KR20210128949A - Novel Pediococcus pentosaseus SEQ0315 strain and use thereof - Google Patents

Abstract

The present invention relates to a novel microorganism capable of bioconversion from legumes to isoflavone metabolites dihydrodaidzein (DHD) or equol ( Pediococcus pentosaseus SEQ0315; Accession number: KACC92051P; SEQ ID NO: 3) relates to a strain and uses thereof.

Description

Novel Pediococcus pentosaseus SEQ0315 strain and use thereof

The present invention relates to a novel Pediococcus pentosaseus SEQ0315 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 Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) of the present invention inhibits mast cell degranulation, and by inhibiting β-hexosaminidase release, has the potential to improve allergy and atopy, and TNF- It can be used as a composition for the treatment of allergic or vascular inflammatory diseases by suppressing the inflammatory response of α-induced blood vessel cells, as well as regulating immune functions such as allergy and atopy, as well as suppressing inflammation of blood vessels, thereby confirming the efficacy of improving hyperlipidemia and cholesterol. can

10-2009-0060510

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, it aims to provide a composition for the treatment of allergic or vascular inflammatory 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 Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) strain.

In one embodiment of the present invention, the "strain" may be represented by SEQ ID NO: 3, 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 pharmaceutical composition for preventing or treating allergic or vascular inflammatory diseases comprising the fermented product prepared by the above method as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of allergic or vascular inflammatory diseases comprising the fermented product prepared by the above method as an active ingredient.

In one embodiment of the present invention, the "vascular inflammatory disease" is arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications occurring after transvascular angioplasty, cerebral infarction, cerebral hemorrhage, and stroke in the group consisting of It may be characterized in that it is selected, but is not limited thereto.

According to the method for producing dihydrodaidzein (DHD) or Equol (Equol) using the Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) strain according to the present invention, It can be cultured even under miracle conditions, which has the effect of having a high production capacity. In the case of using the novel strain, an improvement effect on allergy or vascular inflammatory disease was confirmed, which may be used as a pharmaceutical composition or a health functional food composition.

1 shows the conversion of dihydrodaidzein (DHD) and equol (Equol) of a fermented product (FPE) using a pentosaceus SEQ0315 strain in an arrowroot (Pueraria lobata ) extract according to an embodiment of the present invention; is the measured degree.
2 is a dihydrodaidzein (Dihydrodaidzein, a fermented product (FPE) using a Pentosaceus SEQ0315 strain according to the addition of a concentration (2% or 5%) of an arrowroot (Pueraria lobata ) extract according to an embodiment of the present invention; DHD) and a diagram measuring the conversion of equol (Equol). In the case of dihydrodaidzein, 41.9 mg/g was produced when fermented with 2% of kudzu extract, and when fermented after adding 5% of kudzu extract, the amount of dihydrodaidzein produced was 59.72 mg/g compared to when 2% of kudzu extract was added. It was confirmed that this increased by about 1.42 times.
3 is a diagram illustrating the measurement of cytotoxicity of each sample using the RBL-2H3 cell line according to an embodiment of the present invention (fermented product of Pediococcus pentosaceus strain; FPE, dihydrodaidzein; DHD, equol).
Figure 4 is a diagram measuring the degranulation inhibition rate of each sample according to an embodiment of the present invention (fermented Pediococcus pentosaceus strain; FPE, dihydrodaidzein; DHD, equol, positive control ketotifen).
5 is a diagram showing the expression levels of COX-2, IL-6 and TNF-α in each sample according to an embodiment of the present invention (dihydrodaidzein; DHD, equol).
6 is a diagram illustrating the measurement of cytotoxicity using the MOVAS cell line, which is a mouse vascular cell, of each sample according to an embodiment of the present invention (Pediococcus pentosaceus strain fermented product; EP, arrowroot extract; PL, equol, positive control E2).
7 is a view confirming the expression of VCAM-1, a vascular inflammation index, of each sample according to an embodiment of the present invention (dihydrodaidzein; DHD, equol).
8 is a diagram confirming the expression of Nrf-2 and HO-1, which are indicators of vascular inflammation in each sample according to an embodiment of the present invention (dihydrodaidzein; DHD, equol).

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) strain and using the same This is a method for producing a fermented product containing flavone 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 pedio in the extract obtained in step (a) Caucus pentosaceus SEQ0315 ( Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) inoculated with each strain, followed by agitation 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 temperature at which the strain can grow Cultured by aerobic fermentation in the field 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 ( Pediococcus SEQ0315 ( Pediococcus SEQ0315) 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 is Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) pharmaceutical for preventing or treating allergic or vascular inflammatory disease comprising a fermented product as an active ingredient A composition is provided.

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.

