KR20210022848A - NOVEL STRAIN OF Lactobacillus brevis AND COMPOSITION FOR ENHANCING IMMUNITY USING THE SAME - Google Patents

Abstract

The present invention relates to a Lactobacillus brevis strain (KCCM 12203P) having an immune enhancing effect, and an immune enhancing composition comprising the same as an active ingredient. The strain can have: i) acid-resistant and bile-resistant; and ii) antioxidant activity.

Description

A novel Lactobacillus brevis strain and a composition for enhancing immunity comprising the same {NOVEL STRAIN OF Lactobacillus brevis AND COMPOSITION FOR ENHANCING IMMUNITY USING THE SAME}

The present invention relates to a novel Lactobacillus brevis strain having an immune enhancing effect and a composition for enhancing immunity comprising the same.

In modern people, the intake of animal foods is gradually increasing due to the pursuit of convenience and the westernized diet. As a result, immunity against diseases is gradually decreasing due to complex social stress as well as chronic metabolic syndromes such as various cardiovascular diseases, obesity, diabetes, and cancer. . In general, for the treatment of these diseases, drugs are used as chemical therapy or physical medical techniques such as surgery are widely used, and in particular, in chemical therapy, the trend of preferring functional substances made of natural materials by recognizing the side effects of chemical substances. to be. Accordingly, for more effective treatment, studies for prevention and treatment through various health functional foods as well as improvement of eating habits in parallel with treatment are being conducted.

As the cause of various diseases, free radical substances and peroxides by free radicals such as free radicals are accumulated in the body due to the above eating habits, insufficient exercise amount, and excessive stress, which promotes aging and is derived from this, which leads to exposure to various diseases, especially causing inflammation in the body. do. Therefore, the generation of free radicals beyond the limits is a cause of diseases such as adult diseases and circulatory disorders. At this time, radical substances may increase reactivity by activating other substances, and peroxides are easily generated from unsaturated fatty acids and are promoted by various factors such as oxygen, heating, ultraviolet rays, physical shock, and metal ions. These substances are very unstable and highly reactive, attacking and damaging the DNA in cells, and causing a chain of fat oxidation, leading to diseases such as aging and cancer. The importance of foods with antioxidants added to prevent diseases caused by these free radicals is being emphasized.

Immunity is a self-protection mechanism that occurs in the body to prevent substances other than its own cellular components from affecting or threatening the homeostasis of the body, and is largely divided into innate and acquired immunity. Here, innate immunity (or natural immunity) generates leukocytes and cytokines such as TNF-α, and before acquired immunity occurs, macrophages act not only against infectious pathogens such as microorganisms and viruses, but also aging general cells and cancer cells. By causing the primary immune response.

In innate immunity, macrophages present in the spleen play the most important role. Macrophages are involved in humoral immunity and cellular immunity by first recognizing the invasion of foreign substances, and when activated, cells proliferate and activate macrophages. It occurs, and it is known to secrete cytokines such as NO, TNF-α, IL-β, and IL-6 to inhibit the proliferation of infectious microorganisms such as various microorganisms and viruses, and to exhibit toxicity to cancer cells.

Probiotics are'living bacteria that are beneficial to health' and refer to bacteria that keep the gastrointestinal tract of a host such as a human body or animals healthy. These strains are mainly lactic acid bacteria and are known as Gram-positive bacteria, and are dominant among intestinal microorganisms by attaching to intestinal cells. Naturally-derived lactic acid bacteria are largely classified into Lactobacillus, Bifidus, and Lactococcus, and proline lactic acid bacteria for inhibiting obesity bacteria are also being released. In particular, breast milk lactic acid bacteria that are emerging recently can be found in many infant products containing patented breast milk lactic acid bacteria derived from breast milk. On the other hand, about 70% of the body's immune cells are distributed in the intestine, so if the intestinal health deteriorates, the immune function also decreases. Beneficial bacteria such as lactic acid bacteria in the intestinal mucosa are known to have the effect of blocking pathogens and viruses by stimulating immune cells and activating immune substances. When ingesting lactobacilli from breast milk, it is known that it can improve immunity and prevent various diseases, and it is known that it can help bowel health by promoting bowel movements.

