KR20210128947A - Novel Lactobacillus paracasei JS1 strain and use thereof - Google Patents

Abstract

The objective of the present invention is to develop a novel Lactobacillus strain that is capable of producing dihydrodaidzein (DHD) and dihydrogenistein (DHG), which are precursors of equol, by using legumes containing a large amount of isoflavones, even under aerobic conditions, and at the same time preparing equol, the final product thereof. Accordingly, the present invention relates to a Lactobacillus paracasei JS1 (accession number: KCCM12288P, SEQ ID NO: 2) strain, a novel microorganism that is capable of the bioconversion of legumes into dihydrodaidzein (DHD) or equol, which are isoflavone metabolites, and uses thereof.

Description

Novel Lactobacillus paracasei JS1 strain and use thereof {Novel Lactobacillus paracasei JS1 strain and use thereof}

The present invention relates to a novel Lactobacillus paracasei JS1 strain and uses thereof.

Legumes are plants belonging to the legume family and are widely used as cultivation targets because of their high utility and economical efficiency. Among them, arrowroot ( Pueraria lobata ) is a wild plant distributed in Korea, Japan, and China, and has been used as an old yellow food since ancient times. It is said to relieve dry mouth and headache, and is used for high blood pressure, angina, diabetes, hangover removal, and antioxidant effects.

On the other hand, arrowroot is known to contain a large amount of isoflavone substances such as puerarin, daidzin, daidzein, and genistein. is becoming

In addition, soybeans contain fewer amino acids than animal proteins, but are rich in vegetable protein and dietary fiber, and have excellent nutritional value because they contain unsaturated fatty acids. It is used in a wide range of fields such as anti-lipemic action and kidney disease.

Soybeans contain daidzin, daidzein, genistin, genistein, glycitin, glycitein, biochanin A, formono Contains netin (formononetin).

Dihydrodaidzein, the production material of the present invention, is one of metabolites produced during the process of daidzein, one of the main isoflavones of soybeans, being absorbed and metabolized in the body, and dihydrodaidzein is iso It is a type of flavonoid and has been reported to have antioxidant and analgesic effects. In addition, dihydrogenistein, another product of the present invention, is a metabolite of genistein in soybean isoflavones by intestinal bacteria.

The isoflavones contained in the legumes have a similar structure to estradiol, so their absorption rate is higher than other substances, and it has been reported that they have an anti-estrogen action and an estrogen-like action due to their excellent binding ability with the estrogen receptor, and anti-estrogenic action. It has been reported to be effective in preventing hormone-dependent cancers such as breast cancer and prostate cancer, as well as improving menopausal disorders, postmenopausal osteoporosis, and hyperlipidemia by estrogen-like action.

In particular, as metabolites of isoflavones, there are substances such as equol, which has a function to prevent and treat female hormone-related diseases such as osteoporosis in recent years. It has been reported that the metastasis to target organs such as breast or prostate tissue is remarkably high, and the effect of improving bone density and lipid metabolism after menopause is also excellent. In addition, the improvement effect of osteoporosis, obstetrics and gynecological diseases, prostate cancer, stomach cancer, breast cancer, etc. has been reported. Although these equols are produced through the metabolism of intestinal microbes, there are individual differences in equol production capacity.

However, since the currently reported strains are used only in anaerobic conditions, they have limitations in culture, and there is an urgent need to develop microorganisms that produce equol in aerobic conditions.

On the other hand, recently, the development rate of health functional food that emphasizes immune function regulation or allergy suppression activity is increasing, and it is known that it has the potential of future functional food. Allergy is a hypersensitivity reaction, which is a malfunction of the immune system and has no effect on normal people, but causes abnormal hypersensitivity reactions such as hives, itchiness, runny nose, and cough only in specific people. Recently, the incidence of allergic diseases is increasing worldwide due to the increase in indoor life, the development of new materials, the rapid increase of allergens such as foreign substances from foreign countries, the instability of the immune status due to environmental pollution and stress, and changes in diet. there is a trend

Therefore, the present invention provides three novel microorganisms capable of bioconversion from legumes to isoflavone metabolites, dihydrodaidzein (DHD), dihydrogenistein (DHG) or equol (Equol). Developed, respectively, Lactobacillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; accession number: KACC92053P; SEQ ID NO: 1) strain, Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P; SEQ ID NO: 2) strain and pedio Caucus pentosaceus SEQ0315 ( Pediococcus pentosaseus SEQ0315; accession number: KACC92051P; SEQ ID NO: 3) strain, using the microorganisms dihydrodaidzein (Dihydrodaidzein, DHD), dihydrogenistein (Dihydrogenistein, DHG) or equol (Equol) was confirmed. In addition, the fermented product using the Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain of the present invention has an improving effect on intestinal diseases such as ulcerative colitis, Crohn's disease, irritable bowel syndrome, chronic constipation or short bowel syndrome By confirming, it can be used as a composition for preventing, improving or treating intestinal diseases.