In one aspect, the present invention includes a fermented product prepared using a Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) strain as an active ingredient for the prevention or improvement of allergic or vascular inflammatory diseases. Provided is a nutraceutical composition.

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

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 new strains

The feces of people who ingested arrowroot or soybean extract for about 1 month were collected and collected from gifu anaerobic medium (GAM), trypticase soy agar (TSA), ethylene bromide agar (EMA), brain heart infusion (BHI), reinforced clostridial medium (RCM). ) and Lactobacilli MRS (MRS), each strain was isolated based on the shape and color of the colonies produced while plated 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. For the PCR reaction mixture, PCR 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 the sequence of the strain was analyzed using NCBI BLAST (Basic Local Alignment Search Tool). The final isolated strains are 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 SEQ0315), respectively. pentosaseus SEQ0315; accession number: KACC92051P) strain. The present invention relates to a Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P; SEQ ID NO: 3) strain.

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

The crushed arrowroot or soybean extract was extracted with 20% alcohol to prepare an arrowroot or soybean extract 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 placed in the medium under the conditions shown in Table 1 below, and then Lactobaillus sakei SEQvitaP (Lactobaillus sakei SEQvitaP; Accession No.: KACC92053P; SEQ ID NO: 1) strain, Lactobacillus paracasei. 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) strain was inoculated with 0.1%, respectively, to prepare a culture solution . The present invention relates to a Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P; SEQ ID NO: 3) 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

LC-20AD (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, 4.6×150mm, 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 10 μl were used.

As a result, as can be seen in FIGS. 1 and 2, dihydrodaidzein (DHD) and Equol, which could not be confirmed in the existing arrowroot extract, were Pediococcus pentosaceus strain SEQ0315 of the present invention. It was confirmed in the used arrowroot fermented product. This is a result confirming the production of dihydrodaidzein (DHD) and equol (Equol) through metabolism during fermentation of the Pediococcus pentosaceus SEQ0315 strain of the present invention.

<Experimental Example 1> Measurement of cytotoxicity in RBL-2H3 cell line

Cytotoxicity of each sample was measured using the RBL-2H3 cell line. After dispensing the RBL-2H3 cell line at 1×10 ⁴ cells/well in a 96-well cell culture plate and culturing for 24 hours in a 5% CO2 incubator, the fermented product of the Pediococcus pentosaceus SEQ0315 strain of the present invention, dihydroda Dihydrodaidzein (DHD) and equol (Equol) were treated for each concentration and then cultured for 24 hours. Thereafter, cell viability was measured using MTT assay, and the results are shown in FIG. 3 . As can be seen in FIG. 3 , cytotoxicity was not observed in all three samples except for high concentrations.

<Experimental Example 2> Degranulation inhibition rate measurement of mast cells

In order to confirm the anti-allergic effect, the degranulation inhibition rate of mast cells was measured. In order to measure the degranulation inhibition rate, the RBL-2H3 cell line was seeded in a 24 well plate at 2×10 ⁵ cells/well and cultured for 24 hours. cultured. Each sample was treated for 20 minutes by concentration, treated with DNP-BSA 0.1ug/ml, treated with pNAG as a substrate, reacted for 1 hour, and then 200ul of stop buffer was added. Then, the absorbance was measured at 405 nm using an ELISA reader. The results are shown in Table 1 and FIG. 4 below.

Through the above experimental method, the fermented product of the Pediococcus pentosaceus SEQ0315 strain of the present invention, dihydrodaidzein (DHD), equol, and ketotifen, a positive control, all showed beta-hexosaminidase release in a concentration-dependent manner. was found to be inhibiting.

<Experimental Example 3> Measurement of expression levels of COX-2, IL-6 and TNF-α

Real-time PCR to confirm the inflammatory response inhibitory effect was performed using cDNA, TB Green, and primers (COX-2, IL-6, TNF-α). The PCR composition was used by diluting cDNA with sterile distilled water, and the real-time PCR cycle was performed 40 times with initial denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 1 minute, and elongation at 95°C for 1 minute. did. The melting curve started at 55°C and ended at 95°C, increased by 0.5°C, and reacted 80 times to detect the desired fluorescence value. Fluorescence signal quantification was performed using the MAXPro program. In order to confirm that the same amount of RNA was used in each RT-PCR, RT-PCR for β-actin, a housekeeping gene, was performed together.

As a result, as can be seen in FIG. 5 , it was confirmed that the gene expression of COX-2 was suppressed by dihydrodaidzein (DHD) and the expression of TNF-α was suppressed by equol (Equol). Pediococcus pentosaceus SEQ0315 strain of the present invention producing daidzein (Dihydrodaidzein, DHD) and equol (Equol) and dihydrodaidzein (Dihydrodaidzein, DHD) and pedi comprising equol (Equol) When using the fermented Ococcus pentosaceus SEQ0315 strain, it was predicted that both COX-2 and TNF-α, which are inflammation-related factors, would be inhibited.