Korean Registered Patent No. 10-1884326 (2018.07.26)

The present invention is to provide an immune enhancing effect Lactobacillus brevis (Lactobacillus brevis) strain (KCCM 12203P) and the like.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a Lactobacillus brevis strain (KCCM 12203P) having an immune enhancing effect.

The strain is i) acid-resistant and bile-resistant; And ii) may have antioxidant activity.

The strain may include the 16S rRNA nucleotide sequence of SEQ ID NO: 1.

In one embodiment of the present invention, it provides a composition for enhancing immunity comprising the Lactobacillus brevis strain (KCCM 12203P) as an active ingredient.

The strain may be prepared as a dead cell.

The dead cells may be prepared by culturing the strain at 30°C to 40°C for 5 to 20 hours, and then heat treatment at 70°C to 90°C for 10 minutes to 1 hour.

The concentration of the strain in the composition may be 0.5 × 10 ⁸ CFU / ml to 5.0 × 10 ⁸ CFU / ml.

The composition may increase the secretion of one or more selected from the group consisting of NO, iNOS and pro-inflammatory cytokines by macrophages.

The pro-inflammatory cytokine may include at least one selected from the group consisting of TNF-α, IL-1β, and IL-6.

The novel Lactobaciollus brevis strain (KCCM 12203P) according to the present invention has an immunity enhancing effect, which can be utilized not only as probiotics, but also as a composition for enhancing immunity.

In particular, the dead cells of the strain (KCCM 12203P) are characterized in that the immune enhancing effect is further improved at a specific concentration compared to the viable cells, and has excellent advantages in both safety and stability.

1 is a diagram showing a schematic diagram of a novel Lactobacillus brevis B7 strain (KCCM 12203P).
Figure 2 is a target of the dead cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 and the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1 four immune factors (TNF-α, iNOS, It is a graph comparing the results of measuring the expression of IL-1β and IL-6).

The present inventors isolated and identified Lactobacillus brevis B7 strain (KCCM 12203P) having an excellent immune enhancing effect among the Lactobacillus brevis strains, and evaluated physiological properties such as acid resistance or bile resistance thereof. In addition, the antioxidant and immune enhancing effects on Lactobacillus brevis B7 strain (KCCM 12203P) were confirmed, and the present invention was completed.

In the present specification, the term "immune enhancement" includes an immune modulating effect in addition to the dictionary meaning, and increases the activity of the immune system by increasing the activity of immune modulators, suppresses angiogenesis, increases the growth inhibition against pathogens, etc. It is defined as having an effect of preventing diseases, and in particular, by enhancing the activity of macrophages to produce effects such as defense against external pathogens, removal of dead cells, and secretion of immune mediators.

The present invention provides a Lactobacillus brevis strain (KCCM 12203P) having an immune enhancing effect.

The strain (KCCM 12203P) can be seen as a subspecies of Lactobacillus brevis. Conventional Lactobacillus brevis is a kind of hetero lactic acid bacteria that produce lactic acid, acetic acid, ethanol, carbon dioxide, etc., and it is known to proliferate a lot in the late fermentation of kimchi or cheese, and is generally known to have excellent acid resistance and dung resistance.

The schematic diagram of the strain (KCCM 12203P) is as shown in FIG. 1, and is isolated from various kimchi such as cabbage kimchi, yeolmu kimchi, and kkakdugi. As a result of analyzing the 16S rRNA sequence to identify the isolated strain, the strain (KCCM 12203P) was confirmed to have the 16S rRNA nucleotide sequence of SEQ ID NO: 1, and deposited with the Korea Microbiological Conservation Center on January 18, 2018. Therefore, it was assigned the accession number KCCM 12203P.

The strain (KCCM 12203P) is characterized by having a particularly excellent immune enhancing effect among Lactobacillus brevis strains.