10-2018-0173952

The present invention can produce dihydrodaidzein (DHD) and dihydrogenistein (DHG), which are precursors of equol, even under aerobic conditions using legumes containing a large amount of isoflavones. , also aims to develop a novel strain capable of producing the final product, Equol (Equol).

In addition, an object of the present invention is to provide a method for producing dihydrodaidzein (DHD), dihydrogenistein (DHG) or equol (Equol) using the strain.

In addition, an object of the present invention is to provide a composition for preventing, improving or treating intestinal diseases comprising at least one selected from the group consisting of the strain, the culture of the strain, the lysate of the strain and the extract of the strain as an active ingredient. .

In order to solve the above problems, the present invention provides a Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain.

In one embodiment of the present invention, the "strain" may be represented by SEQ ID NO: 2, but is not limited thereto.

In addition, the present invention provides a food composition comprising as an active ingredient at least one selected from the group consisting of the strain, the culture of the strain, the lysate of the strain and the extract of the strain.

In addition, the present invention provides a composition for food addition comprising at least one selected from the group consisting of the strain, the culture of the strain, the lysate of the strain and the extract of the strain as an active ingredient.

In addition, in the method for producing a legume fermented product containing dihydrodaidzein or equol from legumes by using the strain, the "method" is, (a) extracting legumes with ethanol an extraction step; And (b) the extract obtained in step (a) is inoculated with the strain of claim 1 and then stirred to make a culture solution; dihydrodaidzein or equol from legumes comprising a; It provides a method for producing a legume ferment comprising a.

In an embodiment of the present invention, the "agitation speed" of the (b) culturing step may be characterized in that it is 100 to 300 rpm, but is not limited thereto.

In one embodiment of the present invention, the "temperature" of the culturing step (b) may be characterized in that 20 to 40 ℃, but is not limited thereto.

In one embodiment of the present invention, the "cultivation time" of the (b) culturing step may be characterized in that it is 20 to 80 hours, but is not limited thereto.

In one embodiment of the present invention, the "extract" of the (b) culturing step may be characterized in that it contains 0.2 to 3% by weight based on the total amount of the culture medium, but is not limited thereto.

In addition, the present invention provides a health functional food composition for preventing or improving intestinal diseases comprising the fermented product prepared by the above method as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of intestinal diseases comprising the fermented product prepared by the above method as an active ingredient.

In one embodiment of the present invention, the "intestinal disease" may be characterized in that it is selected from the group consisting of ulcerative colitis, Crohn's disease, irritable bowel syndrome, chronic constipation and short bowel syndrome, but is limited thereto no.

According to the method for producing Dihydrodaidzein (DHD) or Equol using the Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to the present invention, aerobic conditions It can also be cultured, which has the effect of having a high production capacity. When using the novel strain, it was confirmed that the prevention, improvement or therapeutic effect on intestinal diseases such as ulcerative colitis, Crohn's disease, irritable bowel syndrome, chronic constipation or short bowel syndrome, which can be used as a health functional food composition, pharmaceutical composition have.