<Experimental Example 4> Measurement of cytotoxicity in the MOVAS cell line, which is a mouse vascular cell

The cytotoxicity of each sample was measured using the MOVAS cell line, which is a mouse vascular cell. The MOVAS cell line was ^{dispensed in a 96-well culture plate at 1×10 4} cells/well and cultured in a CO2 incubator for 24 hours, and each sample was treated for 24 hours at each concentration after 24 hours. Thereafter, MTT reagent (5mg/ml) was treated and reacted for 3 hours, and after removing the supernatant, cells were dissolved with 100ul of DMSO and then measured at 550 nm using an ELISA reader. MTT assay was performed using the above experimental method, and as a result, cytotoxicity was observed at a high concentration (100 μM) only in Equol, as can be seen in FIG. 6 .

<Experimental Example 5> Measurement of expression of VCAM-1, Nrf-2 and HO-1, which are markers of vascular inflammation

The expression of VCAM-1, Nrf-2 and HO-1, which are markers of vascular inflammation, was checked using dihydrodaidzein (DHD) and Equol. The MOVAS cell line was aliquoted in a 60 mm cell culture plate at 1×10 ⁶ cells/well, and then cultured in a CO2 incubator for 24 hours. After that, dihydrodaidzein (DHD) and Equol were pre-treated for 2 hours at each concentration, and TNF-alpha was treated at a concentration of 10ng/ml in the remaining wells except for the normal control for 24 hours. inflammation was induced. After 24 hours, it was washed with cold PBS, and the cells were lysed using a homogenazer buffer, and then the protein was extracted and used. Protein was quantified using the BCA method, and protein expression of VCAM-1, Nrf-2, and HO-1 was measured. As a result, as can be seen in FIGS. 7 (VCAM-1) and 8 (Nrf-2 and HO-1), the active material of the Pediococcus pentosaceus SEQ0315 strain fermented product of the present invention, Dihydrodaidzein (Dihydrodaidzein) , DHD) and equol (Equol) suppressed the expression of VCAM-1 (see Fig. 7), the expression of the sub-factors Nrf-2 and HO-1 is inhibited by the final metabolite, equol (Fig. 8) was confirmed. Accordingly, the Pediococcus pentosaceus SEQ0315 strain of the present invention producing dihydrodaidzein (DHD) and equol (Equol) and dihydrodaidzein (Dihydrodaidzein, DHD) and equol (Equol) When using the Pediococcus pentosaceus SEQ0315 strain ferment containing

Pediococcus pentosaceus SEQ0315 strain and dihydrodaidzein (DHD) of the present invention to produce dihydrodaidzein (DHD) and equol (Equol) according to the Examples and Experimental Examples Since the Pediococcus pentosaceus SEQ0315 strain fermented product containing Equol and Equol confirmed the anti-allergic effect and the improvement of vascular inflammation, it is a pharmaceutical composition for the prevention or treatment of allergic or vascular inflammatory diseases and allergy or blood vessels. It may be used as a health functional food composition for the prevention or improvement of inflammatory diseases.

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.

Agricultural Biotechnology Research Institute KACC92053 20150409 Korea Microorganism Conservation Center (Overseas) KCCM12288 20180713 Agricultural Biotechnology Research Institute KACC92051 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 (13)

Pediococcus pentosaseus SEQ0315 (Pediococcus pentosaseus SEQ0315; accession number: KACC92051P) strain. According to claim 1,
The strain is represented by SEQ ID NO: 3, 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 10% by weight of the extract of the (b) culturing step based on the total amount of the culture solution . 10. A pharmaceutical composition for the prevention or treatment of allergic or vascular inflammatory diseases comprising the fermented product prepared by any one of claims 5 to 9 as an active ingredient. 11. The method of claim 10,
The vascular inflammatory disease is selected from the group consisting of arteriosclerosis, high blood pressure, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications occurring after carotid arterioplasty, cerebral infarction, cerebral hemorrhage and stroke, Allergy or vascular inflammation A pharmaceutical composition for preventing or treating a disease. A health functional food composition for the prevention or improvement of allergies or vascular inflammatory diseases, comprising the fermented product prepared by any one of claims 5 to 9 as an active ingredient. 13. The method of claim 12,
The vascular inflammatory disease is selected from the group consisting of arteriosclerosis, high blood pressure, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications occurring after carotid arterioplasty, cerebral infarction, cerebral hemorrhage and stroke, Allergy or vascular inflammation A health functional food composition for preventing or improving diseases.

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