KR102244732B1 - Probiotic acetic acid bacteria Acetobacter pasteurianus MGLV and its immunomodulatory effect - Google Patents

Abstract

The present invention relates to an Acetobacter pasteurianus MGLV strain which is deposited with accession number KCCM 12600P, and a use thereof. The strain of the present invention is a probiotic acetic acid strain isolated from naturally fermented food, and has immune enhancing activity, so the strain or a culture medium thereof can be used as a probiotic preparation, a starter composition for fermented food production, a composition for enhancing immunity, and an anti-inflammatory composition.

Description

Probiotic acetic acid bacteria Acetobacter pasteurianus MGLV and its immunomodulatory effect}

The present invention relates to a naturally fermented food-derived Acetobacter pasteurianus MGLV strain and its use.

Probiotics are living microorganisms that are beneficial to the host when ingested in an appropriate amount. Lactobacillus , Lactococcus , etc., soil microorganisms such as Bacillus , intestinal microorganisms such as Bifidobacterium, and yeasts such as Saccharomyces boulardii are known as probiotic microorganisms, and these strains are reported to have various health functions depending on the strain level. Has become. In order for microorganisms to act as probiotics, they must possess viability and beneficial functionality in the host's body.

Mild inflammatory reactions occur every moment in human and animal bodies due to various factors such as imbalance of the flora and fauna and exposure to potentially toxic substances. This phenomenon is the same in healthy humans and animals. Although overt symptoms do not appear, if this mild inflammatory reaction persists, the immune system is unnecessarily activated, and inflammatory metabolites continue to accumulate in the body. It not only puts an immunological burden on the body, but also accelerates the aging phenomenon. Therefore, it is essential to maintain health, well-being, and further extend lifespan to reduce the mild inflammatory response that is constantly present in humans and animals.

Korean Patent Registration No. 10-0824513

It is an object of the present invention to provide an Acetobacter pasterianus MGLV strain deposited with the accession number KCCM 12600P.

Another object of the present invention is to provide a probiotic preparation comprising as an active ingredient the Acetobacter pasterianus MGLV strain or a culture solution thereof consigned to the accession number KCCM 12600P.

Another object of the present invention is to provide a starter composition for preparing fermented foods comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof entrusted with accession number KCCM 12600P as an active ingredient.

Another object of the present invention is to provide a composition for enhancing immunity comprising, as an active ingredient, an Acetobacter pasterianus MGLV strain or a culture solution thereof, entrusted with accession number KCCM 12600P.

Another object of the present invention is to provide an anti-inflammatory composition comprising as an active ingredient the Acetobacter pasterianus MGLV strain or a culture solution thereof entrusted with accession number KCCM 12600P.

Another object of the present invention is a method for reducing the concentration of C-reactive protein comprising the step of treating a sample with an Acetobacter pasterian MGLV strain or a culture solution thereof deposited with the accession number KCCM 12600P. To provide.

In order to achieve the above object, the present invention provides an Acetobacter pasterianus MGLV strain deposited with the accession number KCCM 12600P.

In one embodiment of the present invention, the strain may be isolated from a naturally fermented food.

In one embodiment of the present invention, the strain may have an immunity enhancing activity by reducing the concentration of C-reactive protein in blood.

In one embodiment of the present invention, the strain may have acid resistance and bile resistance.

In addition, the present invention provides a probiotic preparation containing as an active ingredient the Acetobacter pasterianus MGLV strain or a culture solution thereof consigned to the accession number KCCM 12600P.

In addition, the present invention provides a starter composition for manufacturing fermented foods comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof entrusted with accession number KCCM 12600P as an active ingredient.

In addition, the present invention provides a composition for enhancing immunity comprising as an active ingredient the Acetobacter pasterianus MGLV strain or a culture solution thereof entrusted with accession number KCCM 12600P.