Specifically, the immune enhancing effect can be evaluated by confirming whether to increase the secretion of one or more selected from the group consisting of NO, iNOS and pro-inflammatory cytokines of macrophages. At this time, the pro-inflammatory cytokine may include at least one selected from the group consisting of TNF-α, IL-1β, and IL-6.

In particular, the dead cells of the strain (KCCM 12203P) have a specific concentration (0.5 Х 10 ⁸ CFU/ml to 1.0 Х 10 ⁹ CFU/ml, preferably 0.5 Х 10 ⁸ CFU/ml to 5.0 Х) compared to the live cells. 10 ⁸ CFU / ml), the immune enhancing effect is further improved, and has excellent advantages in both safety and stability.

In addition, the strain (KCCM 12203P) has i) acid resistance and bile resistance; And ii) may have antioxidant activity.

Specifically, when the acid resistance is reacted for 3 hours under pH 2.5 conditions, the survival rate of the strain may be 99% or more, and the bile resistance is 0.3% (w/w) when reacting for 24 hours under oxagall conditions, the survival rate of the strain is 105% or more. (Preferably, 107% or more).

In addition, the antioxidant activity can be evaluated through calculation of DPPH free radical scavenging ability. Therefore, it can be of great help in anti-aging, removal of toxins (detox), and the like. At this time, the strain (KCCM 12203P) may have a higher antioxidant effect than the Lactobacillus rhamnosus GG strain (KCTC 5033) corresponding to the commercial probiotic.

In addition, the present invention provides a composition for enhancing immunity comprising the Lactobacillus brevis strain (KCCM 12203P) as an active ingredient.

The strain (KCCM 12203P) is as described above.

In addition, the strain (KCCM 12203P) may be prepared as a live cell, a dead cell or a culture filtrate, and the strain (KCCM 12203P) is preferably prepared as a dead cell, but is not limited thereto.

In the present specification, the term "dead cells" refers to a form produced through heat treatment of a viable cell cultured with a strain, and is a form that prevents the activity of the strain, and contains antimicrobial active substances such as cytoplasm, bacteriocin, and organic acids. Can include.

Specifically, the dead cells may be prepared by culturing the strain (KCCM 12203P) at 30° C. to 40° C. for 5 to 20 hours, and then heat treatment at 70° C. to 90° C. for 10 minutes to 1 hour. Thereby, the dead cells have a specific concentration (0.5 Х 10 ⁸ CFU/ml to 1.0 Х 10 ⁹ CFU/ml, preferably 0.5 Х 10 ⁸ CFU/ml to 5.0 Х 10 ⁸ CFU/ml) compared to the live cells. It is characterized in that the immune enhancing effect is further improved, and has excellent advantages in both safety and stability. Due to such excellent stability against quality changes, it is expected that the shelf life can be significantly extended.

The composition may be used for the prevention, treatment or improvement of immune-related diseases, and immune-related diseases include asthma, seasonal or perennial rhinitis, allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, hemolysis of red blood cells, acute glomerulonephritis, It varies from cold, chronic fatigue, and cancer.

Specifically, the composition may increase the secretion of one or more selected from the group consisting of NO, iNOS and pro-inflammatory cytokines of macrophages. At this time, the pro-inflammatory cytokine may include at least one selected from the group consisting of TNF-α, IL-1β, and IL-6.

More specifically, the concentration of the strain (KCCM 12203P) prepared as dead cells in the composition may be 0.5 Х 10 ⁸ CFU/ml to 1.0 Х 10 ⁹ CFU/ml, and 0.5 Х 10 ⁸ CFU/ml to 5.0 Х It is preferably 10 ⁸ CFU / ml, but is not limited thereto. When the concentration of the strain (KCCM 12203P) prepared as dead cells in the composition is 0.5 Х 10 ⁸ CFU/ml to 5.0 Х 10 ⁸ CFU/ml, compared to Lactobacillus rhamnosus GG strain (KCTC 5033) corresponding to commercial probiotics , In addition to significantly increasing the amount of NO production in macrophages, it has the advantage of being able to express genes significantly higher for immune factors of iNOS, IL-1β, and IL-6.