1 is a diagram showing the results of genetic analysis of Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain of the present invention (a), and GC of Lactobacillus paracasei JS1 and other Lactobacillus strains of the present invention It is a figure (b) comparing the content.
Figure 2 is a fermented product (FPE) using Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention Dihydrodaidzein (Dihydrodaidzein, DHD) and Equol (Equol) ) is a diagram measuring the conversion of (1; Daidzin, 2; daidzein, 3; dihydrodaidzein, 4; equol).
3 is a fermented product (FPE) using Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the body weight of an animal model of intestinal disease It is a measure of change.
Figure 4 is a Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) according to an embodiment of the present invention to confirm the effect of improving intestinal disease of the fermented product (FPE) using the strain, the organs of the intestinal disease animal model It is a measure of weight change.
5 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention. It is a diagram measuring the tissue length.
Figure 6 is the Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) according to an embodiment of the present invention In order to confirm the effect of improving the intestinal disease of the fermented product (FPE) using the strain, the large intestine of the intestinal disease animal model It is a diagram measuring the change of TNF-α expression in RT-PCR.
7 is a fermented product (FPE) using Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, colon of intestinal disease animal model It is a diagram measuring the change of COX-2 expression in RT-PCR.
8 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the large intestine of an animal model of intestinal disease It is a diagram measuring the change of iNOS expression in RT-PCR.
9 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the large intestine of an animal model of intestinal disease It is a diagram measuring the change in IL-1β expression in RT-PCR.
10 is a fermented product (FPE) using Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the large intestine of an animal model of intestinal disease It is a diagram measuring the change in IL-4 expression in RT-PCR.
11 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the large intestine of an animal model of intestinal disease It is a diagram measuring the change in IL-6 expression in RT-PCR.
12 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, colon of an animal model of intestinal disease It is a diagram measuring the change in IL-10 expression through RT-PCR.
13 is a fermented product (FPE) using a Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain according to an embodiment of the present invention to confirm the effect of improving intestinal disease, the large intestine of an animal model of intestinal disease is a diagram confirmed through H&E staining.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings by way of example embodiments of the present invention. However, the following examples are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

In addition, the terms used in this specification are terms used to properly express a preferred embodiment of the present invention, which may vary according to the intention of a user or an operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part "includes" a certain component, it means that other components may be further included, rather than excluding other components, unless otherwise stated.

Throughout this specification, "%" used to indicate the concentration of a specific substance is, unless otherwise stated, solid/solid (w/w) %, solid/liquid (w/v) %, and Liquid/liquid is (v/v) %.

The present invention will be described in detail as follows.

The present invention relates to a method for producing a fermented product containing dihydrodaidzein (DHD), dihydrogenistein (DHG) or equol from a legume extract.

The microorganisms used in the production method of the present invention are as follows.

The present invention provides Lactobacillus sakei SEQvitaP (Lactobaillus sakei ) capable of converting arrowroot or soybean extract into isoflavone metabolites dihydrodaidzein (DHD), dihydrogenistein (DHG) or equol (Equol) SEQvitaP; accession number: KACC92053P) strain, Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain or Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P using the strain) It is a method for producing a fermentation product containing isoflavone metabolites, dihydrodaidzein (DHD), dihydrogenistein (DHG) or equol (Equol).

The method for preparing a fermented product containing the dihydrodaidzein, dihydrogenistein or equol includes: (a) an extraction step of stirring and extracting crushed arrowroot or soybean with ethanol; And (b) the Lactobacillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; accession number: KACC92053P) strain, Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain or pedi in the extract obtained in step (a) Ococcus pentosaceus SEQ0315 ( Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) inoculated with each strain, followed by stirring the culture step to make a culture solution; includes.

In the method for producing a fermented arrowroot or soybean fermented product containing dihydrodaidzein, dihydrogenistein or equol, the raw material used in the extraction step (a) may be used. As for the shape of arrowroot or soybean used in the present invention, it may be powdered, pulverized or crushed, and preferably, powdered arrowroot or soybean is generally used, but the present invention is not limited thereto.

In the method for producing a fermented product containing dihydrodaidzein, dihydrogenistein or equol, ethanol may be used as a solvent used in the extraction step (a), and preferably, for the efficiency of extraction, alcohol-based To use 20% to 90% of ethanol, but is not limited thereto.

In the method for producing a fermented arrowroot or soybean fermented product containing dihydrodaidzein, dihydrogenistein or equol, an aerobic strain is used, so that oxygen is supplied by stirring at a constant speed. To this end, preferably, the stirring speed of the culturing step (b) is 100 to 300 rpm, but is not limited thereto.

The method for preparing the fermented arrowroot or soybean fermented product containing the dihydrodaidzein, dihydrogenistein or equol includes the Lactobaillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; accession number: KACC92053P) strain, Lactobacillus para Casei JS1 ( Lactobacillus paracasei JS1; accession number: KCCM12288P) strain or Pediococcus pentosaseus SEQ0315 ( Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) After inoculating the medium with any one of the strains, the strain is viable Cultured by aerobic fermentation in a temperature range for 20 to 80 hours. Preferably, the temperature is set to 28 to 30° C., but is not limited thereto.

Preferably, the method for producing a fermented arrowroot or soybean fermented product containing dihydrodaidzein, dihydrogenistein or equol is obtained by culturing the extract extracted in the (a) extraction step in the (b) culturing step. It is included in 0.2 to 2% by weight based on the total amount, but is not limited thereto.

