KR20170049216A - Novel Lactobacillus gasseri and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus gasseri strain and uses thereof. Provided are a Lactobacillus gasseri strain having probiotic activity deposited with accession number KCCM11777P; a pharmaceutical composition for the treatment or prevention of inflammatory diseases comprising, as an active ingredient, at least one selected from the group consisting of the strain, a lysate of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture and a fermented product by the strain; a food composition for the improvement or prevention of inflammatory diseases; and a probiotic composition. According to the present invention, the strain has excellent acid resistance, bile resistance and intestinal adherence, and induces an immune response by Th_2 cells to treat and alleviate an inflammatory disease caused by an excessive immune response of Th_1 cells with an inflammation mitigating effect through immunomodulation.

Description

Novel Lactobacillus gasseri and Uses Thereof < RTI ID = 0.0 >

The present invention relates to a novel Lactobacillus taxa strains and uses thereof.

Irritable bowel syndrome or inflammatory bowel disease is a multifactorial disorder characterized by an abnormal immune response to intestinal antigens. Especially intestinal bacteria are thought to play an important role in the development of the disease. Clinical observations such as antibiotic or probiotic strains in patients with inflammatory bowel disease show that fecal diversion is effective in patients with Crohn 's disease, and that intestinal bacteria are associated with bowel disease. It is thought that intestinal disease occurs as a result of abnormal mucosal immune response resulting from breaking of normal interaction between host and bacteria.

In mammals, the immune system consists of innate immunity and adaptive immunity. Acquired immunity is mediated by T lymphocytes and B lymphocytes, which are antigen-specific immune systems found only in vertebrate animals. When specific antigens bind to receptors, clonal expansion of lymphocytes occurs and an immune response is produced. Congenital immunity, on the other hand, serves as an outpost for rapid response when microorganisms are invaded and activates acquired immunity. In the past, abnormal and excessive immune responses to intestinal bacteria mediated by T lymphocyte were known to be the main pathogenesis of inflammatory growth disease. Recently, "PRRs (pattern recognition receptors)" which detect microorganisms such as TLRs (toll- The importance of innate immunity has been focused on.

 In the human intestinal membrane, the immune system and intestinal bacteria of the host face the epithelial layer of the monolayer. When bacteria that enter the body through the PRR are recognized, the transcriptional regulatory proteins and transcription factors are activated through various pathways, and as a result, a variety of cytokines are produced, leading to a mucosal host defense And it also induces the differentiation and proliferation of T lymphocytes and B lymphocytes and activates acquired immunity. PRRs such as TLRs and NOD receptors usually function to maintain mucosal homeostasis, but they are understood to play an important role in the development and progression of disease in pathological conditions such as inflammatory bowel disease. Active studies on congenital immunity are changing the paradigm of understanding of inflammatory bowel disease etiology and may play an important role in establishing future therapeutic strategies.

Helper T (Th) cells, which are cytokine secretory T lymphocytes in T lymphocytes, are divided into Th ₁ and Th ₂ cells. IFN-γ secreted by Th ₁ cells activates macrophages to increase the ability of macrophages to kill microorganisms and to produce IL-12, which promotes the differentiation of Th ₁ cells. In addition, IFN-γ promotes inflammatory responses and also helps to produce cytotoxic T cells. Th ₂ cells secrete IL-4 and IL-5 to stimulate IgE production and induce eosinophil activation to promote an allergic or parasitic immune response. Th ₁ and Th ₂ cells jyeoteuna obtained through the T cell cloning over a long period of time from the original test tube (in vitro), actually (in vivo) These cells are also found in certain inflammatory diseases. In most healthy individuals, many helper T cells do not show cytokine production of Th ₁ and Th ₂ cells, but produce both cytokines of these two cells.