At this time, iNOS is a NO synthase that produces NO from L-arginine. NO produced by iNOS exhibits various physiological actions such as antithrombosis, anti-inflammatory, and antibacterial action. IL-1β is a monokine produced in macrophages and various cells. Unlike IL-1α, which is active only in precursors, IL-1β also exhibits activity in mature organisms. IL-1β is involved in several biological reactions, such as the production of T cells, B cells, and PGE _2. In addition, it has been reported that IL-1 family cytokines are also related to chronic inflammation such as chronic rheumatoid arthritis. IL-6 is a B cell stimulating factor that induces the final differentiation of B cells into antibody-producing cells. It is involved in immune response, proliferation and differentiation of hematopoietic and nervous system cells, and acute reactions. It is known to be related to the occurrence of abnormalities, inflammatory diseases, and lymphatic tumors.

On the other hand, the composition may be provided as a pharmaceutical composition, a health functional food, a composition for feed, and the like.

Each of the pharmaceutical compositions can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method, For formulation, a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions may be included.

Examples of the carrier or excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided And various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, weight agents, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration may be prepared by mixing at least one excipient, for example, starch, calcium bonate, sucrose or lactose, gelatin, and the like with the strain (KCCM 12203P). In addition to simple excipients, lubricants such as magnesium stearate and talc can also be 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, sweetening agents, fragrances, and preservatives may be included. .

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous agents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, 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, glycerol gelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, administration route and duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably at 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be divided several times. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, in the health functional food, when the strain (KCCM 12203P) is used as an additive to a health functional food, it can be added as it is or used with other foods or food ingredients, and can be appropriately used according to a conventional method. . The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as general foods or beverages.

There is no particular limitation on the type of food, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , Various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes, and may include all foods in the usual sense.

In general, when preparing food or beverage, the strain (KCCM 12203P) may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less based on 100 parts by weight of raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

Among the health functional foods, beverages may contain various flavoring agents or natural carbohydrates as an additional component, like ordinary beverages. The natural carbohydrates described above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food 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, alcohols, It may contain carbonation agents used in carbonated beverages. In addition, the health functional food may contain pulp for the manufacture of natural fruit juice, fruit juice beverage, and vegetable beverage. These ingredients may be used independently or in combination. The ratio of these additives is not limited, but it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food.

In addition, the feed composition may additionally include a known feed supplement, food additive, or feed additive, and may be prepared in the form of fermented feed, blended feed, pellet form, and silage.

In addition, the present invention can provide a starter for fermentation, a seed composition or a fermented food comprising a Lactobaciollus brevis strain (KCCM 12203P).

In addition, the present invention can provide a method of activating the macrophages by treating the macrophages with a Lactobaciollus brevis strain (KCCM 12203P) in vitro.

As described above, the novel Lactobaciollus brevis strain (KCCM 12203P) according to the present invention has an immunity enhancing effect, which can be utilized not only as probiotics, but also usefully as a composition for enhancing immunity.

In particular, the dead cells of the strain (KCCM 12203P) are characterized in that the immune enhancing effect is further improved at a specific concentration compared to the viable cells, and has excellent advantages in both safety and stability. Due to such excellent stability against quality changes, it is expected that the shelf life can be significantly extended.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example>

Example 1: Lactobacillus brevis Isolation and identification of B7 strain

In order to isolate the new strain, various kimchi broths, such as cabbage kimchi, yeolmu kimchi, and kkakdugi made at home, were sampled and used as samples. Kimchi broth was smeared on MRS agar medium, LBS agar medium, and PES agar medium using a 10-fold dilution method, streaked and cultured several times, and then the colony morphology was observed to isolate a new strain. 16S rRNA sequencing was requested to Bionics (Seoul, Korea) for the identification of the isolated new strain. Specifically, 16S rRNA sequencing was performed, and PCR was performed on the 16S rRNA gene using 27F and 1492R primers. Based on the results of nucleotide sequence analysis, the BLAST program was used to compare homology with other standard strains registered in Genbank's database (Lactobacillus spp., Leuconostoc spp. , and Pediococcus spp.). The new strain was identified as Lactobacillus brevis B7 strain, and the similarity was shown in %. In addition, homology was analyzed using the Mega 7 program and a schematic diagram was obtained (FIG. 1). At this time, the Lactobacillus brevis B7 strain was deposited with the Korea Microbial Conservation Center on January 18, 2018, and was given the accession number KCCM 12203P.