In addition, the present invention is the Lactobacillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; accession number: KACC92053P) strain, Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain or Pediococcus pentosaceus SEQ0315 ( Provided is a fermented arrowroot or soybean fermented product using any one of Pediococcus pentosaseus SEQ0315;

Dihydrodaidzein, dihydrogenistein, or equol is generated and accumulated in the fermented arrowroot or soybean fermented product obtained by the fermentation process through the above steps. The fermented arrowroot or soybean fermented product obtained under the above conditions can be used as a material for food, medicine, cosmetics, etc. as it is after fermentation, and if necessary, it is made into a dry solid state through a drying treatment step to produce food, pharmaceuticals, cosmetics, etc. It can also be used as a material.

Preferably, in order to improve the storage stability of the fermented arrowroot or fermented soybean, the fermented arrowroot or fermented soybean is stored in a solid state by heat-drying treatment. may be, but is not limited thereto.

As described above, the fermented arrowroot or fermented soybean of the present invention can express various physiological and pharmacological activities because it contains dihydrodaidzein or equol, which are useful physiologically active substances. For example, the fermented arrowroot or fermented soybean of the present invention is superior to the general fermented arrowroot or soybean extract in high concentration in the modulating effect of female hormone receptors, and has a property of alleviating cytotoxicity, and thus is safe for the human body, It is useful for preventing or improving diseases or symptoms such as osteoporosis. In addition, the fermented arrowroot or soybean fermented product of the present invention is useful for preventing or improving symptoms accompanying menopause or menopausal disorders, which are diseases caused by female hormone dysregulation, particularly in middle-aged and elderly women.

In addition, when the fermented arrowroot or fermented soybean of the present invention is used as a pharmaceutical material, it is prepared as a pharmaceutical preparation in the form of tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories, and the like. The pharmaceutical preparation containing the fermented arrowroot or fermented soybean of the present invention is useful as an agent for preventing or ameliorating diseases or symptoms such as menopausal disorders and osteoporosis. In particular, it is preferably used for the prevention or treatment of complaints of malaise or symptoms accompanying menopause in middle-aged and elderly people.

In order to more effectively apply the fermented arrowroot or fermented soybean, the present invention may prepare and provide a fermented arrowroot or fermented soybean by freeze-drying the fermented arrowroot or fermented soybean.

In one aspect, the present invention provides a health functional food composition for preventing or improving intestinal disease comprising a fermented product prepared using a Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain as an active ingredient .

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional food composition in addition to containing the extract as an active ingredient.

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 conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The above-described flavoring agent may advantageously use a natural flavoring agent (taumatine), a stevia extract (eg, rebaudioside A, glycyrrhizin, etc.), and a synthetic flavoring agent (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the following pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, in addition to extracts as active ingredients. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.

The health functional food composition of the present invention may be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, and the like. In the present invention, the term 'health functional food composition' refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 of the Health Functional Food Act, and It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects. The health functional food of the present invention may contain normal food additives, and whether or not it is suitable as a food additive is evaluated on the item according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the relevant standards and standards. The items listed in the 'Food Additives Codex' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, a noodle-added alkali agent, a preservative preparation, and a tar dye preparation, etc. are mentioned. For example, a health functional food in tablet form is granulated by a conventional method by mixing the active ingredient of the present invention with an excipient, a binder, a disintegrant and other additives, followed by compression molding with a lubricant, etc., or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a corrosive agent or the like, if necessary. Among health functional foods in the form of capsules, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients in ordinary hard capsules. It can be prepared by filling the mixture mixed with the capsule base such as gelatin. The soft capsules may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. A health functional food in the form of a ring can be prepared by molding a mixture of the active ingredient of the present invention with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and can be coated with sucrose or other peeling agent if necessary, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules can be prepared in a granular form by a conventionally known method by mixing a mixture of the active ingredient excipients, binders, disintegrants, etc. of the present invention, and may contain flavoring agents, flavoring agents, etc. can

In one aspect, the present invention provides a pharmaceutical composition for preventing or treating intestinal diseases comprising a fermented product prepared using a Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain as an active ingredient.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in the active ingredient, for example, starch, calcium carbonate, sucrose, lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppository, Witepsol, TweenW 61, cacao butter, laurin, glycerogelatin, etc. may be used.