Although it is not known exactly how Th ₁ and Th ₂ cells are obtained, the form in which the immune response appears is closely related to the induction of Th ₁ or Th ₂ cells. In addition, it is investigated whether certain types of helper T cells are activated by cytokines. In particular, IL-4 is important for the activation of Th ₂ cells, and IL-12 is known to be important for activation of Th ₁ cells. The macrophage-activated macrophages that are proliferating in the cell are known to be the major producers of IL-12, and it is understood that the T cells activated by these cells are mainly differentiated into Th ₁ cells. On the other hand, the production of IL-4 was found to be very important for Th ₂ cell differentiation. In fact, complex cytokine networks are thought to be involved in the activation of Th ₁ and Th ₂ cell cells, and it is understood that the balance between these two groups of cells maintains a healthy immune state.

Indeed, it is known that inflammatory bowel disease is induced by excessive immune response of Th ₁ cells, and allergy is induced by excessive immune response of Th ₂ cells. Because Th ₁ and Th ₂ cells antagonize each other, therapies for treating diseases induced by excessive immune responses of Th ₁ cells by inducing immune responses by Th ₂ cells have been developed, and Th _{2 < /} RTI _> cells, an immune response by Th1 cells is induced to treat diseases caused by excessive immune responses.

On the other hand, among microorganisms, some microorganisms such as lactic acid bacteria are very useful as microorganisms having a very beneficial effect on human health. In recent years, studies on such strains have been actively carried out, and their application range has been expanded not only as general foods but also as health foods and medicines.

The present inventors have made an effort to develop therapeutic agents for inflammatory diseases. As a result, Lactobacillus gasseri , a novel lactic acid bacterium strain having acquired acid resistance, resistance to intestines, intestinal adhesiveness and immunoregulation ability among the probiotics candidate strains was selected. Through the control of immune cells using the strain, inflammatory The present inventors have completed the present invention by actually confirming the effect of inhibiting and alleviating intestinal diseases in a cell level, a protein level and an animal test.

Accordingly, an object of the present invention is to provide a novel strain of Lactobacillus gasseri .

It is another object of the present invention to provide a pharmaceutical composition for the treatment or prevention of inflammatory diseases.

It is another object of the present invention to provide a food composition for preventing or ameliorating an inflammatory disease.

It is still another object of the present invention to provide a probiotic composition.

Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, the present invention provides a Lactobacillus gasseri strain having probiotic activity deposited with Accession No. KCCM11777P.

Among the candidate strains (512, 564, 597, 709, 710, 806, and 808) isolated from the feces of the newborn infants, the acidity and resistance of the candidate strains (QSH334, QWW311, QMY337, QMS339) , Intestinal adhesiveness and immune control ability were selected. The molecular biology of the isolated strain was analyzed by 16S rDNA gene sequence, and the isolated strain was identified as a novel strain having high homology with Lactobacillus gasseri. The strain was named Lactobacillus gasseri strain 808 and deposited at the Korean Microorganism Conservation Center (KCCM), an international microorganism depository, on Oct. 1, 2015, and received the deposit number KCCM11777P.

According to a preferred embodiment of the present invention, the strain has excellent immunoregulatory ability. In addition, the strain has excellent resistance to acid and excellent resistance to bile, and has excellent intestinal adhesiveness and excellent survival rate in digestive organs.

Further, according to a preferred embodiment of the present invention, the strain can regulate the expression of Th ₁ and Th ₂ cytokines.

That is, the gene expression levels of major Th ₁ type cytokines IFN-y and major Th ₂ type cytokines IL-4 and IL-10 associated with inflammation causing antagonism to each other; Or the level of expression of the protein of interest.

More preferably, said strain is an expression level of IFN-y gene which is a Th ₁ type cytokine; On the other hand, or to increase the level of expression of the protein thereof, Th ₂ type cytokines IL-4 and a gene expression level of IL-10; Or the level of expression of the protein of interest.

According to a preferred embodiment of the present invention, the strain is an expression level of at least one gene selected from the group consisting of inflammatory cytokines TNF-a, IFN-y and IL-1 ?. Or the level of expression of the protein of interest.

The strain of the present invention has an inflammation-relieving effect through immunomodulation and is characterized by alleviating inflammatory bowel disease by inducing an immune response by Th ₂ cells in an inflammatory disease caused by an excessive immune response of Th ₁ cells.