In addition, the present invention provides a composition for anti-inflammatory comprising as an active ingredient the Acetobacter pasterian MGLV strain or a culture solution thereof consigned to the accession number KCCM 12600P.

In addition, the present invention provides a method for reducing the concentration of C-reactive protein, comprising the step of treating a sample with the Acetobacter pasterian MGLV strain or a culture solution thereof deposited with the accession number KCCM 12600P. .

The Acetobacter pasterianus MGLV strain entrusted with the accession number KCCM 12600P according to the present invention is a probiotic acetic acid strain isolated from natural fermented foods and has immunity enhancing activity.This is a probiotic preparation, a starter composition for fermented food production. , It can be used as a composition for enhancing immunity and an anti-inflammatory composition.

The present invention provides a Acetobacter pasteurianus (Acetobacter pasteurianus) MGLV strain deposited with the accession number KCCM 12600P.

The present inventors have identified strains isolated from naturally fermented foods such as kimchi or vinegar, and identified Acetobacter pasterianus MGLV strains as strains having acid resistance, bile resistance, and immunity enhancing activity, and this was sent to the Korea Microbiology Conservation Center in 2019. It was deposited on October 02, 2010 and was given the deposit number KCCM 12600P.

In the present invention, "acid resistance" and "bile resistance" are the ability to survive by reaching the intestine without being killed by digestive juices containing digestive enzymes such as bile, without being killed by gastric acid present in the stomach. And the strain having bile resistance has a high digestive tract survival rate. In the present invention, acid resistance may be acid resistance at pH 3-6, and bile resistance may be bile resistance at 0.1 to 0.3%.

Immunity is divided into innate immunity and acquired immunity. Innate immunity is a function that protects against antigen invasion by reacting non-specifically without a special memory function for the antigen. There are many anatomical defense and biological actions such as skin, mucous tissue, strong acidic stomach acid, and complement present in the blood. Cells responsible for the innate immunity include macrophages, polymorphonuclear leukocytes, and NK cells that kill infected cells and cancer cells. Acquired immunity refers to a stronger immune response, including immunological memory, which means the function of remembering antigenic markers, and is specific to the antigen and requires recognition of non-self antigens through the process of antigen presentation. .

In the present invention, "immune enhancing activity" refers to enhancing the biological defense ability against an antigen, and increasing cellular and humoral immunity against the antigen. The mechanism of immunity enhancement is not limited, but may include, for example, promoting the activity of antigen-presenting cells such as macrophages or promoting specific activity for lymphocytes, and preferably, a C-reactive protein in blood (C -reactive protein) may have an immunity enhancing activity by reducing the concentration.

In addition, the present invention provides a probiotic preparation comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof as an active ingredient.

The probiotic formulation may be prepared and administered in various formulations and methods according to methods known in the art. For example, the strain of the present invention or a culture solution thereof is mixed with a carrier commonly used in the pharmaceutical field and mixed with powders, liquids and solutions, tablets, capsules, and syrup. , Suspension or granules may be prepared and administered. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent, but is not limited thereto. In addition, the dose to be administered can be appropriately selected according to the degree of absorption, inactivation rate, excretion rate of the active ingredient in the body, the age, sex, breed, condition, and severity of disease of the recipient.

The method of culturing the strain of the present invention may be cultured according to a method commonly used in the art, and is not limited to a specific method.

When the strain or a culture thereof obtained in the step of culturing the strain of the present invention is used as an additive, the strain or a culture thereof may be added as it is or other additives may be used together, and may be appropriately used according to a conventional method. . The mixing amount of the active ingredient can be appropriately determined according to the purpose of use.

In addition, the present invention provides a starter composition for producing fermented foods comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof as an active ingredient.

