KR102456356B1 - Composition for relieving premenstrual syndrome comprising mixture of lactobacillus strains as an active ingredient - Google Patents

Abstract

The present invention provides a composition for relieving a premenstrual syndrome, which comprises as an active ingredient a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) or at least one of the culture, the crushed product, and the extract of the mixture. The mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) according to one embodiment of the present invention reduces the generation amount of NO in blood and expression of IL-6, iNOS and COX-2 genes and increases a production ratio of prostaglandin E1 to prostaglandin E2 in blood and the levels of GLA and DGLA in blood, thereby relieving the premenstrual syndrome. Therefore, the strain mixture can be applied to a food composition, a functional health food composition, a pharmaceutical composition, and the like.

Description

A composition for relieving premenstrual syndrome, comprising a Lactobacillus mixed strain as an active ingredient

The present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of LM1071 strain (KCCM12650P), or one or more of the culture, lysate, and extract of the mixed strain is effective It relates to a composition for alleviating premenstrual syndrome comprising as a component.

Premenstrual syndrome (PMS) is a group of symptoms characterized by emotional, behavioral, and physical symptoms that occur repeatedly before menstruation. psychological changes, etc. These symptoms become progressively worse after ovulation, are most severe one week before menstruation, and disappear within a few days after menstruation begins.

Among the characteristics of typical physical symptoms, menstrual pain is a common gynecological symptom that any woman of childbearing experience periodically experiences every month until she reaches menopause, and is very common in the female menstrual cycle.

According to the results of a study on Korean women, 77-94% complained of menstrual pain. Of these, 47% experienced menstrual cramps every month, 53.2% experienced severe cramps, and 46% of production women reported that they were restricted from their activities due to menstrual cramps. In foreign countries, the prevalence of menstrual cramps was high at 60-93%, and 42% experienced severe menstrual cramps. 10-50% of female students said that menstrual cramps limited their daily activities, including school life. There was a loss of productivity.

Therefore, the study results on the changes or mechanisms of menstrual pain-related factors can be used as sufficient evidence for the development of premenstrual syndrome-related food materials, health functional food materials, and pharmaceuticals.

As an example of developing a composition for alleviating premenstrual syndrome through the change of prolactin, there is a composition for improving premenstrual syndrome symptoms (Korean Patent No. 10-2187335) comprising a compound isolated from malt extract as an active ingredient. . However, there is still a need for in-depth development and various studies on compositions showing excellent effects in relation to the alleviation of premenstrual syndrome.

Accordingly, the present inventors have made diligent efforts to develop an excellent composition capable of alleviating premenstrual syndrome, resulting in reducing NO, IL-6 gene expression and increasing blood prostaglandin E ₁ / prostaglandin E ₂ production rate and DGLA level. By developing a mixed strain, the present invention was completed.

Korean Patent Publication No. 10-2187335

The present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of LM1071 strain (KCCM12650P), or one or more of the culture, lysate, and extract of the mixed strain is effective An object of the present invention is to provide a composition for alleviating premenstrual syndrome comprising as a component.

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

The first aspect of the present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of the LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a pharmaceutical composition for preventing or treating premenstrual syndrome, comprising one or more as an active ingredient.

The second aspect of the present application is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a food composition for relieving menstrual pain, comprising one or more as an active ingredient.

A mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) according to an embodiment of the present application reduces blood NO production and the expression of IL-6, iNOS and COX-2 genes, and By increasing the prostaglandin E ₁ / prostaglandin E ₂ production rate and blood GLA and DGLA levels, premenstrual syndrome can be alleviated, and the mixed strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like.