According to another aspect of the present invention, the present invention relates to a Lactobacillus gasseri strain having probiotic activity deposited with the above-mentioned accession number KCCM11777P, a lysate of the strain, a culture of the strain, There is provided a pharmaceutical composition for treating or preventing an inflammatory disease comprising, as an active ingredient, at least one selected from the group consisting of a concentrate of the cultured product, a dried product of the cultured product, and a fermented product by the strain.

In addition, the culture may be obtained by removing the novel Lactobacillus gasseri strain of the present invention, or may be a supernatant obtained by centrifuging the culture.

As used herein, the term "comprising as an active ingredient" refers to a strain of the present invention, a lysate of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, Means that the at least one member selected from the group includes an amount sufficient to achieve the desired effect or activity in the present invention and can be selected and carried out by a person skilled in the art within a suitable range.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral formulations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the individual, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention may be administered in an amount of 1 mg / kg to 10000 mg / kg per day, or may be administered once a day or divided into several doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of inflammatory diseases.

According to a preferred embodiment of the present invention, the inflammatory disease is inflammatory bowel disease, allergy, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, , Rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, Inflammatory bowel disease, allergies, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, stiffness Spondylitis, rheumatic fever, acute and chronic inflammatory diseases, most preferably inflammatory The disease.

According to another aspect of the present invention, the present invention provides a method for producing Lactobacillus gasseri strain having probiotic activity deposited with the above-mentioned accession No. KCCM11777P, a lysate of the strain, a culture of the strain, And a food composition for preventing or ameliorating an inflammatory disease comprising, as an active ingredient, at least one selected from the group consisting of a concentrate of the culture, a dried product of the culture, and a fermented product by the strain.

The food composition is food, beverage or fermented milk.

In addition, the composition of the present invention may be added to a health functional food for the purpose of preventing or improving an inflammatory disease. In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of disease, bio-defense, immunity, recovery after disease and aging inhibition.

When the composition of the present invention is used as a food additive, the fermentation broth or the fermentation broth and loess powder can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharine and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention may further contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, A carbonating agent used in a carbonated beverage, and the like. In addition, the composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

According to another aspect of the present invention, the present invention provides a method for producing Lactobacillus gasseri strain having probiotic activity deposited with the above-mentioned accession No. KCCM11777P, a lysate of the strain, a culture of the strain, And at least one selected from the group consisting of a concentrate of the cultured product, a dried product of the cultured product, and a fermented product by the strain as an active ingredient.

As used herein, the term " probiotic "or" probiotics "refers to a living organism that improves intestinal microbial environment by enhancing resistance to harmful microorganisms in the gastrointestinal tract of an animal, including humans, Means microorganisms. Probiotics may be fed in human or animal-derived cell or fermented product form in the form of single or complex strains.

Therefore, when the probiotic agent is prepared using the strain of the present invention, it has the effect of improving the inflammatory response in the body and producing the probiotic agent capable of effectively improving intestinal environment.

A Lactobacillus gasseri strain having probiotic activity deposited with Accession No. KCCM11777P according to the present invention, a lysate of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture And a fermented product of the strain may be usefully used as a safe and non-toxic probiotics with excellent immunoregulatory effect.

Therefore, a strain of Lactobacillus gasseri having a probiotic activity deposited with Accession No. KCCM11777P, a lysate of the strain, a culture of the strain, a concentrate of the culture, a dried product of the culture, And a fermented product by a strain may be usefully used for a prophylactic agent, especially a formulant, a lactic acid bacterial preparation, a feed additive, and a health food, which are human pharmaceuticals.

In addition, the probiotics of the present invention can be used for animals including livestock and can be added to food or animal feed. It can also be used in animal medicine.

Since the biocidal composition of the present invention uses the above-mentioned strain and the product thereof, common description thereof will be omitted in order to avoid the excessive complexity of the present specification according to the repeating material.

The Lactobacillus gasseri strain of the present invention has excellent acid resistance, resistance to intestines and intestinal adherence, and inflammatory diseases caused by an excessive immune reaction of Th ₁ cells with inflammation-modulating effect through immunomodulation are called Th ₂ cells Lt; RTI ID = 0.0 > inflammatory < / RTI > disease.