The pharmaceutical composition according to the present invention may be administered to an individual by various routes. Any mode of administration can be envisaged, for example, by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The pharmaceutical composition may be variously formulated in an oral or parenteral dosage form.

Formulations for oral administration include, for example, tablets, pills, hard, soft capsules, solutions, suspensions, emulsifiers, syrups, granules, and the like. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (eg silica, talc, stearic acid and its magnesium or calcium salts and/or polyethylene glycol). In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and optionally starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, coloring, flavoring and sweetening agents. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, representative formulations for parenteral administration are injection formulations, and water, Ringer's solution, isotonic physiological saline, or suspension can be exemplified as a solvent for the injection formulation. The sterile, fixed oil of the injectable preparation may be used as a solvent or suspending medium, and any non-irritating fixed oil including mono- and di-glycerides may be used for this purpose.

In addition, the injection preparation may use a fatty acid such as oleic acid.

Hereinafter, the present invention will be described in more detail through examples. However, the above embodiments and experimental examples are presented as examples for the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. , and the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

<Example 1> Identification of strains

The feces of people who ingested arrowroot or soybean extract for 1 month were collected and prepared using gifu anaerobic medium (GAM), trypticase soy agar (TSA), ethylene bromide agar (EMA), brain heart infusion (BHI), and reinforced clostridial medium (RCM). , Lactobacilli MRS (MRS) Each strain was isolated on the basis of the shape and color of the colonies produced while plated on 6 types of medium and cultured at 28°C for 24 hours.

The isolated strains were analyzed using HPLC to confirm isoflavone-producing strains. To identify each isolated strain, genomic DNA was extracted using Solgent's genomic DNA prep kit. ) and reverse primer 1492R (5'-ggYtaccttg tacgactt-3') were used. PCR reaction mixture was performed after mixing 1 μl of template, 10 μl of 2X Taq PCR premix (QIAGEN), 2 μl of forward primer (10 pmole/μl), 2 μl of reverse primer (10 pmole/μl), and 5 μl of dDW. .

The PCR cycle was performed at 94° C. for 10 seconds, 45° C. for 20 seconds, and 72° C. for 1 minute, and the PCR process was repeated 30 times. In addition, the pre-denaturation process was performed at 94°C for 2 minutes, the post-elongation process was performed at 72°C for 5 minutes, and the PCR results were confirmed on 0.7% agarose gel. For the purification of the PCR product, QIAquick Gel Extraction kit was used, and for strain identification, the purified DNA was requested to Solgent for sequencing, and then the sequence of the strain was analyzed using NCBI BLAST (Basic Local Alignment Search Tool). The final isolated strains were Lactobacillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; accession number: KACC92053P) strain, Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain and Pediococcus pentosaceus SEQ0315 ( Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) strain. The present invention relates to a Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P; SEQ ID NO: 2) strain (see FIG. 1).

<Example 2> Preparation of arrowroot or soybean extract and preparation of culture solution

A kudzu or soybean extract was prepared by using the crushed arrowroot or soybean as 20% alcohol under the conditions shown in Table 1 below, followed by filtration under reduced pressure, and the solvent was removed using a rotary vacuum concentrator. Thereafter, 100 mL of GAM medium was added to a 1L Erlenmeyer flask, and the extract was added to the medium under the conditions shown in Table 1 below, and then Lactobaillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; Accession Number: KACC92053P; SEQ ID NO: 1) strain, Lactobacillus paraca J. JS1 ( Lactobacillus paracasei JS1; accession number: KCCM12288P; SEQ ID NO: 2) strain and Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P; SEQ ID NO: 3) strains were each inoculated with 0.1% to prepare a culture solution did. The present invention relates to Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P; SEQ ID NO: 2) strain.

At this time, the culture time, culture temperature, agitation speed, and content of the extract at the time of preparing the culture solution were prepared so as to satisfy the conditions shown in Table 1 below.

