

The contents of the present invention described above are applied equally to each other unless they contradict each other, and implementation by a person skilled in the art with appropriate changes is also included in the scope of the present invention.

Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited to the following examples.

Experimental Example 1 . Strain preparation

Among the Lactobacillus strains isolated from the vagina of Korean women to identify lactic acid bacteria with antioxidant activity , Lactobacillus salivarius MG242 deposited with accession number (KCTC 18554P) and Lactobacillus deposited with accession number (KFCC 11650P) Plantarum ( Lactobacillus plantarum ) MG989 was selected. The strains were cultured at 37°C using MRS (de Man, Rogosa, and Sharpe) medium (BD Biosciences, Frankin Lakes, NJ, USA) and cultured at -80°C using 20% (v/v) glycerol. It was stored.

Experimental Example 2. Confirmation of antioxidant properties of strains

The antioxidant properties of the strains of Experimental Example 1 were confirmed by DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging analysis. First, the OD600 value of the strain culture was adjusted to approximately 1.0 using PBS (pH 7.4), 0.05 mM DPPH solution was added, and mixed well (1:2 v/v). The mixture was then left at room temperature in the dark for 30 minutes. The control reaction included ethanol added to the DPPH solution. The absorbance of each mixture was measured at 517 nm. The analysis of each sample was repeated three times, and the results were compared with 10 μg/mL ascorbic acid. In addition, antioxidant activity was calculated using Equation 1 below.

[Equation 1]

Scavenging effect (%) = (Ac-As)/Ac × 100

In Equation 1, As is the absorbance of the sample at 517 nm, and Ac is the absorbance of the control group at 517 nm.

In addition, ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity was confirmed. First, 7mM ABTS and 2.45mM potassium persulfate were mixed (1:1 v/v) and left at room temperature for 24 hours in the dark to prepare radical cations. Afterwards, 50 μL of the selected strain culture medium and 100 μL of ABTS solution were mixed and reacted at room temperature for 10 minutes. The absorbance of the mixture was measured at 734 nm. The analysis of each sample was repeated three times, and the scavenging rate was calculated using Equation 2 below.

[Equation 2]

Scavenging rate (%) = (Ac-As)/Ac × 100

In Equation 2, As is the absorbance of the sample at 734 nm, and Ac is the absorbance of the control group at 734 nm.

Experimental Example 3. Confirmation of the effect of the strain on macrophages

3-1. Macrophage culture

The RAW 264.7 cell line, a murine macrophage, was obtained from KCLB (Korean Cell Line Bank, (Korea)). This was incubated at 5% concentration using DMEM medium (BD Biosciences) supplemented with 10% FBS (Fetal bovine serum, (Gibco)) and 1% 100 units/mL penicillin/100 μg/mL streptomycin. Cultured under conditions of CO2 and 37°C. Cells were sub-cultured every 3 days until 95% confluency.

3-2. Nitrogen oxide (NO) production

NO production was detected based on the accumulation of nitrite, an indicator of NO synthesis, in the culture medium through the Griess reaction.

First, the strains were cultured overnight and then heat-killed by heating at 90°C for 30 minutes. After _{centrifugation} at 12,000 It was resuspended to a concentration of 10 ⁸ cells/mL. RAW 264.7 cells were placed in a 96-well plate at 2 × 10 ⁵ cells/well, stimulated by treatment with 1 μg/ml LPS (lipopolysaccharide), and then 10 ⁹ cells/well of each strain prepared as above were added. After culturing it for 24 hours, the NO concentration was determined by measuring the amount of nitrite from the cell culture supernatant using Grease's reagent. In addition, the absorbance was confirmed at a wavelength of 550 nm using an Epoch 2 microplate reader (BioTek, Winooski, VT, USA). Fresh culture medium was used as a blank control for all experiments.

3-3. Cytotoxicity confirmed

MTT (3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (Sigma, USA)) analysis was performed to confirm the survival rate of RAW 264.7 cells treated with the strain according to the present invention. After washing the RAW 264.7 cells twice with PBS, 100 μL of MTT reagent (0.5 mg/mL) dissolved in PBS was added to each well. After incubation for 1 hour, the MTT reagent was removed and 100 Μl of DMSO (dimethyl sulfoxide, (Sigma, USA)) was added to dissolve formazan (reactant) formed as a reactant between the MTT reagent and the metabolites of viable cells. formazan) was dissolved. Absorbance was measured at a wavelength of 570 nm, and compared with the results of the negative control group (untreated strain medium), it was calculated using Equation 3 below.