1 is an experiment for examining the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome, according to an embodiment of the present application. It is a diagram showing the comparison of NO production capacity between the strain-treated group and the control group and the results of measuring the NO production capacity by mixing ratio of the mixed strain.
Figure 2 shows the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome, according to an embodiment of the present application. In the experiment, it is a diagram showing the results of comparing and measuring IL-6, iNOS and COX-2 gene expression ability between the strain-treated group and the control group.
Figure 3 shows the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome, according to an embodiment of the present application. In the experiment, it is a diagram showing the results of analyzing PGE ₁ /PGE ₂ values by ELISA analysis of blood collected from rats 3 days before the last administration of the test substance.
Figure 4 shows the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome, according to an embodiment of the present application. In this experiment, the concentration of GLA and DGLA in the blood was analyzed using the LC-MS/MS (Liquid Chromatograph-Tandem Mass Spectrometer; Liquid Chromatograph-Multi-Mass Spectrometer) method for the serum obtained by collecting blood on the last day of administration of the test substance. is a diagram showing

Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art to which the present application pertains can easily implement them. However, the present application may be implemented in several different forms and is not limited to the embodiments described herein. And in order to clearly explain the present application in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout this specification, when a member is said to be located “on” another member, this includes not only a case in which a member is in contact with another member but also a case in which another member is present between the two members.

Throughout this 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.

As used throughout this specification, the terms "about," "substantially," and the like are used in a sense at or close to the numerical value when the manufacturing and material tolerances inherent in the stated meaning are presented, and are intended to enhance the understanding of this application. To help, precise or absolute figures are used to prevent unfair use by unscrupulous infringers of the stated disclosure. The term "step of" or "step of" as used throughout this specification does not mean "step for".

Throughout this specification, the term "combination(s) of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means to include one or more selected from the group consisting of the above components.

Throughout this specification, reference to “A and/or B” means “A or B, or A and B”.

Hereinafter, embodiments and examples of the present application will be described in detail with reference to the accompanying drawings. However, the present application may not be limited to these embodiments and examples and drawings.

The first aspect of the present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) One of the mixed strains or cultures, lysates, and extracts of the LM1071 strain (KCCM12650P) It provides a pharmaceutical composition for preventing or treating premenstrual syndrome, comprising the above as an active ingredient.

In one embodiment of the present application, the mixed strain may be to reduce the amount of NO production in the blood.

The term “nitric oxide (NO)” used throughout the present specification refers to a compound in which nitrogen is oxidized, called “nitric oxide” or “nitrogen monoxide”. It is formed from arginine, an amino acid in cells, and is involved in various physiological activities such as immune function, vasodilation and signal transduction as a kind of signal transduction material. It is also known to induce inflammation and pain by promoting the secretion of inflammatory cytokines such as TNF-α and IL-6 (Hu et al., 2020).

Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) The mixed strain of the LM1071 strain (KCCM12650P) reduces the amount of NO production, which can exhibit a menstrual pain relief effect.

The term “Griess reagent” used throughout the present specification refers to a reagent used in a Griess test for confirming the presence of nitrite ions NO ₂ ⁻ . In general, it is prepared by mixing a solution of 0.5 g of sulfanilic acid in 150 ml of diluted acetic acid and a solution of 0.1 g of a-naphthylamine in 20 ml of water and 150 ml of acetic acid. The NO production ability measurement experiment is an experiment using the above grease reagent, and through this, the expression amount of NO can be measured.

The Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of LM1071 strain (KCCM12650P) reduces the expression of IL-6, iNOS and COX-2 genes, thereby As a result, it may have an effect of relieving menstrual cramps.

The term "IL-6 (InterLeukin-6)" used throughout the present specification refers to a physiologically active protein called a cytokine together with TNF-α (Tumor Necrosis Factor-α). IL-6 is secreted from various cells, such as T lymphocytes and macrophages, and promotes immune responses. In particular, IL-6 is involved in inflammatory responses, causing inflammation and pain in the process of regulating high fever or acute phase proteins.

The term "iNOS (inducible NO synthase)" used throughout the present specification is an inducible NO synthase, and expression is induced by cytokine stimulation in immune system cells to use L-arginine as a substrate to synthesize nitric oxide (NO). it means. The gene expression of iNOS is closely related to the inflammatory response, and the gene is induced in cells such as macrophages, smooth muscle cells, and hepatocytes by stimulation of inflammatory cytokines to generate NO.

As used throughout this specification, the term “COX-2 (cyclooxygenase-2)” refers to an enzyme that produces prostaglandins, and the expression level is increased along with an inflammatory reaction.