Incubation time (h) Incubation temperature (℃) Stirring speed (rpm) Extract content (%) Example 1 24 28 250 0.2 Example 2 24 28 250 0.4 Example 3 24 28 250 0.6 Example 4 24 28 250 0.8 Example 5 24 28 250 1.0 Example 6 24 28 250 2.0 Example 7 24 28 100 2.0 Example 8 24 28 150 2.0 Example 9 24 28 200 2.0 Example 10 24 28 300 2.0 Example 11 24 37 250 2.0 Example 12 48 28 250 2.0 Example 13 48 37 250 2.0 Example 14 72 28 250 2.0 Example 15 72 37 250 2.0

<Example 3> Analysis conditions of the culture medium

DGU-20A3R (Shimadzu, Japan) HPLC was used for the analysis of the culture medium, and the detector was measured at 280 nm with a PDA detector (Shimadzu, SPD-M20A). Column is Skypack C18 (SKchemical, 4.6*250mm, 5micron), Mobile phase A is water:acetic acid = 100: 1 (v/v%), B is water: acetonitrile: acetic acid = 50: 50:1 (v/ v%), flow rate of 1mL/min, column oven at 40℃, and injection volume of 50 μl were used. Injection samples were used by separating the supernatant under the conditions of 12000 rpm, 15 min, and 4°C using a centrifuge. As a result, as can be seen in FIG. 2, dihydrodaidzein (DHD) and Equol, which could not be confirmed in the conventional arrowroot extract, were Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number) of the present invention. : KCCM12288P) was confirmed in the fermented arrowroot using the strain. It can be confirmed that the production of dihydrodaidzein (DHD) and equol (Equol) through metabolism during fermentation of the Lactobacillus paracasei JS1 (accession number: KCCM12288P) strain of the present invention It is the result.

<Experimental Example 1> Measurement of the efficacy of improving Crohn's disease in an animal model of intestinal disease

In order to confirm the efficacy of improving Crohn's disease in the intestinal disease animal model according to the treatment of the Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain of the present invention, the experiment was conducted as follows. 8-week-old C57BL/6J male mice were obtained through Raon Bio, and were acclimatized for 1 week while freely supplying general solid feed and water. The environmental conditions of the animal room were set to temperature 23±3℃, relative humidity 50±10%, lighting time 12 hours (8 am to 8 pm), ventilation frequency 10 to 20 times/hour, and illumination intensity 150 to 300 Lux. During the test period, the temperature and humidity of the animal room were automatically controlled by a thermo-hygrostat. To measure the efficacy of improving ulcerative colitis, experimental animals were 1) normal control group, 2) DSS induction group 3) DSS + strain culture 4) DSS + legume extract 5-7) DSS + fermented legume (low, medium, high) ) 8) as a positive control group (ASA). Except for the normal control group, the remaining experimental groups were induced with 5% DSS for 7 weeks to induce ulcerative colitis, and then each sample was orally administered daily for 3 weeks. After the end of the experiment, in order to measure the efficacy of improving Crohn's disease in the experimental animal, the length of the colon tissue of the experimental animal was measured. As a result, it was confirmed that the length of the colon tissue reduced by DSS induction was recovered similarly to the positive control group in the strain culture solution and the strain fermented product of the present invention (see FIGS. 4 and 5).

<Experimental Example 2> Measurement of the efficacy of improving intestinal health in an animal model of intestinal disease

In order to confirm the effect of improving intestinal health in an animal model of intestinal disease according to the treatment of the Lactobacillus paracasei JS1 (Accession No.: KCCM12288P) strain of the present invention, the experiment was carried out as follows. Four-week-old C57BL/6J male mice were obtained through Raon Bio, and were acclimatized for 1 week while freely supplying general solid feed and water. The environmental conditions of the animal room were set to temperature 23±3℃, relative humidity 50±10%, lighting time 12 hours (8 am to 8 pm), ventilation frequency 10 to 20 times/hour, and illumination intensity 150 to 300 Lux. During the test period, the temperature and humidity of the animal room were automatically controlled by a thermo-hygrostat. To measure the effect of improving intestinal health of fermented legumes prepared using the JS1 strain, experimental animals were 1) normal control group (CON) 2) leguminous plant extract 200 mg/kg administered group (PE200) 3) fermented legumes Water was separated into 50 mg/kg administration group (FPE 50) 4) 100 mg/kg legume fermented product group (FPE100) 5) 200 mg/kg legume fermented product administration group (FPE200). For a total of 4 weeks, the control group received physiological saline and the sample intake group orally administered the legume extract or fermented product daily.

After the end of the experiment, in order to measure the effect of improving intestinal health of the experimental animal, the expression of inflammation-related genes was measured from the colon tissue of the experimental animal. When the legume extract was administered for 4 weeks, the expression of TNF-α, IL-4, and IL-6 genes was decreased, but the gene expression of COX-2, iNOS, and IL-1β was increased by 0.2 to 2.5 times. Contrary to this, when the fermented legume using the strain of the present invention was administered for 4 weeks, the gene expression of TNF-α, COX-2, iNOS, IL-1β, IL-4, and IL-6 was all concentration-dependently inhibited. was confirmed (see FIGS. 6 to 12).