[Equation 3]

Cell viability (%) = (A sample/A negative control) × 100

In Equation 3 above, A is the absorbance measured at 570 nm.

3-4. Semi-quantitative RT-PCR (reverse transcription-polymerase chain reaction)

RT-PCR was performed to analyze the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and tumor necrosis factor-α (TNF-α). Total RNA was extracted from RAW 264.7 cells using TRI REGENT™ (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions. In addition, to perform RT-PCR, primers for iNOS, COX-2, and TNF-α were designed. At this time, GAPDH (Glyceraldehydes-3-phosphate dehydrogenase) was used as a housekeeping gene.

RT-PCR was performed using the ONE-STEP RT-PCR PreMix kit™ (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's instructions. Each primer and 1 μg of RNA template were mixed with ONE-STEP RT-PCR PreMix™. These samples were amplified through RT-PCR, and the reaction was performed under the following conditions.

Reverse transcription reaction at 95°C for 5 minutes; 30 cycles of 95°C for 45 seconds, 60°C for 45 seconds (COX-2 and GAPDH), and 72°C for 1 minute or 95°C for 45 seconds and 63°C for 45 seconds (iNOS and TNF-α). 35 cycles of and 72°C for 1 minute; and a final elongation step at 72°C for 5 minutes. iNOS, COX-2, and TNF-α mRNA expression levels were quantified using a densitometer and Quantity One software (Bio-Rad, Hercules, CA, USA).

Experimental Example 4. Confirmation of presence of SCFA (short-chain fatty acid) in strain culture medium

SCFA of the strain culture was analyzed through gas chromatography-mass spectrometry (QP2020 NXW/ORP230; Shimadzu, Japan) using the headspace solid-phase microextraction method.

First, the samples were separated using a Stabilwax-DA column (60 m × 0.32 mm × 0.25 μm, Shimadzu). Then, the analysis was performed under the following conditions: the oven was maintained at 50°C for 2 minutes, The temperature was raised to 100°C at 10°C/min, 200°C at 2°C/min, then raised to 220°C at 20°C/min and maintained for 2 minutes. At this time, splitless mode was used, and helium was It was used as a carrier gas at a flow of 2 mL/min. The mass spectrometer was operated in electron-impact mode at 65 eV. The scan range was 40 -200 m/z, scan rate was 0.2 s/scan, electron energy was 70 eV, ionization source and quad temperature were 200℃ and 150℃, respectively. The peak area of each extracted sample was quantified and normalized to the initial volume of the sample. The linear regression equation for each analyte was calculated as the peak area. It was calculated using a calibration curve of analyte concentration (μmol) compared to area.

Experimental Example 5. Statistical analysis

The results were expressed as the mean ± standard deviation of three experiments. Differences between experimental groups were evaluated using the student's test, and a P value of <0.05 was considered significant.

Example 1. Confirmation of in vitro antioxidant properties of strains according to the present invention

By measuring the DPPH and ABTS radical scavenging activities of Lactobacillus salivarius MG242 deposited with accession number ( KCTC 18554P) and Lactobacillus plantarum MG989 deposited with accession number (KFCC 11650P), the above The antioxidant activity of the strain was confirmed.

As shown in Table 2, the DPPH radical scavenging activity of Lactobacillus salivarius MG242 deposited under the accession number (KCTC 18554P) was 56.9 ± 6.0, and the ABTS radical scavenging activity was 97.1 ± 0.4. In addition, the DPPH radical scavenging activity of Lactobacillus Plantarum MG989 deposited with the deposit number (KFCC 11650P) was 59.4 ± 3.8, and the ABTS radical scavenging activity was 98.9 ± 0.5. Therefore, it was confirmed that the strains according to the present invention have excellent ABTS radical scavenging ability.

Example 2. Confirmation of effect on NO production and cytotoxicity of strains according to the present invention

NO is a multi-functional mediator and plays a pivotal role in the immune response to inflammatory activities. Therefore, the effect of the two Lactobacillus strains according to the present invention on NO production in macrophages in which an inflammatory response was induced in an oxidative stress environment was confirmed. As a result, as shown in Figure 1, it was confirmed that NO in macrophages treated with LPS significantly increased. However, when treating the Lactobacillus strain according to the present invention, it was confirmed that NO production was significantly reduced. More specifically, Lactobacillus salivarius MG242 deposited with accession number (KCTC 18554P) showed NO production of 14.60 μM, and Lactobacillus plantarum MG989 deposited with accession number (KFCC 11650P) showed NO production of 19.60 μM.