FIG. 1 shows PCR results confirming mRNA expression of IFN-y, IL-4, and IL-10.
Fig. 2 shows the results of comparing cytokine protein expression in mouse spleen cells and macrophage cells.
Figure 3 shows the results of weight and feed intake analyzes of intestinal inflammatory animals.
Fig. 4 shows the result of measurement of intestinal length of intestinal inflammatory animal model mice.
Figure 5 shows the histological results of intestinal inflammatory animal model mouse intestines.
Figure 6 shows the results of inflammatory cytokine mRNA expression of mesenteric lymph nodes.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the appended claims. It will be obvious to you.

Example  1. Isolation of candidate strains

Among the candidate strains, QSH334, QWW311, QMY337 and QMS339 were isolated from Kimchi and 512, 564, 597, 709, 710, 806 and 808 from the feces of newborn. Kimchi samples and neonatal fecal samples were diluted in 0.001% peptone solution and homogenized. One milliliter of the diluted sample was dropped into the BCP solid medium, spread widely by a flat plate method, and cultured in a 37 DEG C incubator for 48 hours. Colonies grown in solid medium were inoculated on MRS medium and cultured in a 37 ° C incubator for 18-24 hours. The isolated strains were able to be preserved by lyophilization preservation or cell suspension freezing.

Example  2. Selection of candidate strains

2-1. Acid resistance  Experiment

The probiotic candidate strains isolated and retained in Example 1 were cultured in MRS medium at 37 DEG C for 18 hours and precipitated by centrifugation (8000 x g, 10 min). After washing twice with sterile saline (0.85% NaCl), the cell suspensions were inoculated at about 10 ⁶ CFU / ㎖ in the control and artificial gastric juice, respectively, and viable counts were measured at intervals of 0, 3 hours at 37 ° C. The artificial gastric juice was prepared by adjusting the pH of the medium to 2.5 with 1N HCl, adding pepsin to 1000 units / ml, sterilized, and using KH ₂ PO ₄ , Na ₂ HPO, L-cystein HCl, And the total number of bacteria was measured by diluting with phosphate buffer (pH 6.8). When the survival count from the acid solution after 3 hours was converted to log value, it was evaluated that the bacterial count was decreased to 0.5 log or less, or the acid was abundant when the growth rate was compared with the original number (Table 1).

As a result, it was confirmed that the log value dropped to less than 0.4 than the untreated group in the 3-hour treatment, and it was confirmed that the stored bacteria had acid resistance.

2-2. Tolerance  Experiment

The probiotic candidate strains isolated and retained in Example 1 were cultured in MRS medium at 37 ° C. for 18 hours and precipitated by centrifugation (8000 × g, 10 min). After washing twice with sterilized saline (0.85% NaCl), the cell suspension was inoculated at about 10 ⁶ CFU / ml into the control and artificial tobacco leaves, respectively, and viable cells were counted at intervals of 0, 24 hours . MRS medium containing 0.3% oxgall was prepared and sterilized. As in the case of the acid-abilities evaluation standard, when survival counts from 24-hour post-mortem solutions were converted to log values, it was evaluated that the number of bacteria decreased to less than 0.5 log, Table 2).

As a result, it was confirmed that the log value was not decreased in the treatment group for 24 hours, and it was confirmed that the retained bacteria had resistance to bacteria.

2-3. Intestinal epithelial cells of microorganisms Attachment  Verification

Intestinal epithelial cells were used as HT-29 cells in Korean Cell Line Bank (KCLB). HT-29 cells were cultured in RPMI 1640 (Gibco, USA) supplemented with heat inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin G (100 IU / ml), and streptomycin (100 mg / ) Medium in the presence of 5% CO ₂ at 37 ° C. For adhesion and adhesion inhibition experiments, HT-29 cells were plated in 12-well plates at a density of 1.0 × 10 ⁵ cells / ml per well, and the medium was replaced every other day until a complete monolayer was formed And cultured.