In addition, colonic tissues were analyzed histopathologically via H&E staining. As a result, as can be seen in FIG. 13 , the number of goblet cells increased in a dose-dependent manner during fermented product (FPE) treatment using the strain of the present invention, confirming that FPE had an inhibitory effect on pro-inflammatory cytokines.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Name of deposit institution: Agricultural Biotechnology Research Institute

Accession number: KACC92053

Deposit date: 20150409

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

Accession number: KCCM12288

Deposit date: 20180713

Name of deposit institution: Agricultural Biotechnology Research Institute

Accession number: KACC92051

Deposit date: 20150409

<110> University-Industry Cooperation Group of Kyung Hee University <120> New microorganisms for producing dihydrodaidzein, dihydrogenistein or equol, and its use <130> PN2004-169 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 1002 <212> DNA <213> Unknown <220> <223> Lactobaillus sakei SEQvitaP <400> 1 gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct tccacaatgg 60 acgaaagtct gatggagcaa cgccgcgtga gtgaagaagg ttttcggatc gtaaaactct 120 gttgttggag aagaatgtat ctgatagtaa ctgatcaggt agtgacggta tccanccaga 180 aagccacggc taactacgtg ccaccagccg cggtaatacg taggtggcaa gcgttgtccg 240 gatttattgg gcgtaaagcg agcgcaggcg gtttcttnag tcagatgtga aagccttcgg 300 ctcaaccgaa gaagtgcatc ggaaactgga gttgcagcct acaatccgaa ctnagaatgg 360 ttttaagaga ttagctaaac ctcgcggtct cgcaactcgt tgtaccatcc attgtagcac 420 gtgtgtagcc cangtcatan ggggnatgat gatttgacgt cgtccccacc ttcctccggt 480 ttgtcaccgg cagtctcact anagtgccca mcttaatgct ggcaactagt aataagggtt 540 gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgaca accatgcacc 600 acctgtcact ttgtccccga agggaaagct ctatctctag agtggtcaaa ggatgtcaag 660 acctggtaag gttcttcgcg ttgcttcgaa ttaaaccaca tgctccaccg cttgtgcggg 720 cccccgtcaa ttcctttgag tttcaacctt gcggtcgtac tccccaggcg gagtgcttaa 780 tgcgttagct gcggcactga agggcggaaa ccctccaaca cctagcactc atcgtttacg 840 gcatggacta ccagggtatc taatcctgtt tgctacccat gctttcgagc ctcagcgtca 900 gttacasacc agacagccrc cttcgccact ggtgttcttc catatatcta cgcatttcac 960 cgctacacat ggagttccac tgtccacttc tgcactcaag tt 1002 <210> 2 <211> 1117 <212> DNA <213> Unknown <220> <223> Lactobacillus paracasei JS1 <400> 2 tgggggataa catttggaaa cagatgctaa taccgcatag atccaagaac cgcatggatt 60 cttggctgaa agatggcgta agctatagct tttggatgga cccgcggcgt attagctagt 120 tggtgaggta atggctcacc aaggcgatga tacgtagccg aactgagagg ttgatcggcc 180 acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg aatcttccac 240 aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaaggctttc gggtcgtaaa 300 actctgttgt tggagaagaa tggtcggcag agtaactgtt gtcggcgtga cggtatccaa 360 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 420 atccggattt attgggcgta aagcgagcgc aggcggtttt ttaagtctga tgtgaaagcc 480 ctcggcttaa ccgaggaagc gcatcggaaa ctgggaaact tgagtgcaga agaggacagt 540 ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag tggcgaaggc 600 ggctgtctgg tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga 660 taccctggta gtccatgccg taaacgatga atgctaggtg ttggagggtt tccgcccttc 720 agtgccgcag ctaacgcatt aagcattccg cctggggagt acgaccgcaa ggttgaaact 780 caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagcaacg 840 cgaagaacct taccaggtct tgacatcttt tgatcacctg agagatcagg tttccccttc 900 gggggcaaaa tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt 960 aagtcccgca acgagcgcaa cccttatgac tagttgccag catttagttg ggcactctag 1020 taagactgcc ggtgataaac cggaggaagg tggggatgac gtcaaatcat catgcccctt 1080 atgacttggg ctacacacgt gttacaatgg atggtac 1117 <210> 3 <211> 1289 <212> DNA <213> Unknown <220> <223> Pediococcus pentosaceus SEQ0315 <400> 3 acttccgtta attgattatg angtacttgt actgattgag attttaacac gaagtgagtg 60 gcgaacgggt gagtaacacg atgggttaac ctgcccagaa gtaggggata acacctggaa 120 acagatgcta ataccgtata acagagaaaa ccgcatggtt ttcttttaaa agatggctct 180 gctatcactt ctggatggac ccgcggcgta ttagctagtt ggtgaggtaa aggctcacca 240 aggcagtgat acgtagccga cctgagaggg taatcggcca cattgggact gagacacggc 300 ccagactcct acgggaggca gcagtaggga atcttccaca atggacgcaa gtctgatgga 360 gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt aaagaagaac 420 gtgggtaaga gtaactgttt accccagtga cggtatttaa ccagaaagcc acggctaact 480 acgtgccagc agccgcggta atacgtaggt ggcaagcgtt atccggattt attgggcgta 540 aagcgagcgc aggcggtctt ttaagtctaa tgtgaaagcc ttcggctcaa ccgaagaagt 600 gcattggaaa ctgggagact tgagtgcaga agaggacagt ggaactccat gtgtagcggt 660 gaaatgcgta gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgcaactg 720 acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg 780 taaacgatga tactaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt 840 aagtaatccg cctggggagt acgaccgcaa ggttgaaact caaaagaatt gacggggggcc 900 cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct 960 tgacatcttc tgacagtcta agagattaga ggttcccttc ggggacagaa tgacaggtgg 1020 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 cccttattac tagttgccag cattaagttg ggcactctag tgagactgcc ggtgacaaac 1140 cggaggaagg tggggacgac gtcaaatcat catgcccctt atgacctggg ctacacacgt 1200 gttacaatgg atggtacaac gagtcgcgag accgcgaggt taagctaatc tcttaaaacc 1260 attctcagtt cggactgtag gctgcaact 1289

