KR102268122B1 - A novel strain of lactobacillus paracasei lm1014 separated from kimchi, and composition for relieving premenstrual syndrome comprising the strain or its culture fluid - Google Patents

Abstract

The present invention relates to a novel strain of Lactobacillus paracasei LM1014 (KCCM12917P) and a composition for relieving premenstrual syndrome including the same or culture fluid thereof. According to one embodiment of the present invention, a Lactobacillus paracasei LM1014 strain (KCCM12917P) reduces blood NO, prostaglandin E2, and leukotriene, increases the production rate of prostaglandin E1/prostaglandin E2, and increases epoxyeicosatrienoic acid (EET) to alleviate premenstrual syndrome so that the strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like.

Description

A novel Lactobacillus paracasei LM1014 strain derived from kimchi, and a composition for alleviating premenstrual syndrome comprising the strain or a culture thereof ITS CULTURE FLUID}

The present application, Kimchi-derived Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), relates to a composition for alleviating premenstrual syndrome comprising the same.

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 and are most severe one week before menstruation and disappear within a few days after menstruation begins.

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

According to the study results of 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. Among female students, 10-50% said that menstrual cramps limited their daily activities, including school life. In the case of industrial settings, an estimate of the damage caused by menstrual cramps resulted in the loss of 600 million hours of work and $2 billion in lost work every year. There was a loss of productivity.

Therefore, the research 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 PMS.

Accordingly, the present inventors made diligent efforts to develop an excellent composition capable of alleviating premenstrual syndrome. As a result, NO, prostaglandin E2, and leukotriene were reduced, and the production rate of prostaglandin E1/prostaglandin E2 in the blood and epoxyeicosatrienoic acid (EET) ) to develop a new strain to increase, completed the present invention.

The present application is to provide a novel Kimchi-derived Lactobacillus paracasei (Lactobacillus paracasei) LM1014 strain (KCCM12917P), a composition for alleviating premenstrual syndrome comprising the strain.

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

A first aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P) is provided.

The second aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, and an extract comprising at least one of the extract as an active ingredient, provides a food composition for relieving menstrual pain.

The third aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, a pharmaceutical composition for the prevention or treatment of premenstrual syndrome comprising one or more of the extract as an active ingredient provides

Lactobacillus paracasei LM1014 strain (KCCM12917P) according to an embodiment of the present application reduces blood NO, prostaglandin E2, and leukotriene, and the production rate of prostaglandin E1/prostaglandin E2 and epoxy eicosatrienoic acid (EET) By increasing, it is possible to alleviate premenstrual syndrome, the strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like.

1 is a diagram showing the change in the amount of NO (nitric oxide) secretion by the Lactobacillus paracasei LM1014 strain in macrophages.
2 is a view showing the change in the amount of PGE2 secretion by the Lactobacillus paracasei LM1014 strain in macrophages.
3 is a view showing the change in the amount of PGE1 secretion by the Lactobacillus paracasei LM1014 strain in macrophages.
Figure 4, Lactobacillus paracasei in macrophages ( Lactobacillus paracasei ) It is a view showing the change of the PGE1/PGE2 ratio by the LM1014 strain.
5 is. Lactobacillus paracasei ( Lactobacillus paracasei ) It is a diagram showing the change in the amount of LTB4 secretion by the LM1014 strain.
6 is a view showing the change in the amount of EET secretion by the Lactobacillus paracasei LM1014 strain.

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 embodied 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 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 the present application. To help, precise or absolute figures are used to prevent unfair use by unscrupulous infringers of the stated disclosure. As used throughout this specification, the term “step of (to)” or “step of” does not mean “step for”.

Throughout this specification, the term “combination(s)” 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.

A first aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P) is provided.

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

The term "NO (nitric oxide)" used throughout the present specification refers to a compound in which nitrogen is oxidized, which is called "nitrogen oxide" or "nitrogen monoxide." It is formed from arginine, an amino acid in cells, and is involved in various physiological activities such as immune action, 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).