To confirm the ability of the probiotic candidate strains isolated in Example 1 to adhere, HT-29 cells formed with a complete monolayer were washed 6 times with PBS buffer at 25 ° C and cultured in RPMI 1640 medium without antibiotics 0.5 ml was added. Eleven kinds of probiotic strains were suspended in RPMI to a concentration of 1.0 x 10 ⁹ CFU / ml, and then 0.5 ml was inoculated on a single layer of HT-29 and cultured in the presence of 5% CO ₂ at 37 ° C for 2 hours Respectively. After cultivation was completed, washing was carried out 6 times with PBS buffer while stirring at 200 rpm for 3 minutes to confirm the removal of the unbound probiotic strains and the ability to adhere to the washing. After the washing was completed, 0.2% trypsin-EDTA was dispensed, and the attached cells were detached. Plates were plated on MRS-agar using continuous peptone using peptone water and cultured at 37 ° C for 24 hours. In order to confirm a part of the attachment, a cover glass which had been completely immersed in 70% alcohol for one day was adhered to a petri dish bottom, and HT-29 cells were cultured, and the same amount of the probiotic strain having the same amount was added. Probiotic strains attached to HT-29 cells without washing by washing were dried and then stained with Gram and observed with an optical microscope (Table 3).

As a result, the experimental strains showed similar values when compared with the GG bacteria used as the control group. Therefore, it was confirmed that the ability of the deposited bacteria to adhere to the sheet was excellent.

2-4. immune Control ability  Evaluation (RT- PCR )

The cytokine secretion ability of mouse spleen cells was measured by RT-PCR to construct an immunoreactive screening system. First, the immune cells from which red blood cells have been removed from spleen cells of mouse (B6 mouse) were separated and the number of cells was adjusted to RPMI1640 medium containing 10% FBS (fetal bovine serum). The immune cells were then seeded into 96 well plates at 1 x 10 ⁶ cells / well (total volume 200 μl). After the seeding of the immune cells was completed, 1 × 10 ⁵ CFU / ml of the probiotic strain heat-treated at 80 ° C. for 60 minutes was added and incubated in a CO ₂ incubator at 5% CO ₂ at 37 ° C. for 1 day. After that, the cells were taken out and the cells were dissolved in 1 ml of Trizol (Invitrogen), and then 200 μl of chloroform (Sigma) was added to separate total RNA. CDNA was synthesized by reverse transcription polymerase using Superscript III (Invitrogen) reverse transcriptase with 1 ㎍ of mRNA extracted as a template. After that, primers were prepared according to the nucleotide sequence obtained from the gene database using the cDNA as a template, and PCR was performed to analyze each gene. The positive control used was con A (concanavalin A), PHA (phytohemaglutinin) and LPS (lipopolysaccharide), which are major mitogens, and 10 ㎍ / ㎖, respectively. As a control for the bacteria, L. rhamnosus GG was used. Thereafter, mRNA expression levels of major Th ₁ type cytokines, IFN-y, and major Th ₂ type cytokines, IL-4 and IL-10, were compared. The primer sets used at this time are shown in Table 4.

Gene Size Sequence IFN-y 432 bp forward 5'-CTG AGA CAA TGA ACG CTA CAC ACT GC-3 '(SEQ ID NO: 1) reverse 5'-AAC AGC TGG TGG ACC ACT CGG AT-3 '(SEQ ID NO: 2) IL-4 366bp forward 5'-GCT AGT TGT CAT CCT GCT CTT C-3 '(SEQ ID NO: 3) reverse 5'-TCA GTG ATG TGG ACT TGG ACT C-3 '(SEQ ID NO: 4) IL-10 572bp forward 5'-AGC TCC ATC ATG CCT GGC TCA-3 '(SEQ ID NO: 5) reverse 5'-CAG ACT CAA TACA CTG CAG GT-3 '(SEQ ID NO: 6) * GAPDH 271 bp forward 5'-ATG ACC ACA GTC CAT GCC ATC-3 '(SEQ ID NO: 7) reverse 5'-CCT GCT TCA CCA CCT TCT TG-3 '(SEQ ID NO: 8)

* Housekeeping gene GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

As a result, the expression of IFN-γ was not expressed in the negative control (NC) but was induced in all the positive control (LPS, ConA, PHA) and lactobacillus treatment groups (1-11) . In the case of IL-4, expression was induced in the positive control ConA and PHA, but almost no expression was observed in the lactobacillus treatment group. In addition, in the case of IL-10, it was confirmed that mRNA expression was increased in all the groups except the 5th and 6th lactobacillus treatment groups (Fig. 1).