Thereafter, the Lactobacillus brevis B7 strain was incubated in lactobacilli MRS broth at 37°C for 15 hours and then centrifuged (12,000 rpm, 4°C, 5 minutes) to separate the precipitate (cells) and the supernatant (culture filtrate). After the precipitate was freeze-dried, live cells were prepared, and dead cells were prepared through additional heat treatment (85° C., 30 minutes). Then, the live cells and dead cells were washed and resuspended three times with 0.1% peptone water and used in the experiment.

Comparative Example 1: Lactobacillus rhamnosus Preparation of GG strain (KCTC 5033)

Lactobacillus rhamnosus GG strain (KCTC 5033) known as a commercial probiotic was prepared. Culture conditions were the same as the Lactobacillus brevis B7 strain.

Experimental Example 1: Evaluation of physiological properties of strains

As physiological characteristics of the Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1, acid resistance and bile resistance were evaluated.

(1) Acid resistance evaluation

The Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 was cultured for 24 hours. 3 mg/mL of pepsin was added to the culture solution test tube adjusted to pH 2.5, and Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1, which had been cultured in advance, was inoculated into each test tube by 1 mL. 0 hours was used as a control, and acid resistance was evaluated by checking the total number of bacteria after 0 hours and 3 hours.

(2) Bile resistance evaluation

The Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 was cultured for 24 hours. 0.3% (w/v) Oxgall was added to the culture solution test tube, and 1 mL of Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 previously cultured was inoculated into each test tube. 0 hour was used as a control, and the total number of bacteria after 0 and 24 hours was checked to evaluate bile resistance.

Experiment contents Number of bacteria (Log CFU/mL) Example 1 Comparative Example 1 Acid and bile resistance Initial bacterial count 8.08±0.08 8.62±0.05 pH 2.5 0.3% (w/v) pepsin 8.08±0.02
(99.71%) 8.73±0.01
(101.31%) 0.3% (w/v) Oxgall 8.66±0.01 (107.04%) 8.83±0.02
(102.51%)

As shown in Table 1, as a result of acid resistance evaluation, the Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 was confirmed to be excellent in acid resistance, as the number of bacteria was hardly decreased compared to the initial number of bacteria. In addition, as a result of evaluation of bile resistance, the Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 was confirmed to be excellent in bile resistance as the number of bacteria increased compared to the initial number of bacteria. This can be seen to be much better than the bile resistance of the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1.

Therefore, the Lactobacillus brevis B7 strain (KCCM 12203P) isolated in Example 1 is excellent in both physiological properties such as acid resistance and bile resistance, and thus can be regarded as a lactic acid bacteria beneficial to the human body.

Experimental Example 2: Evaluation of antioxidant effect on strain

To evaluate the antioxidant effect on live and dead cells of the live and dead cells of Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 and the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1, DPPH Free radical scavenging ability was calculated.

Specifically, DPPH free radical scavenging ability is one of the antioxidant measurement methods to observe the color change of DPPH solution by removing DPPH free radical, and the scavenging ability of free radicals was measured as follows. 150 μL of each sample and control was mixed with 150 μL of 40 μM DPPH (1,1-diphenyl-2-picrylhydrazyl), left for 30 minutes in a dark room at 37° C., and absorbance (optical density, OD) was measured at 517 nm. . At this time, the DPPH free radical scavenging ability was evaluated as follows:

DPPH free radical scavenging ability (%) = (1-absorbance of sample/absorbance of control) × 100.

Experiment contents DPPH free radical scavenging activity (%) Example 1 Comparative Example 1 Antioxidant
effect Live cells 25.66±2.30 23.55±1.90 Dead cells 22.48±2.13 20.82±1.61

As shown in Table 2, as a result of the antioxidant effect evaluation, it was confirmed that the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 has a high antioxidant effect compared to the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1. do.