The term "lipopolysaccharide (LPS)" used throughout this specification refers to a representative pathogenic substance that induces a macrophage-mediated inflammatory response. LPS binds to Toll-like receptor 4 (TLR4) expressed on the surface of macrophages and induces an inflammatory response through the secretion of inflammatory cytokines and substances. This inflammatory response is also induced by the inflammatory complex (inflammasome) present in the cell. When the inflammatory complex is activated, pyroptosis, a cell death caused by the inflammatory response, and interleukin-1 beta, an inflammatory cytokine, 1β, IL-1β) and interleukin-18 (IL-18) secretion is increased. In macrophage-mediated inflammatory responses, mouse caspase-11 and its human homologues, caspase-4/5, are also known to induce pyroptosis and secretion of IL-1β and IL-18, which are derived from Gram-negative bacteria. It was found to induce an inflammatory response by directly recognizing intracellular LPS (LPS) in a cell (Lee, 2017).

Therefore, the Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of LM1071 strain (KCCM12650P) reduces the expression of IL-6, iNOS and COX-2 genes , it can be seen that it can be effective in relieving menstrual pain and treating or preventing premenstrual syndrome.

Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) The mixed strain of the LM1071 strain (KCCM12650P) increases the production rate of prostaglandin E ₁ / prostaglandin E ₂ in the blood due to this It may have the effect of relieving menstrual cramps and premenstrual syndrome.

The term "prostaglandin" as used throughout the present specification refers to a kind of hormone secreted during menstruation, which contracts the uterus to smoothly push the menstrual blood when the endometrium is peeled off to the outside of the body.

Prostaglandins are converted from arachidonic acid to prostaglandins using a cyclooxygenase (COX) system, and arachidonic acid is converted to prostaglandin G2 and then to prostaglandin H2. Prostaglandins with various physiological activities can be produced. In addition, prostaglandins have been reported as substances that cause inflammation and pain (Jayesh et al., 2020).

As used throughout this specification, the term “PGE ₂ (prostaglandin E ₂ )” is also referred to as “dinoprostone” and refers to a naturally occurring prostaglandin having oxytocin properties.

PGE ₂ induces pain through inflammation mediating in tissues or cells damaged by G protein bound to PGE ₂ receptor (Treutlein et al., 2018), and stimulates the central nervous system and peripheral nervous system (Grφsch et al., 2017).

In one embodiment of the present application, the mixed strain may increase the blood prostaglandin E ₁ / prostaglandin E ₂ ratio, and specifically, the strain may increase the production ratio of prostaglandin E ₁ to prostaglandin E ₂ .

As used throughout this specification, the term "PGE ₁ (prostaglandin E ₁ )" is also referred to as "alprostadil" and refers to a naturally occurring prostaglandin that expands blood vessels in the body, relaxes smooth muscle and reduces the secretion of inflammatory cytokines. It is known that it has a pain-relieving effect (Gezginci-Oktayoglu et al., 2016).

The ratio of PGE ₁ /PGE ₂ (PGE ₂ to PGE ₁ ) production is a major indicator within the guidelines of the Ministry of Food and Drug Safety on premenstrual syndrome in women.

Therefore, the Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) The mixed strain of the LM1071 strain (KCCM12650P) increases the prostaglandin E ₁ / prostaglandin E ₂ production rate in the blood. It can be seen that it can be effective in relieving menstrual pain and premenstrual syndrome.

In one embodiment of the present application, the mixed strain may increase the level of GLA or DGLA in the blood.

As used throughout the present specification, the term "GLA (Gamma-linolenic acid)" refers to fatty acids mainly found in vegetable oils.

In women with premenstrual syndrome, metabolic disorders of prostaglandins are found. Because prostaglandins are involved in central nervous system function, fluid balance, and uterine contractility regulation, premenstrual syndrome may occur if there is a prostaglandin metabolism disorder. Therefore, it is known that if women who have a defect in the process of converting linoleic acid into gamma-linolenic acid in the prostaglandin synthesis process can consume GLA, they can synthesize prostaglandin immediately, which can help alleviate the symptoms of PMS.

The term "DGLA (Dihomo-Gamma-linolenic acid)" used throughout this specification refers to an omega-6 fatty acid composed of 20 carbons and is mainly contained in seed oil of plants. When linoleic acid, an essential fatty acid, is consumed, GLA is synthesized slowly, and then GLA is rapidly converted to DGLA.