2-5. immune Control ability  Evaluation (ELISA)

To establish an immune response screening system, cytokine secretion ability of mouse spleen cells was measured by ELISA. First, the red blood cells were removed from the spleen cells of the mouse (B6 mouse), and then the number of cells was adjusted in RPMI1640 medium containing 10% FBS (fetal bovine serum). The immune cells were then seeded into 96 well plates at 1 x 10 ⁶ cells / well (total volume 200 μl). After completion of seeding of the immune cells, 1 x 10 ⁵ CFU / ml of the probiotic strain heat-treated at 80 ° C for 60 minutes was added and cultured in a CO ₂ incubator at 5% CO ₂ and 37 ° C for 3 days. After 3 days, 50 [mu] l of the culture was dispensed and the amount of IL-4 and IFN- [gamma] contained in the culture solution was measured by ELISA assay. These experiments were performed at least 3 repetitions. The positive control used was con A (concanavalin A), PHA (phytohemaglutinin) and LPS (lipopolysaccharide), which are major mitogens, respectively, and 10 ㎍ / ㎖, respectively. As a control for the bacteria, L. rhamnosus GG was used. Then, the ratio of the major Th ₁ type cytokine, IFN-y, and the major Th ₂ type cytokines, IL-4 and IL-10, to the secretion amount was determined to select strains that maximize Th ₂ type cytokine secretion .

The expression of IFN-γ was not expressed in the negative control (NC) but was induced in all the positive control (LPS, ConA, PHA) and lactobacillus treatment groups (1-11) And the amount of protein secretion was increased (Fig. 2).

In the case of IL-4, expression was induced in the positive control, ConA, but almost no protein expression was observed in the lactic acid bacteria-treated group as in the mRNA expression amount (FIG.

Expression of IL-10 was most induced in LPS, a positive control, and it was confirmed that ConA and PHA were induced to some extent. As a result of analysis of the graphs except for the positive control for comparison of lactic acid bacteria, it was confirmed that the protein expression level of IL-10 was increased in all the samples (1-11) than the GG sample used as the control of the bacteria, The highest expression level was confirmed in the sample and the 11th sample (Fig. 2).

Example  3: Safety screening and genetic identification of selected lactic acid bacteria

Based on the results shown in Example 2, the present inventors selected lactic acid bacteria of QSH334, 808 and QMS339. Gelatin liquefaction test and confirmation test for production of harmful metabolites (ammonia) were carried out to check the stability of the selected lactic acid bacteria. In addition, rRNA cloning and sequencing were performed to identify selected lactic acid bacteria.

3-1. Gelatin liquefaction test

One of the important reasons why bacteria become pathogenic is related to their ability to penetrate cells. In order to invade the cell, it must have proteolytic ability, and one way to investigate the proteolytic ability is to judge whether gelatin liquefaction is developing. The gelatin liquefaction was measured by culturing the lactic acid bacteria to be measured in a gelatin nutrient medium for a certain period of time using a MRS gelatin nutrient culture medium medium and then observing the solidification of the medium in a refrigerator.

3-2. harmfulness Metabolite (Ammonia) production confirmation test

The production of ammonia, which is known as a harmful metabolite produced by intestinal microorganisms, is an important indicator for evaluating the stability of the selected lactic acid bacteria. Too much ammonia accumulates in the body to treat the liver and kidneys and can cause abnormalities, especially in patients with hepatitis may cause coma. Urea agar base medium is used. When the target microorganism produces urease, the urea decomposes and the pH of the medium becomes high. The phenol red used as an indicator by the increased pH changes from yellow to red.