Therefore, the Lactobacillus paracasei of the present application ( Lactobacillus paracasei ) LM1014 strain reduces the amount of NO production, it can be seen that this can exhibit a menstrual pain relief effect.

In one embodiment of the present application, the strain may be to reduce prostaglandin E2 in the blood.

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

Prostaglandin is the conversion of arachidonic acid to prostaglandin using the cyclooxygenase (COX) system, and arachidonic acid is converted to prostaglandin G2 and then to prostaglandin H2. In addition, prostaglandins have been reported as substances that cause inflammation and pain (Jayesh et al., 2020).

As used throughout this specification, the term "PGE2 (prostaglandin E2)" is also referred to as "dinoprostone" and refers to a naturally occurring prostaglandin with oxytocin properties.

PGE2 induces pain through inflammatory mediation in damaged tissues or cells by G protein bound to the PGE2 receptor (Treutlein et al., 2018), and stimulates the central nervous system and peripheral nervous system (Grφsch et al., 2017).

Therefore, it can be seen that the Lactobacillus paracasei LM1014 strain of the present application reduces prostaglandin E2 in the blood, thereby exhibiting an effect in relieving menstrual pain.

In one embodiment of the present application, the strain may increase the ratio of prostaglandin E1/prostaglandin E2 in the blood, and specifically, the strain may increase the ratio of prostaglandin E1 to prostaglandin E2 production.

The term "PGE1 (prostaglandin E1)" as used throughout this specification is also called "alprostadil" and refers to a naturally occurring prostaglandin that expands blood vessels in the body, and causes pain through relaxation of smooth muscle and reduction of secretion of inflammatory cytokines. It is known to have a relieving effect (Gezginci-Oktayoglu et al., 2016).

The ratio of PGE1/PGE2 (PGE2 to PGE1) production is a major indicator within the guidelines of the Ministry of Food and Drug Safety for women with premenstrual syndrome.

Therefore, it can be seen that the Lactobacillus paracasei LM1014 strain of the present application increases the production rate of prostaglandin E1/prostaglandin E2 in the blood, thereby exhibiting an effect on alleviating menstrual pain and premenstrual syndrome.

In one embodiment of the present application, the strain may be to reduce blood leukotriene.

As used throughout this specification, the term "leukotriene (LT)" refers to an inflammatory mediator of the eicosanoid family produced by metabolizing arachidonic acid by 5-lipoxygenase, and inflammation It is known as a substance that induces reactions and vascular damage (Tunctan et al., 2019). Accordingly, studies for alleviating inflammation and pain through inhibition of arachidonic acid metabolism have been continuously reported (Cheng et al., 2019; Jayesh et al., 2020).

Therefore, it can be seen that the Lactobacillus paracasei LM1014 strain of the present application reduces leukotriene in the blood, thereby exhibiting an effect in relieving menstrual pain.

In one embodiment of the present application, the strain may be to increase the blood epoxy eicosatrienoic acid (EET).

As used herein, the term "epoxyeicosatrienoic acid (EET)" refers to a substance formed through two pathways in an unsaturated fatty acid having 20 carbons, such as arachidonic acid, as a signaling molecule. Epoxyeicosatrienoic acid is known to mediate inflammation by inhibiting the activity of NK-kB, preventing the increase of inflammatory cytokines such as IL-6, and promoting the activity of the peroxisome proliferator-activated receptor (PPAR) (Tunctan et al. , 2019).

Therefore, it can be seen that the Lactobacillus paracasei LM1014 strain of the present application increases epoxy eicosatrienoic acid in the blood, thereby exhibiting an effect in alleviating premenstrual syndrome.

In one embodiment of the present application, the strain may be to alleviate premenstrual syndrome, specifically, the strain may be included in various compositions such as a food composition for premenstrual syndrome alleviation, a health functional food composition, a pharmaceutical composition, and the like.

The second aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, comprising at least one of an extract as an active ingredient, provides a food composition for alleviating premenstrual syndrome do. 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 “alleviation” refers to any action in which premenstrual syndrome is alleviated or related symptoms are improved by administration of the composition.