DGLA is metabolized to produce PGE ₁ and 15-OH-DGLA. As described above, PGE ₁ is effective in inhibiting inflammation, and 15-OH-DGL inhibits 5-lipoxygenase and 12-lipoxygenase to produce pro-inflammatory metabolites such as PGE ₂ and LTB4 produced from arachidonic acid. interfere

Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) The mixed strain of the LM1071 strain (KCCM12650P) improves the production of GLA and DGLA, which can exhibit a menstrual pain relief effect have.

The term "treatment" used throughout the present specification is Lactobacillus gasseri of the present application ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) Pharmaceutical containing a mixed strain of LM1071 strain (KCCM12650P) as an active ingredient By administering the composition to an individual with premenstrual syndrome, it means any action to improve or benefit the symptoms of premenstrual syndrome.

In one embodiment of the present application, the composition may be to treat or prevent premenstrual syndrome, specifically, reduces blood NO production and the expression of IL-6, iNOS and COX-2 genes, and blood prostaglandin E ₁ / prostaglandin It may be to treat or prevent PMS by increasing the E ₂ production rate and the levels of GLA and DGLA.

As used throughout this specification, the term "premenstrual syndrome" refers to a series of symptoms characterized by emotional, behavioral, and physical symptoms that occur repeatedly before menstruation, and includes edema, breast pain, digestive disorders, headache, back pain, lower abdominal pain, and abdominal pain. These include bloating, constipation, diarrhea, depression and insomnia.

In one embodiment of the present application, the pharmaceutical composition is in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, respectively, according to a conventional method. It may be formulated and used, but may not be limited thereto.

In one embodiment of the present application, when formulating the pharmaceutical composition, it may be prepared using a diluent or excipient such as a generally used filler, extender, binder, wetting agent, disintegrant, or surfactant, but limited thereto it may not be

In one embodiment of the present application, solid preparations for oral administration include tablets, pills, powders, granules, or capsules, and these solid preparations contain at least one excipient, for example, in the dead cells of the strain. It may be prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used, but may not be limited thereto.

In one embodiment of the present application, liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, for example, wetting agents, Sweeteners, fragrances, preservatives, etc. may be included, but may not be limited thereto.

In one embodiment of the present application, preparations for parenteral administration may include, but may not be limited to, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. For example, the non-aqueous solvent or suspending agent may include, but may not be limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. For example, as the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, etc. may be used, but may not be limited thereto.

The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

As used throughout this specification, the term "quasi-drug" refers to articles with a milder action than pharmaceuticals among articles used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases of humans or animals, for example, the Pharmaceutical Affairs Act. According to the Act, quasi-drugs exclude products used for pharmaceutical purposes, and include products used for the treatment or prevention of diseases in humans and animals, and products with minor or no direct action on the human body.

The quasi-drug composition of the present application consists of a body cleanser, disinfectant cleaner, detergent, kitchen cleaner, cleaning agent, toothpaste, gargle, wet tissue, detergent, soap, hand wash, hair cleaner, hair softener, humidifier filler, mask, ointment, and filter filler. It may be prepared in a formulation selected from the group, but is not limited thereto.

In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" as used herein means treating a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. or an amount sufficient to prevent, the effective dose level is the severity of the disease, the drug activity, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and excretion rate, treatment period, factors including drugs used in combination with or concurrently with the composition of the present invention, and other factors well known in the medical field. The pharmaceutical composition of the present application may be administered alone or in combination with a component known to exhibit a therapeutic effect on a known intestinal disease. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects.

In one embodiment of the present application, the dosage of the pharmaceutical composition can be determined by those skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient, etc. have. For example, the pharmaceutical composition of the present invention may be administered in an amount of about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present invention is particularly limited thereto. However, it can be administered once a day or administered several times by dividing the dose. The dosage or frequency of administration is not intended to limit the scope of the present application in any way.

The pharmaceutical composition of the present application is not particularly limited thereto, but as desired, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. It can be administered through the route of However, when administered orally, it can be administered in an unformulated form, and since the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain may be denatured or degraded by gastric acid, the oral composition contains the active agent. It can also be administered orally in the form of a coating or formulated to be protected from degradation in the stomach or in the form of an oral patch. In addition, the composition may be administered by any device capable of transporting the active substance to the target cell.