The "starter for fermented food production" means a preparation or composition containing microorganisms involved in fermentation for the production of fermented food. It is used to provide microorganisms that can grow in fermented foods or as dominant species by adding them in the manufacture of fermented foods. In the case of manufacturing food using the food fermentation starter, by the microorganisms included in the food fermentation starter, the quality of the food is regulated to be constant, or for a specific purpose, for example, it does not cause off-flavor in food, The purpose of reducing can be achieved. In the present invention, a fermented food having acid resistance, bile resistance and immunity enhancing activity can be prepared.

The fermented food may be kimchi, paste, or fermented milk, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for enhancing immunity or a pharmaceutical composition for anti-inflammatory, comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof as an active ingredient.

The “anti-inflammatory” refers to an action of inhibiting or reducing inflammation, and the “inflammation” is a protective reaction that occurs in the body when a living tissue is damaged, and is a cause of causing an inflammatory disease. In the present invention, the anti-inflammatory composition may be used to prevent, treat, or improve inflammatory diseases by exhibiting an activity of inhibiting or reducing inflammation.

The Acetobacter pasterianus MGLV strain or a culture medium thereof may be one that exhibits an immunity enhancing activity or an inflammatory inhibitory activity by reducing the concentration of C-reactive protein in blood.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the present invention, the term "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art such as a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, a stabilizer, a base agent, an excipient, and a lubricant.

In addition, the pharmaceutical composition of the present invention is formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. I can. Further, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel or gel for external skin. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the Cycotria rubra extract, It is prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the route of administration of the composition may be administered through any general route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto.

The term "individual" refers to all animals including humans, such as rats, mice, and domestic animals. Preferably, it may be a mammal including a human.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not cause side effects, and the effective dose level is the sex, age, and Weight, health condition, type of disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration, and rate of excretion, duration of treatment, factors including drugs used in combination or concurrently, and other fields of medicine. It can be readily determined by a person skilled in the art according to well-known factors. Administration may be administered once a day at the recommended dosage, or may be divided several times.

In addition, the present invention provides a health functional food for enhancing immunity or a health functional food for anti-inflammatory, comprising the Acetobacter pasterianus MGLV strain or a culture solution thereof as an active ingredient.

The food composition of the present invention may include all foods in a conventional sense, and may be mixed with terms known in the art, such as functional foods and health functional foods.

The term "functional food" of the present invention refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body according to the Health Functional Food Act No.6727, and the term "functional" It refers to ingestion for the purpose of obtaining useful effects for health purposes such as controlling nutrients for structure and function or for physiological effects.

The term "health functional food" of the present invention refers to a food manufactured and processed by extracting, concentrating, refining, mixing, or extracting, concentrating, refining, and mixing specific ingredients contained in food ingredients or using specific ingredients as a raw material for the purpose of supplementing health. It refers to foods designed and processed to sufficiently exert biological control functions such as biological defense, biological rhythm control, disease prevention and recovery, etc., by ingredients, and the health food composition is used to prevent diseases and recover diseases. Can perform functions related to etc.

There is no limitation on the kind of food in which the composition of the present invention can be used. In addition, the composition of the present invention can be prepared by mixing suitable other auxiliary ingredients that may be included in food and known additives according to the choice of a person skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and There are vitamin complexes and the like, and can be prepared by adding the extract according to the present invention and a fraction thereof as a main component, such as juice, tea, jelly, and juice.

In addition, foods that can be applied to the present invention include, for example, special nutritional foods (e.g., formulas, infant foods, etc.), processed meat products, fish meat products, tofu, rice cakes, noodles (e.g., ramen, noodles, etc.), health supplement foods. , Seasoned foods (e.g. soy sauce, miso, red pepper paste, mixed sauce, etc.), sauces, sweets (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.) ), beverages (eg, fruit, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (eg, ramen soup, etc.).

When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweetening agents, flavoring agents, natural carbohydrates, and the like as an additional component, as in ordinary beverages. In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin. , Alcohol, carbonated beverages, etc. may contain. In addition, it may contain flesh for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of acetic acid bacteria

The acetic acid bacteria used in the present invention were isolated from various natural fermented foods in Korea. Briefly, foods such as kimchi or vinegar were diluted with sterile physiological saline, and the dilution was spread on a selective medium for acetic acid bacteria to recover specific colonies of acetic acid bacteria, and the strain was identified at the species level level through 16S rRNA sequencing.