In one embodiment of the present application, the composition may be to relieve premenstrual syndrome, specifically, reduce blood NO, prostaglandin E2, and leukotriene, and the production rate of prostaglandin E1/ prostaglandin E2 in blood and epoxyeicosatrienoic acid ( EET) may be alleviating premenstrual syndrome.

In one embodiment of the present application, the composition may include a Lactobacillus paracasei LM1014 strain, a live cell thereof, a dead cell thereof, a culture thereof, a lysate thereof, and/or an extract thereof.

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 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 high, 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".

As used throughout this specification, the term "food" 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 in the human body according to Act No. 6727 of the Health Functional Food Act, 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 consumed 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 food prepared by adding the Lactobacillus paracasei LM1014 strain of the present application to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc. It means that it brings a specific effect on health, but unlike general drugs, it has the advantage of not having side effects that can occur when taking a drug for a long time using food as a raw material.

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). It may also 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) , sodium, etc.), flavorings (vanillin, lactones, etc.), expanding 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 Lactobacillus paracasei LM1014 strain 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, in the production of 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 consumed for a long period of time for health and hygiene purposes, it may contain an amount less than 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 taumatin, 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 regulators, 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 the Lactobacillus paracasei LM1014 strain of the present application in various weight % if it can exhibit an effect of improving the intestinal environment, specifically, the Lactobacillus paracasei LM1014 strain of the present application relative to the total weight of the food composition It may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight, but is not limited thereto.

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

The third aspect of the present application, Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, a pharmaceutical composition for the prevention or treatment of premenstrual syndrome comprising one or more of the extract as an active ingredient provides The contents overlapping with the first aspect and the second aspect are equally applied to the pharmaceutical composition of the third aspect.

In one embodiment of the present application, the composition may include a Lactobacillus paracasei LM1014 strain, a live cell thereof, a dead cell thereof, a culture thereof, a lysate thereof, and/or an extract thereof.

As used throughout this specification, the term "treatment" refers to administering a pharmaceutical composition comprising the Lactobacillus paracasei LM1014 strain of the present application as an active ingredient to an individual with PMS to improve or benefit from the symptoms of PMS. means all actions

In one embodiment of the present application, the composition may be for treating or preventing premenstrual syndrome, and specifically, it reduces blood NO, prostaglandin E2, and leukotriene, and the production rate of prostaglandin E1/ prostaglandin E2 and epoxyeicosatrienoic acid by increasing (EET) may be treating or preventing premenstrual syndrome.

As used throughout this specification, the term "premenstrual syndrome" refers to a group 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, There are 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 is 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 such solid preparations include at least one excipient in the dead cells of the strain, for example, It may be prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. Also, for example, lubricants such as magnesium stearate and talc may be used in addition to simple excipients, 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 water and liquid paraffin, which are commonly used simple diluents, 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" herein refers to treatment of 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 activity of the drug, 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 duration, 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 capable of obtaining the maximum effect with a minimum amount without side effects.

In one embodiment of the present application, the dosage of the pharmaceutical composition may be determined by those skilled in the art in consideration of the purpose of use, the degree of disease addiction, 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 may 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 depending on the purpose, 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 may be administered in an unformulated form, and since the Lactobacillus paracasei LM1014 strain may be denatured or degraded by gastric acid, the oral composition may be coated with an active agent or protected from degradation in the stomach. It can also be administered orally in a form formulated to be protected or in the form of an oral patch. In addition, the composition may be administered by any device capable of transporting an active substance to a target cell.

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 understanding of the present application, and the content of the present application is not limited to the following examples.

[Example]

Preparation of premenstrual syndrome relief composition comprising Lactobacillus paracasei LM1014

The Lactobacillus paracasei LM1014 cells according to this example were prepared by a batch culture method.