As a result, it was confirmed that the experimental strains did not produce gelatin liquefaction reaction and ammonia production, indicating that the ability as a probiotic is excellent.

3-3. Of selected strains rRNA Cloning  And sequencing

From the microbial cultures cultured in MRS medium, the genomic DNA prep. Genomic DNA was extracted using a kit (INTRON, Korea) and the extracted genomic DNA was amplified using a universal primer (27F 5'-AGA GTT TGA TCM TGG CTC AG-3 'and 1492R 5'-TAC GGH TAC CTT GTT ACG ACT T-3 ') was used to amplify the 16S rRNA gene sequence. The PCR product was purified using PCR Purification Kit (Qiagen, Germany) and used for DNA sequencing to confirm the DNA sequence of the PCR product. DNA sequences were determined by ABI PRISM 3700 DNA analyzer (Perkin Elmer, USA) and compared with the GenBank database using NCBI BLAST (Table 5).

Strains ID (DNA Sequencing) QSH334 Lactobacillus fermentum 808 Lactobacillus gasseri QMS339 Lactobacillus gasseri

As a result of the analysis, it was confirmed that Lactobacillus fermentum and Lactobacillus gasseri were tested . Among the three selected lactic acid bacteria, the 808 lactic acid bacteria strains which showed the most excellent immunomodulating effect were used in the model of the intestinal inflammatory animal.

The 808 lactic acid bacteria strain was designated as Lactobacillus gasseri 808 strain and deposited on October 01, 2015 at the Korean Microorganism Conservation Center (KCCM), which is an international microorganism depository, and received the deposit number KCCM11777P.

Example  4: Intestinal inflammation  Anti-inflammatory effects in animal models

4-1. Intestinal inflammation  Animal experiment design

8-week-old female Balb / c mice were given a 12-hour, 12-hour day and 22-day incubation period in which the day and night changed. The mice were divided into five groups. The first group is a control group that did not cause intestinal inflammation. The second group was 4% DSS (molecular weight 5000, Wako Pure Chemical Industries, Ltd , Osaka, Japan) to supply free mixing in water, until the third of five from the second group, the group with water free of 4% DSS each probiotic strains 10 ⁹ CFU / ml and sulfasalazine (SIGMA, USA) at a concentration of 50 mg / kg.

The probiotic samples were orally administered for 7 days for pretreatment and 7 days for DSS treatment for 14 days, and DSS was treated for 7 days to induce inflammation. The length of the intestine was measured by sacrificing at week 3 after 21 weeks of observation. Table 6 shows the intestinal inflammatory animal model experimental design.

4-2. Intestinal inflammation  Animal weight and feed intake analysis

The body weight and feed intake of each group of mice were measured for a total of 14 days from the pretreatment of the ProBodyitics samples (FIG. 3).

As a result of the measurement, it was confirmed that the body weight was slightly recovered at the 14th day in the group treated with sulfasalazine and lactic acid bacteria than the DSS-treated group (FIG. Feed intake was similar to that of the normal group and lactobacillus group, and the sulfasalazine group showed higher feed intake than the DSS alone group (FIG. 3).

4-3. Intestinal inflammation  Animal Janggil  Measurement and histological analysis

Intestinal Inflammatory Animal Model We measured the body length and feed intake of the mice and sacrificed the length of the intestines. Each group was photographed with a representative photograph of the intestines and the length of the intestines of each group of animals (FIG. 4).

As a result, it was confirmed that the length of the sulfasalazine treated group was increased by 31%, 224% for the 334 probiotics and 12% for the 808 probiotics than the DSS-treated group which caused only intestinal inflammation.

The intestinal end portion of each animal was sampled for histological examination of intestinal inflammatory animal models. Samples were fixed in 10% formalin solution, dehydrated in ethanol and fixed in paraffin. Subsequently, 4 micron-thick sections were prepared and stained with hematoxylin and eosin to confirm histologic lesions (Fig. 5).