Experimental Example 3: Evaluation of immune enhancing effect on strains (1): NO assay and MTT assay

To evaluate the effect of enhancing immunity against live and dead cells of the live and dead cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1, and the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1, First, NO assay and MTT assay were performed.

Specifically, RAW 264.7 cells previously cultured in a 96 well plate were inoculated at a concentration of 2 ^{Х10 6} /mL and incubated for 2 hours under conditions of _{5% CO 2 and 37°C to attach to the plate.} After 2 hours, 50 μL of each sample diluted in stages and 50 μL of an LPS (1 μg/mL) solution were treated. After incubation for 24 hours, 100 μL of the supernatant was transferred to a new well plate, 100 μL of griess reagent was mixed, reacted for 10 minutes, and measured at 540 nm. The NO production capacity was calculated using a standard curve using sodium nitrite. In addition, after removing the medium remaining in the existing well plate and washing the cells with PBS buffer, 100 μL of MTT solution at a concentration of 0.5 mg/mL was treated and incubated for 30 minutes. After removing the MTT reagent, 100 μL of DMSO was added to react for 10 minutes, and then absorbance was measured at 570 nm to confirm cell viability.

Experiment contents Cell viability (%) Example 1 Comparative Example 1 10 ⁹ CFU/mL Live cells - - Dead cells 62.54 ± 5.65 76.27 ± 2.55 10 ⁸ CFU/mL Live cells 28.45 ± 3.30 33.58 ± 6.04 Dead cells 99.81 ± 1.59 104.68 ± 5.46 10 ⁷ CFU/mL Live cells 94.89 ± 5.11 41.67 ± 1.32 Dead cells 114.39 ± 2.85 103.85 ± 5.89 10 ⁶ CFU/mL Live cells 111.92 ± 2.53 101.82 ± 3.76 Dead cells - - Experiment contents NO generation (μM) Example 1 Comparative Example 1 10 ⁹ CFU/mL Live cells - - Dead cells 13.38 ± 1.75 18.46 ± 0.51 10 ⁸ CFU/mL Live cells -0.07 ± 0.38 -1.01 ± 0.14 Dead cells 26.09 ± 2.40 22.06 ± 1.23 10 ⁷ CFU/mL Live cells 20.94 ± 3.86 0.96 ± 1.68 Dead cells 2.91 ± 1.87 0.46 ± 0.64 10 ⁶ CFU/mL Live cells 7.98 ± 1.99 15.14 ± 2.31 Dead cells - -

* LPS (+), 1ng/mL: 19.95 ± 2.1 / LPS (-): 3.39 ± 0.81

As shown in Table 3, according to the results of the MTT assay, the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 was confirmed to have a higher cell viability of macrophages of dead cells compared to live cells at the same concentration. However, in the case of the dead cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1, it was confirmed that the cell viability of macrophages was significantly reduced at a concentration of more than ^{10 9 CFU/mL.}

In addition, according to the NO assay results, it was confirmed that the live cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 were difficult to produce stable NO in macrophages, but the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 was confirmed to be difficult. ) Of the dead cells at ^{a concentration of 10 8} ~ 10 ⁹ CFU/mL (especially 10 ⁸ CFU/mL), it is confirmed that the amount of NO production in macrophages increases significantly, and it can be seen that the immunity enhancing effect is excellent. .

Experimental Example 4: Evaluation of the effect of enhancing immunity against strains (2): Experiment of expression of immune factors

In order to evaluate the immunity enhancing effect of the dead cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 and the dead cells of the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1, additionally, RT-PCR Through the immunological factor expression experiment was performed.