The lactic acid bacteria culture was carried out in a 100 L fermenter (Cobiotech) with a total amount of 60 L of the culture solution, the stirring speed was 30 rpm, the temperature was 30 ° C. to 37 ° C., and the pH was maintained at 5 to 6.5. After incubation, the culture medium was centrifuged at 13,000 rpm for 30 minutes to recover the cells. The recovered cells were mixed with a cryoprotectant and frozen in a cryogenic freezer for 24 hours, and finally the cells (Lactobacillus casei LM1014) were obtained through a freeze-drying process.

RAW 264.7 Cell Culture and Cytotoxicity Experiment

RAW 264.7 macrophages were cultured in RPMI 1640 containing 10% FBS. In addition, for cytotoxicity experiments, it was seeded in a 96-well plate at a concentration of ^{1Х10 5 cells/well.} LPS-stimulated RAW 264.7 cells were treated with Lactobacillus paracasei LM1014 at four different concentrations (10, 20, 30, 40 ug/mL). The cell culture medium was discarded, and a cytotoxicity experiment was performed using the cultured cells, and for this, the EZ-Cytox Cell Viability Assay Kit (Daeil Labservice, Korea) was used. Cell proliferation ratio (%) was determined by the following formula.

[Equation]

In order to confirm the effect of relieving menstrual pain, the new strain of the present application, Lactobacillus paracasei LM1014 ( Lactobacillus paracasei LM1014, deposit institution: Korea Microbial Conservation Center, accession number: KCCM12917P, deposit date: December 23, 2020) The same experiment was performed.

Example 1: Confirmation of change in the amount of nitric oxide (NO) produced by lactic acid bacteria treatment

To measure the NO production ability using macrophage RAW 264.7 cells, RAW 264.7 cells ^{were aliquoted at 1×10 5} cells/well in a 96 well plate, and cultured in an incubator at 37° C. and 5% CO _{2 conditions for 24 hours.} After 24 hours, each concentration of Lactobacillus paracasei LM1014 was added, and _{incubated in an incubator at 37° C. and 5% CO 2} conditions for 24 hours, Griess reagent was added to 100 uL of the culture supernatant, and left at room temperature for 10 minutes. Then, the absorbance was measured at 540 nm using an ELISA plate reader. The concentration of nitrite, a metabolite of NO, was calculated from a standard curve obtained using sodium nitrite.

As a result of comparing the positive control group (LPS-treated group) with the Lactobacillus paracasei LM1014 (KCCM12917P) treatment group with 100%, it was confirmed that the significant reduction in NO generation caused by the Lactobacillus paracasei LM1014 strain treatment was confirmed. (p<0.05). As Lactobacillus paracasei LM1014 was treated with 10, 20, 30 and 40 μg/mL, respectively, it was confirmed that NO production was reduced to 87.57, 63.28, 14.26 and 8.22% compared to the positive control group, respectively (see FIG. 1). .

Based on the above results, Lactobacillus paracasei of the present application ( Lactobacillus paracasei ) LM1014 It was confirmed that the strain showed an effect on relieving menstrual pain by reducing the amount of NO production.

Example 2: Confirmation of changes in prostaglandin secretion by lactic acid bacteria treatment

RAW 264.7 macrophages were cultured in RPMI 1640 containing 10% FBS for PGE1 and PGE2 production ability and PGE1/PGE2 ratio analysis. In addition, for ELISA analysis, it was seeded in a 96-well plate at a concentration of ^{1×10 5 cells/well.} LPS-stimulated RAW 264.7 cells were treated with Lactobacillus paracasei LM1014 at four different concentrations (10, 20, 30, 40 ug/mL). The production and ratio of PGE1 (prostaglandin E1) and PGE2 (prostaglandin E2) (PGE1/PGE2) were analyzed by ELISA kit (Enzo Life Sciences, USA) using cultured cell cultures.

Experimental results In the Lactobacillus paracasei LM1014 strain test group, the secretion of PGE2 was 291.16, 224.23, 135.13, and 71.06 pg/mL, respectively (10, 20, 30 and 40 μg/mL lactic acid bacteria powder treatment), respectively. It was confirmed that there was a significant decrease (see FIG. 2 ).