The second aspect of the present application is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a food composition for relieving menstrual pain, comprising one or more as an active ingredient. The contents overlapping with the first aspect are equally applied to the food composition of the second aspect.

As used throughout this specification, the term “relief” refers to any action in which menstrual pain is relieved or related symptoms are improved by administration of the composition.

In one embodiment of the present application, the composition may be to relieve menstrual pain, specifically, reduces the amount of NO production in the blood and the expression of IL-6, iNOS and COX-2 genes, and the blood prostaglandin E ₁ / prostaglandin E ₂ production ratio and by increasing the levels of GLA and DGLA, it may be to relieve menstrual cramps.

In one embodiment of the present application, the composition is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or live cells, dead cells, cultures, lysates and/or its It may contain an extract.

As used throughout this specification, the term "dead cells" is the opposite concept of live cells, and refers to a form in which the growth of live cells and metabolites obtained through fermentation is prevented from occurring by heat treatment or the like. The dead cells may include cytoplasm, cell wall, antibacterial active substances such as bacteriocin, polysaccharide, organic acid, and the like. The product using the dead cells has high stability compared to the live cell product, has excellent heat resistance, and has high stability to the external environment, so that it is easier to store than the existing live cell product and has the advantage of being able to extend the shelf life. In addition, as regulations on the use of antibiotics are being strengthened, the marketability and growth potential are very large, as there are only a handful of companies that have yet to enter into full-scale production of dead cells and their utility as an alternative.

As used throughout this specification, the term "culture" refers to a thing obtained by culturing the strain of the present application in a known liquid medium or solid medium, and may be used interchangeably with "culture medium".

The term "food" used throughout this specification refers to dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, and ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the ordinary sense.

As used throughout this specification, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients useful for the human body according to Act No. 6727 on Health Functional Foods, and is referred to as 'functionality'. means to obtain useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body.

The food of the present application can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, the formulation of the food may also be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage that there are no side effects that may occur during long-term administration of the drug using food as a raw material, and is excellent in portability, so the present invention Foods of can be ingested as a supplement to enhance the effect of improving the intestinal environment.

The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement may be used interchangeably. Specifically, the health functional food is a mixture of the Lactobacillus gasseri LM1065 strain (KCCM13018P) and the Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application is added to food materials such as beverages, teas, spices, gum, confectionery, or the like, It is a food manufactured by encapsulation, powder, suspension, etc., which means that certain health effects are obtained when ingested. There is this.

Since the food composition of the present application can be consumed on a daily basis, a high effect on the improvement of depression can be expected, and thus it can be very usefully used.

The food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. Also, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr). In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, D-potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, improvers, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less with respect to the food or beverage. However, when ingested for a long period of time for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

As an example of the food composition of the present application, it may be used as a health drink composition, and in this case, it may contain various flavoring agents or natural carbohydrates as an additional component like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as taumatine, stevia extract; A synthetic sweetener such as saccharin or aspartame may be used. The ratio of the natural carbohydrate may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid, pectic acid salts, alginic acid, alginic acid salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol, a carbonation agent, and the like. In addition, it may contain the pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These components may be used independently or in combination. Although the ratio of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The food composition of the present application may include a mixed strain of the Lactobacillus gasseri LM1065 strain (KCCM13018P) and the Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application in various weight % if it can exhibit an effect of improving the intestinal environment, specifically, the present application The mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto.

In one embodiment of the present application, the food composition may be a health functional food composition.

Hereinafter, the present invention will be described in more detail through examples of the present application, but the following examples are merely illustrative to aid the understanding of the present application, and the content of the present application is not limited to the following examples.

Example 1: NO production ability measurement experiment after LPS treatment (NO assay)

The following experiment was conducted to confirm the ability to produce NO, an important biomarker in the immune response.

RAW 264.7 cells, which are macrophages of mice, were inoculated into a 96-well plate at a concentration of 1x10 ⁵ cell/well. The culture plate was incubated for 24 hours at 37° C., 5% CO ₂ environment.