Specifically, RAW 264.7 cells were inoculated in a ^{6-well plate at a concentration of 1Х10 6} _{and incubated for 24 hours at 5% CO 2} , 37°C, and then 1 mL of each sample at a concentration of ^{10 8 CFU/mL was treated.} PBS buffer was treated. In order to confirm the immune response, LPS was treated at a concentration of 1 ng/mL to the control group, and DMEM medium was dispensed to the other treatment groups. RNA was extracted from RAW 264.7 cells cultured for 24 hours using an RNA extraction kit, and then converted to cDNA using a cDNA synthesis kit, followed by an RT-PCR experiment or stored in a deep freezer. Through RT-PCR, the level of gene expression was measured for the four immune factors, TNF-α, iNOS, IL-1β, and IL-6. Β-actin was used as a reference gene, and SYBR fluorescent material was used to detect the PCR reaction. The primer sequences of the four immune factors are shown in Table 4 below, and RT-PCR proceeded to the following steps: 95° C., 2 minutes (polymerase activation; polymerase activation); 40 cycle-95°C, 5 seconds (denaturation); 65° C., 30 seconds (annealing/extension).

Primer sequence TNF-α Forward direction GTGGGCCGCCCTAGGCACCAG Reverse GGAGGAAGAGGATGCGGCAGT iNOS Forward direction CCCTTCCGAAGTTTCTGGCAGC Reverse GGCTGTCAGAGCCTCGTGGCTTTGG IL-1β Forward direction TTGACCTCAGCGCTGAGTTG Reverse CCTGTAGCCCACGTCGTAGC IL-6 Forward direction CAGGATGAGGACATGAGCACC Reverse CTCTGCAGACTCAAACTCCAC β-actin Forward direction GTACTCCAGAAGACCAGAGG Reverse TGCTGGTGACAACCACGGCC

As shown in FIG. 2, when the dead cells of the Lactobacillus brevis B7 strain (KCCM 12203P) according to Example 1 were at a concentration ^{of 10 8} CFU/mL, compared to the Lactobacillus rhamnosus GG strain (KCTC 5033) according to Comparative Example 1, Except for TNF-α, it was confirmed that genes can be expressed significantly higher for immune factors of iNOS, IL-1β, and IL-6, and a slightly lower or similar level than that of the positive control group was shown, which greatly affected macrophages. It can be seen that it has an immunity-enhancing effect within the range that does not.

Hereinafter, an example of the preparation of a composition containing the strain of the present invention as an active ingredient is described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1: Preparation of powder

Lactobacillus brevis B7 strain 20 mg

100 mg lactose hydrate

10 mg of talc

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2: Preparation of tablets

Lactobacillus brevis B7 strain 10 mg

100 mg corn starch

100mg lactose hydrate

2mg magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3: Preparation of capsules

Lactobacillus brevis B7 strain 10 mg

Microcrystalline cellulose 3 mg

Lactose Hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above ingredients, the capsules were prepared by filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4: Preparation of injection

Lactobacillus brevis B7 strain 10 mg

180mg mannitol

2974 mg of sterile distilled water for injection

Sodium monohydrogen phosphate 26 mg

After mixing the above ingredients, it was prepared in the amount of the above ingredients per ampoule (2 mL) according to a conventional method for preparing injections.

Formulation Example 5: Preparation of liquid formulation

Lactobacillus brevis B7 strain 10 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

Lemon flavor appropriate amount

The above ingredients are dissolved by adding each ingredient to purified water according to a conventional manufacturing method, added lemon flavor, and adjusted to a total of 100 mL by adding purified water, sterilized, and filled in a brown bottle to prepare a solution.

Formulation Example 6: Preparation of health functional food

Lactobacillus brevis B7 strain 10 mg

The right amount of vitamin mixture

Vitamin A acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

Nicotinic acid amide 1.7 mg

Folic acid 50 ㎍

Calcium pantothenate 0.5 mg

Mineral mixture Appropriate amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium Phosphate Monobasic 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for the health functional food, but it is possible to arbitrarily modify the composition, and the above ingredients are mixed according to the general health functional food manufacturing method. Then, the granules are prepared, and can be used in the manufacture of health functional foods according to a conventional method.