As a result, 6 strains of acetic acid bacteria were isolated, and the isolated acetic acid bacteria were as follows: Gluconacetobacter europaeus BBV, Gluconacetobacter europaeus RV, Gluconacetobacter kakiaceti PSV, Gluconobacter frateurii KC, Acetobacter cibinongensis GJ and Acetobacter pasteurianus MGLV.

Example 2. Experiment to confirm the growth temperature of acetic acid bacteria

The present inventors suspended the acetic acid bacteria in glucose-yeast extract broth for the growth temperature test of acetic acid bacteria, and then cultured the acetic acid bacteria at 30, 34, 37 and 40 ℃ for 48 hours. The growth of the strain was confirmed through the increase in turbidity generated during the culture process.

As a result, as shown in Table 1, it was confirmed that all the acetic acid strains of 6 strains isolated from fermented foods were all grown at 30 °C, and at 34 °C, all strains except Gluconacetobacter europaeus RV were grown, and at 37 °C, Gluconacetobacter europaeus It was found to be grown only BBV and Acetobacter pasteurianus MGLV, between 40 ℃ was found to be only Acetobacter pasteurianus MGLV grown.

From the above results, the present inventors confirmed that the Acetobacter pasteurianus MGLV strain has the widest growth temperature range of 30 to 40 °C.

Strain Growth at 30 ℃ 34 ℃ 37 ℃ 40 ℃ Gluconacetobacter europaeus BBV + + + - Gluconacetobacter europaeus RV + - - - Gluconacetobacter kakiaceti PSV + + - - Gluconobacter frateurii KC + + - - Acetobacter cibinongensis GJ + + - - Acetobacter pasteurianus MGLV + + + +

Example 3. Confirmation of gastrointestinal viability of acetic acid bacteria

The present inventors were subcultured twice under aerobic conditions at 30° C. for 48 hours in GYC agar to restore the vitality of the acetic acid strain before the experiment. The test for confirming the viability in artificial gastric juice was performed using MRS broth (pepsin-MRS broth) with the pH adjusted to 2.5, 4.5, 5.5, and 6.8, respectively, and 1,000 units/mL pepsin. Acetic acid bacteria were added to 3 mL of pepsin-MRS broth and cultured at 37° C., and viable cells were counted at 0 and 2 hours to confirm the survival rate.

As a result, as shown in Table 2, the acetic acid bacteria surviving at pH 6.8, pH 5.5 and pH 4.5 were identified as Gluconacetobacter europaeus BBV and Acetobacter pasteurianus MGLV.

Strain Growth in 37 ℃ at pH 6.8 pH 5.5 pH 4.5 pH 2.5 Gluconacetobacter europaeus BBV + + + - Gluconacetobacter europaeus RV - - - - Gluconacetobacter kakiaceti PSV - - - - Gluconobacter frateurii KC - - - - Acetobacter cibinongensis GJ - - - - Acetobacter pasteurianus MGLV + + + -

In addition, in order to confirm the viability in the artificial intestine, the pH was adjusted to 8 and MRS broth (oxgall-MRS broth) to which 0.1, 0.3, 0.5, and 1.0% of oxgall (bile) was added was used. Acetic acid bacteria were added to 3 mL of oxgall-MRS broth and cultured at 37° C., and viable cells were counted at 0 and 24 hours to confirm the survival rate.

As a result, as shown in Table 3, the acetic acid bacteria surviving in 0.1% and 0.3% bile salt were identified as Gluconobacter frateurii KC and Acetobacter pasteurianus MGLV.