Therefore, it was confirmed that the two Lactobacillus strains according to the present invention can significantly reduce NO production increased by oxidative stress stimulation of macrophages.

Additionally, as shown in Figure 2, it was confirmed that the two Lactobacillus strains according to the present invention did not show cytotoxicity to macrophages (Cell viability: 80% or more).

Example 3. Confirmation of the effect of strains according to the present invention on the expression of inflammation-related factors

Through RT-PCR, the effects of the two strains according to the present invention on the expression of inflammation-related factors iNOS, COX-2, and TNF-α mRNA in macrophages induced with an inflammatory response in an oxidative stress environment were confirmed. As a result, as shown in Figure 3, the expression of iNOS, COX-2, and TNF-α mRNA was increased by LPS treatment, but Lactobacillus salivarius MG242 deposited with the accession number (KCTC 18554P) according to the present invention and accession number ( It was down-regulated by treatment of Lactobacillus plantarum MG989 deposited as KFCC 11650P). At this time, the housekeeping gene GAPDH was not affected by the strain treatment according to the present invention.

Therefore, it was confirmed that the two Lactobacillus strains according to the present invention can suppress NO production through downregulation of iNOS and COX-2.

Example 4. Analysis of SCFAs in strain culture according to the present invention

SCFA, the main product of microbial fermentation of carbohydrates, can inhibit the growth of pathogenic intestinal bacteria and regulate lipid metabolism and the immune system. SCFAs also inhibit bacterial growth by lowering intestinal pH and promoting the bioavailability of minerals such as magnesium and calcium.

Accordingly, SCFAs in the strain culture according to the present invention were analyzed and are shown in Table 3 below.

In Table 3, AA is acetic acid, PA is propionic acid, and BA is butyric acid. The data are expressed as mean ± standard deviation (n=3). As confirmed above, among the SCFAs in the strain culture according to the present invention, AA was the most abundant.

Overall, the present invention relates to the activity of Lactobacillus salivarius MG242 deposited with accession number (KCTC 18554P) and Lactobacillus plantarum MG989 deposited with accession number (KFCC 11650P), and the two types of lactobacillus according to the present invention. Bacillus strains not only show excellent antioxidant activity, but also suppress NO production and expression of inflammation-related factors in macrophages induced by inflammatory responses in an oxidative stress environment, making them useful for various purposes for antioxidant or anti-inflammatory purposes. there is.

Korea Microbial Conservation Center KFCC11650P 20160120

Claims (12)

An antioxidant food composition comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
According to clause 1,
The composition is a food composition characterized by having DPPH (2,2-diphenyl-1-picrylhydrazyl) or ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging activity. .
According to clause 1,
A food composition, characterized in that the composition has an effect of inhibiting the production of nitric oxide.
According to clause 1,
The composition is a food composition characterized in that it inhibits the expression of one or more genes selected from the group consisting of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2).
Antioxidant health supplement containing at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain, its culture, its extract, and its cell-free supernatant, with accession number KFCC11650P.
An antioxidant health functional food composition comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
An antioxidant cosmetic composition comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
A composition for adding feed to increase antioxidant activity, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
A food composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
A cosmetic composition for preventing or improving inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
A pharmaceutical composition for the prevention or treatment of inflammatory diseases, comprising at least one selected from the group consisting of Lactobacillus plantarum MG989 ( Lactobacillus plantarum MG989) strain with accession number KFCC11650P, its culture, its extract, and its cell-free supernatant.
According to clause 11,
The inflammatory diseases include allergic asthma, allergic rhinitis, allergic mucositis, allergic diseases including urticaria and anaphylax, systemic sclerosis, dermatomyositis, and inclusion body myositis. ), including myopathies, arthritis, atopic dermatitis, psoriasis, asthma, multiple sclerosis, sepsis, polychondritis, scleroderma, eczema, gout, periodontal disease, Behcet syndrome, edema, vasculitis, Kawasaki disease, diabetic retinitis, autoimmune A pharmaceutical composition, characterized in that it is one or more selected from the group consisting of pancreatitis, vasculitis, glomerulonephritis, acute and chronic bronchitis, and influenza infection.