Formulation Example 7: Preparation of health drink

Lactobacillus brevis B7 strain 10 mg

Vitamin C 15 g

100 g of vitamin E (powder)

19.75 g of iron lactate

Zinc oxide 3.5 g

3.5 g of nicotinic acid amide

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g of vitamin B2

Purified water quantification

After mixing the above ingredients according to the normal health drink manufacturing method, stirring and heating at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and then stored in a refrigerator. It is used in the manufacture of the health beverage composition of the invention.

The composition ratio is composed of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Korea Microorganism Conservation Center (overseas) KCCM12203P 20180118

<110> Konkuk University Industrial Cooperation Corp <120> NOVEL STRAIN OF Lactobacillus brevis AND COMPOSITION FOR ENHANCING IMMUNITY USING THE SAME <130> 2019-I213 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1494 <212> RNA <213> Lactobacillus brevis <400> 1 cgctgcgctg cctatacatg cagtcgaacg agcttccgtt gaatgacgtg cttgcactga 60 tttcaacaat gaagcgagtg gcgaactggt gagtaacacg tgggaaatct gcccagaagc 120 aggggataac acttggaaac aggtgctaat accgtataac aacaaaatcc gcatggattt 180 tgtttgaaag gtggcttcgg ctatcacttc tggatgatcc cgcggcgtat tagttagttg 240 gtgaggtaaa ggcccaccaa gacgatgata cgtagccgac ctgagagggt aatcggccac 300 attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360 tggacgaaag tctgatggag caatgccgcg tgagtgaaga agggtttcgg ctcgtaaaac 420 tctgttgtta aagaagaaca cctttgagag taactgttca agggttgacg gtatttaacc 480 agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggatttat tgggcgtaaa gcgagcgcag gcggtttttt aagtctgatg tgaaagcctt 600 cggcttaacc ggagaagtgc atcggaaact gggagacttg agtgcagaag aggacagtgg 660 aactccatgt gtagcggtgg aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720 ctgtctagtc tgtaactgac gctgaggctc gaaagcatgg gtagcgaaca ggattagata 780 ccctggtagt ccatgccgta aacgatgagt gctaagtgtt ggagggtttc cgcccttcag 840 tgctgcagct aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagctacgcg 960 aagaacctta ccaggtcttg acatcttctg ccaatcttag agataagacg ttcccttcgg 1020 ggacagaatg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttattatca gttgccagca ttcagttggg cactctggtg 1140 agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggac ggtacaacga gtcgcgaagt cgtgaggcta 1260 agctaatctc ttaaagccgt tctcagttcg gattgtaggc tgcaactcgc ctacatgaag 1320 ttggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380 cacaccgccc gtcacaccat gagagtttgt aacacccaaa gccggtgaga taaccttcgg 1440 gagtcagccg tctaaggtgg gacagatgat tagggtgaag tcgtaacaag agcc 1494

Claims (9)

Lactobacillus brevis having an immune enhancing effect (Lactobacillus brevis) strain (KCCM 12203P).
The method of claim 1,
The strain is i) acid-resistant and bile-resistant; And ii) having antioxidant activity, Lactobacillus brevis strain (KCCM 12203P).
The method of claim 1,
The strain comprises the 16S rRNA nucleotide sequence of SEQ ID NO: 1, Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12203P).
Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12203P) a composition for enhancing immunity comprising as an active ingredient.
The method of claim 4,
The strain is made of dead cells, the composition for enhancing immunity.
The method of claim 5,
The dead cells are prepared by culturing the strain at 30°C to 40°C for 5 to 20 hours, and then heat treatment at 70°C to 90°C for 10 minutes to 1 hour, the composition for enhancing immunity.
The method of claim 4,
The concentration of the strain in the composition is 0.5 × 10 ⁸ CFU / ml to 5.0 × 10 ⁸ CFU / ml, the composition for enhancing immunity.
The method of claim 4,
The composition increases the secretion of any one or more selected from the group consisting of NO, iNOS and pro-inflammatory cytokines of macrophages, a composition for enhancing immunity.
The method of claim 8,
The pro-inflammatory cytokine comprises at least one selected from the group consisting of TNF-α, IL-1β, and IL-6, a composition for enhancing immunity.

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