In addition, the Lactobacillus paracasei LM1014 strain experimental group decreased the secretion of PGE1 to 3467.35, 3062.71, 2668.48 and 2434.98 pg/mL at the treatment concentrations of 10, 20, 30 and 40 µg/mL, and was positive at the treatment concentration of 40 µg/mL. It was reduced by 28.37% compared to the control group (LPS-treated control group) (see FIG. 3).

Finally, a relative change in secretion of PGE2 was confirmed through the ratio of PGE1 and PGE2, and as a result, it was confirmed that Lactobacillus paracasei LM1014 could increase the PGE1/PGE2 ratio to 34.31 (see FIG. 4 ).

Therefore, it was confirmed that the Lactobacillus paracasei LM1014 strain was effective in alleviating menstrual pain and premenstrual syndrome by reducing PGE2, a pain-causing substance, and increasing the production ratio of PGE1/PGE2, a major indicator related to PMS.

Example 3: Confirmation of changes in secretion amount of leukotriene (LTB4) and epoxyeicosatrienoic acid (EET) by lactic acid bacteria treatment

RAW 264.7 macrophages were cultured in RPMI 1640 containing 10% FBS for analysis of pain-inducing substance leukotriene (LTB4) and inflammation-reducing substance epoxyeicosatrienoic acid (EET). In addition, for ELISA analysis, it was seeded in a 96-well plate at a concentration of ^{1×10 5 cells/well.} LPS-stimulated RAW 264.7 cells were treated with Lactobacillus paracasei LM1014 at four different concentrations (10, 20, 30, 40 ug/mL). LTB4 and EET-containing cultured cell culture was analyzed by ELISA kit (Enzo Life Sciences, USA).

As a result of the experiment, the Lactobacillus paracasei LM1014 strain test group showed a significant decrease in LTB4 compared to the positive control group (LPS-treated group) at the concentrations of 20, 30 and 40 μg/mL, and the secretion amount of LTB4 was 91.29 at the concentration of 40 μg/mL. % decreased to 15.75 pg/mL (see FIG. 5).

In addition, the Lactobacillus paracasei LM1014 strain experimental group showed a higher concentration of EET secretion than the positive control group (LPS-treated group), confirming the possibility of pain relief through inhibition of the inflammatory response. Lactobacillus paracasei LM1014 increased the amount of EET secretion to 184.79, 190.25, 218.98 and 268.41 μg/mL according to the treatment concentration (see FIG. 6 ).

Therefore, it was confirmed that the Lactobacillus paracasei LM1014 strain suppressed leukotriene production and increased the amount of epoxy eicosatrienoic acid (EET) secretion, thereby showing efficacy in alleviating inflammation and pain.

Overall, it was confirmed that the Lactobacillus paracasei LM1014 strain can contribute to menstrual pain relief by inhibiting NO and PGE2 and LTB4, which are pain-causing substances, increasing the PGE1/PGE2 ratio, and promoting the production of EET, an inflammatory substance.

The above 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 dispersed form, and likewise components described as distributed may 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 equivalents should be construed as being included in the scope of the present application.

Korea Microorganism Conservation Center (Overseas) KCCM12917P 20201223

Claims (9)

Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, and a pharmaceutical composition for preventing or treating menstrual cramps comprising at least one of an extract as an active ingredient.
The method of claim 1,
The 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 strain is to reduce the secretion amount of prostaglandin E2 in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The strain is to increase the prostaglandin E1/prostaglandin E2 ratio in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The strain is to reduce the production of leukotriene in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
The method of claim 1,
The strain is to increase the production of epoxy eicosatrienoic acid (EET) in the blood, a pharmaceutical composition for preventing or treating menstrual pain.
Lactobacillus paracasei ( Lactobacillus paracasei ) LM1014 strain (KCCM12917P), a culture of the strain, a lysate, a food composition for relieving menstrual pain comprising at least one of the extract as an active ingredient.
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