Next, after removing the supernatant of the culture plate after culturing, a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) was treated at a concentration of 1x10 ⁸ CFU/ml for 2 hours Cultured in an incubator.

Then, it was treated with LPS at a concentration of 1 ug/ml and further cultured for 24 hours.

Finally, 100ul of the supernatant of the culture plate was dispensed into a new culture plate, and the absorbance at 540 nm was measured with an ELISA reader machine after reaction with Griess reagent.

As a result, compared to the positive control (PC; Positive Control) treated only with LPS, when a mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was additionally cultured, NO production at 1x10 ⁸ CFU/ml concentration was statistically significant. was confirmed to be significantly reduced (see FIG. 1).

In addition, in the mixing ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain, experiments were performed at a ratio of 5:5, 4:6, 3:7, 2:8, and 1:9, respectively. As the ratio of the Bacillus reuteri LM1071 strain increased, it was confirmed that the amount of NO production decreased. However, when the mixing ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain was 3:7 or more, there was no significant difference. Therefore, it could be confirmed that the optimal ratio was 3:7 to 1:9, and all subsequent experiments were performed with the ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain being 3:7 (see FIG. 1). ).

Through the above results, it was confirmed that the mixed strain of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain of the present application exhibits an effect of alleviating menstrual pain and premenstrual syndrome by inhibiting the production of NO.

Example 2: Experiment for measuring anti-inflammatory-related gene expression using real-time PCR

In order to confirm the expression level of the anti-inflammatory genes IL-6, iNOS and COX-2, the following experiment was conducted.

RAW 264.7 cells, which are macrophages of mice, were inoculated into a 24 well plate at a concentration of 1x10 ⁵ cell/well. The culture plate was incubated for 24 hours at 37° C., 5% CO ₂ environment.

Next, after removing the supernatant of the culture plate after the culture was completed, a mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was treated at a concentration of 1x10 ⁸ CFU/ml and then cultured in an incubator for 2 hours.

Then, it was treated with LPS at a concentration of 1 ug/ml and further cultured for 24 hours.

Then, RNA was extracted using an RNA extraction kit (RNA extraction kit [TaKaRa]), and cDNA was synthesized and extracted using a high-capacity cDNA Reverse Transcription kit [Applied Biosystems]. It was diluted to a final concentration of 5 ng/ul.

Finally, the expression levels of IL-6, iNOS and COX-2 genes were compared and confirmed by performing real-time PCR using SYBR Green PCR Master Mix [Applied Biosystems].

As a result, when a mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was additionally cultured compared to the positive control (PC; Positive Control) treated only with LPS, IL-6 at a concentration of 1x10 ⁸ CFU/ml, It was confirmed that the iNOS and COX-2 gene expression levels decreased to a statistically significant level (see FIG. 2 ).

Based on the above results, it can be confirmed that the mixed strain of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain of the present application suppresses IL-6, iNOS and COX-2 gene expression, thereby exhibiting the effect of alleviating dysmenorrhea and premenstrual syndrome. there was.

Example 3: PMS improvement effect evaluation experiment using SD Rat

The following experiment was performed to evaluate the improvement effect when the mixed strain of the present application was repeatedly administered daily for 4 weeks to a SD (Sprague Dawley) rat model induced by PMS (premenstrual syndrome).

The rats used in this example are specific pathogen-free (SPF) rats produced and supplied by Coretech Co., Ltd. For 63 6-week-old females, the temperature was 23 ± 3 °C, the relative humidity was 55 ± 15%, the number of ventilation was 10 to 20 times/hr, and the lighting time was 1 hour (lights from 8:00 am to pm to 8:00 pm) and Experiments were performed in a laboratory set with an illumination intensity of 150 to 300 Lux.

In the case of administration of the test substance, an appropriate amount of the test substance was weighed and diluted in sterile water for injection to prepare a prescribed concentration. In the case of administration method, it was administered once/day by oral administration method for 4 weeks, and the amount of the administration liquid was calculated as 10 mL/kg based on the body weight measured on the latest weighing day.