Claims (11)

Lactobacillus paracasei JS1 (Lactobacillus paracasei JS1; accession number: KCCM12288P) strain. According to claim 1,
The strain is represented by SEQ ID NO: 2, the strain. A food composition comprising as an active ingredient at least one selected from the group consisting of the strain of claim 1, the culture of the strain, the lysate of the strain and the extract of the strain. The composition for food addition comprising at least one selected from the group consisting of the strain of claim 1, the culture of the strain, the lysate of the strain and the extract of the strain as an active ingredient. Using the strain of claim 1, in a method for producing a legume ferment containing dihydrodaidzein or equol from legumes,
The method is
(a) an extraction step of extracting legumes with ethanol; and
(b) the extract obtained in step (a) is inoculated with the strain of claim 1 and then stirred to make a culture solution; dihydrodaidzein or equol from legumes comprising a; A method for producing a legume ferment comprising a. 6. The method of claim 5,
The method for producing a fermented legume containing dihydrodaidzein or equol from legumes, characterized in that the stirring speed of the (b) culturing step is 100 to 300 rpm. 6. The method of claim 5,
The (b) method for producing a fermented legume containing dihydrodaidzein or equol from legumes, characterized in that the temperature of the culturing step is 20 to 40 ℃. 6. The method of claim 5,
The method for producing a fermented legume containing dihydrodaidzein or equol from legumes, characterized in that the incubation time of the (b) culturing step is 20 to 80 hours. 6. The method of claim 5,
Method for producing a fermented legume containing dihydrodaidzein or equol from legumes, characterized in that it comprises 0.2 to 3% by weight of the extract of the culturing step based on the total amount of the culture solution (b) . In the health functional food composition for the prevention or improvement of intestinal disease comprising the fermented product prepared by any one of claims 5 to 9 as an active ingredient, the intestinal disease is ulcerative colitis, Crohn's disease, irritable bowel syndrome , which is any one of chronic constipation and short bowel syndrome, a health functional food composition for the prevention or improvement of intestinal disease. In the pharmaceutical composition for the prevention or treatment of intestinal disease comprising the fermented product prepared by any one of claims 5 to 9 as an active ingredient, the intestinal disease is ulcerative colitis, Crohn's disease, irritable bowel syndrome, chronic Any one of constipation and short bowel syndrome, a pharmaceutical composition for the prevention or treatment of intestinal disease.

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