Strain Growth in 37 ℃ at Bile salt 0.1% Bile salt 0.3% Bile salt 0.5% Bile salt 1.0% Gluconacetobacter europaeus BBV - - - - Gluconacetobacter europaeus RV - - - - Gluconacetobacter kakiaceti PSV - - - - Gluconobacter frateurii KC + + - - Acetobacter cibinongensis GJ - - - - Acetobacter pasteurianus MGLV + + - -

From the above results, the present inventors confirmed that the Acetobacter pasteurianus MGLV strain is the strain having the best viability in the gastric/intestinal environment.

Example 4. Confirmation of immunomodulatory ability through animal testing of selected acetic acid strain

The present inventors selected the Acetobacter pasteurianus MGLV strain, which showed the most excellent effect in the above example, and conducted an animal experiment for immunomodulatory ability. Animal experiments were conducted under the approval of Konkuk University's Animal Experimental Ethics Committee (KU19120). Briefly, female Balb/c mice were prepared and acclimatized in laboratory animal facilities for one week before entering the experiment. The temperature was maintained at 21.5 ± 1 ℃, the humidity was maintained at 52 ± 10%, and the lighting time was adjusted in a 12-hour cycle (08 to 20:00). Feeding boxes, feeders, and water bottles were measured for dietary and drinking water, and were washed and replaced every week. Animal room disinfection was performed after the work was completed.

The experimental group was set as a control group and a acetic acid administration group, and 8 mice were used for each experimental group. All experimental groups were supplied with negative water and chow diet autonomously. In the acetic acid administration group, acetic acid bacteria were administered orally to 4-week-old female Balb/c mice at 10 ⁷ CFU/head each day between 15:00 and 17:00 for 2 weeks. To the control group, sterilized physiological saline was administered. Thereafter, on the day of the end of the experiment, the body weight and the concentration of C-reactive protein in blood were measured. The concentration of C-reactive protein in the blood is an immune indicator that levels rise due to an inflammatory reaction.

As a result, it was confirmed that in the group administered with the selected Acetobacter pasterian MGLV strain, there was no significant difference in weight loss, sensory deterioration of hair, behavioral abnormalities, changes in intake and drinking water. Thus, it was confirmed that the administered Acetobacter pasterianus MGLV strain had no acute toxic effect.

In the result of measuring the level of C-reactive protein, the concentration of C-reactive protein in the blood compared to the control group when the Acetobacter Pasterian MGLV strain was administered to mice for 2 weeks. Was found to be significantly reduced (Table 4).

density Control A. pasteurianus MGLV administration group Conc. Average [ng/ml] 2637.82 1935.06

From the above results, the present inventors found that the Acetobacter pasterianus MGLV strain of accession number KCCM 12600P isolated from the naturally fermented food can be used as a starter composition for preparing probiotics preparations and fermented foods, and the value is increased by an inflammatory reaction. It was confirmed that it can be used as an anti-inflammatory composition or a composition for enhancing immunity from immunomodulatory effects such as anti-inflammatory effect or immunity enhancing effect by reducing the concentration of C-reactive protein in blood, which is an immune indicator.

Korea Microorganism Conservation Center (overseas) KCCM12600P 20191002

Claims (9)

As the Acetobacter pasteurianus MGLV strain deposited with the accession number KCCM 12600P,
The strain is characterized in that it is isolated from kimchi,
The strain is characterized in that it has immunity enhancing activity by reducing the concentration of C-reactive protein in blood,
Acetobacter pasteurianus (Acetobacter pasteurianus) MGLV strain deposited with the accession number KCCM 12600P. delete delete The method of claim 1,
The strain is a strain characterized in that it has acid resistance and bile resistance. A probiotic preparation comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A starter composition for producing fermented food comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A composition for enhancing immunity comprising the strain of claim 1 or a culture solution thereof as an active ingredient. An anti-inflammatory composition comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A method for reducing the concentration of C-reactive protein, comprising the step of treating a sample with the strain of claim 1 or a culture solution thereof.