Vaginal cornification (Smear cytology) examination was performed at the 4th week of administration of the test substance. Blood collection (Day26, Day27, Day28) was performed once/day for 3 days from 3 days before the last administration of the test substance. Blood was collected through the jugular vein, and the collected blood was injected into a vacuum tube containing a clot activator, left at room temperature for about 15 minutes to coagulate, and then centrifuged at 3,000 rpm for 10 minutes. Serum was isolated. Serum was stored in a deep freezer set at -70° C. or lower until analysis, and used for ELISA analysis. At each blood sampling point, PGE ₁ and PGE ₂ concentrations and PGE ₁ /PGE ₂ concentrations were analyzed using a portion of the serum obtained by centrifugation using an ELISA kit. For the analysis, a commercially available ELISA kit was used.

The concentrations of GLA and DGLA in the blood were analyzed using the LC-MS/MS method using the serum collected on Day 26 from the last administration day of the test substance.

Normality of the data was assumed for the results of this example, and analysis was performed using prametric multiple comparison procedures or non-parametric multiple comparison procedures. If the results of the one-way ANOVA were significant, a post hoc test was performed using Dunnett's multiple comparison test, and a non-parametric Kruskal-Wallis H test (Kruskal-Wallis' H-test) was performed. When the analysis result was significant, a post hoc test was performed using the Mann-whitney test. Statistical analysis was performed using Prism 7.04 (GraphPad Software INC., San Diego, CA, USA), and when the p-value was less than 0.05, it was determined to be statistically significant.

As a result of the examination using a vaginal cornification, no difference was observed in the keratinocyte change patterns observed in estrus between all experimental groups.

As a result of ELISA analysis, the prostaglandin E ₁ / prostaglandin E ₂ ratio of the mixed strain administration group of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain was significantly higher than that of the normal control group on Day 28 (p<0.05, FIG. 3 ) Reference).

Also, as a result of GLA and DGLA analysis, it was observed that the GLA and DGLA levels were also significantly higher than that of the normal control group (p<0.05, see FIG. 4 ).

When the test substance was repeatedly administered to the SD Rat model induced by PMS under these experimental conditions for 4 weeks, the ratio of prostaglandin E ₁ / prostaglandin E ₂ increased in the mixed strain administration group of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain and increased plasma concentrations of GLA and DGLA. It appears that the administration of the test substance induced an increase in PGE ₁ and a decrease in PGE ₂ to induce symptom improvement in the PMS model.

As such, the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain increases the PGE ₁ /PGE ₂ ratio and promotes the production of GLA and DGLA, which are anti-inflammatory substances, and can contribute to alleviating menstrual pain and premenstrual syndrome. could confirm that there was

Overall, the mixed strain of the present application, or a composition comprising a culture of the mixed strain, suppresses the production of NO, which is an inflammatory and pain-inducing substance, and inhibits the expression of anti-inflammatory-related genes IL-6, iNOS and COX-2, , It was confirmed that PGE ₁ /PGE ₂ ratio, which is a major indicator related to PMS, was increased, and the production of GLA and DGLA, which are anti-inflammatory substances, was stimulated, thereby showing an effect in relieving PMS.

The foregoing description of the present application is for illustration, and those of ordinary skill in the art to which the present application pertains will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present application.

Korea Microorganism Conservation Center (Overseas) KCCM13018P 20210622 Korea Microorganism Conservation Center (Overseas) KCCM12650P 20191231

Claims (10)

Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of the LM1071 strain (KCCM12650P) or a culture of the mixed strain, a lysate, comprising at least one of an extract as an active ingredient , a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain is to reduce the amount of NO (nitric oxide) production in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain is to reduce the expression of IL-6, iNOS and COX-2 genes, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain is prostaglandin E ₁ / prostaglandin E ₂ (PGE ₁ /PGE ₂ ) in the blood to increase the ratio, the pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain increases the level of DGLA in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain increases the GLA level in blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The mixed strain is Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P) is a mixture of 3:7 to 1:9 ratio, menstrual pain prevention or treatment pharmaceutical composition for use.
delete Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) A mixed strain of the LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain. A food composition for relieving menstrual pain.
10. The method of claim 9,
The mixed strain is Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P) is a mixture of 3:7 to 1:9 ratio, menstrual pain relief food composition.

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