Histological examination revealed that the epithelial layer almost completely disappeared in the DSS-treated group compared with the control group. On the other hand, the sulfasalazine treated group showed slight damage to the intestinal epithelial cells and 334 and 808 probiotics treated groups showed some degree of damage but relatively relaxed compared to the DSS treated group.

4-4. Intestinal inflammation  Cytokines in animal mesenteric lymph nodes mRNA  Confirmation of expression

To measure the expression of the inflammatory cytokines TNF-a, IFN-y and IL-1β, mesenteric lymph nodes of intestinal inflammatory animal models were detached and homogenized into single cells. Thereafter, 200 μl of chloroform per 1 ml of Trizol was added to separate total RNA, and 1 μg of the extracted mRNA was used as a template to synthesize cDNA by reverse transcription polymerase using Superscript III reverse transcriptase . Thereafter, the cDNA was used as a template and primers were prepared according to the nucleotide sequence obtained from the gene database, and the respective genes were analyzed by PCR (FIG. 6). The primer sets used at this time are shown in Table 7.

Gene Size Sequence TNF-a 502bp forward 5'-GAC CTG GAA CTG GCA GAA GAG G-3 '(SEQ ID NO: 9) reverse 5'-TGA CGG CAG AGA GGA GGT TGA C-3 '(SEQ ID NO: 10) IFN-y 433bp forward 5'-CTG AGA CAA TGA ACG CTA CAC ACT GC-3 '(SEQ ID NO: 11) reverse 5'-AAC AGC TGG TGG ACC ACT CGG AT-3 '(SEQ ID NO: 12) IL-1? 358bp forward 5'-CCA GGA TGA GGA CAT GAG CAC C-3 '(SEQ ID NO: 13) reverse 5'-ATC CAC ACT CTC CAG CTG CAG G-3 '(SEQ ID NO: 14) * GAPDH 271 bp forward 5'-ATG ACC ACA GTC CAT GCC ATC-3 '(SEQ ID NO: 7) reverse 5'-CCT GCT TCA CCA CCT TCT TG-3 '(SEQ ID NO: 8)

* Housekeeping gene GAPDH: Glyceraldehyde 3-phosphate dehydrogenase

As a result, it was confirmed that the expression of inflammatory cytokines was lowered in the mesangial lymph nodes of the sulfasalazine treated group and the probiotic treated group than the DSS treated group (FIG. 6).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Microorganism Conservation Center (overseas) KCCM11777P 20151001

<110> Korea University Research and Business Foundation <120> Novel Lactobacillus gasseri and Uses Thereof <130> KU1-57P <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> IFN-r F <400> 1 ctgagacaat gaacgctaca cactgc 26 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IFN-r R <400> 2 aacagctggt ggaccactcg gat 23 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-4 F <400> 3 gctagttgtc atcctgctct tc 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-4R <400> 4 tcagtgatgt ggacttggac tc 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IL-10 F <400> 5 agctccatca tgcctggctc a 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IL-10R <400> 6 cagactcaat acacactgca ggt 23 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GAPDH F <400> 7 atgaccacag tccatgccat c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH R <400> 8 cctgcttcac caccttcttg 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TNF-a F <400> 9 gacctggaac tggcagaaga gg 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TNF-a R <400> 10 tgacggcaga gaggaggttg ac 22 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> IFN-r F <400> 11 ctgagacaat gaacgctaca cactgc 26 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IFN-r R <400> 12 aacagctggt ggaccactcg gat 23 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-1b F <400> 13 ccaggatgag gacatgagca cc 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IL-1b R <400> 14 atccacactc tccagctgca gg 22

Claims (10)

Lactobacillus gasseri strain having probiotic activity deposited with Accession No. KCCM11777P.
The strain according to claim 1, wherein the strain has immunomodulating ability.
The strain according to claim 1, wherein the strain has acid resistance, resistance to fungi and intestinal adhesiveness.
The method of claim 1, wherein the strains are strains, characterized in that to control the expression of the Th ₁ and Th ₂ cytokine.
5. The method according to claim 4, wherein the strain expresses the expression level of at least one gene selected from the group consisting of TNF-a, IFN-y and IL-1 ?. Or &lt; / RTI &gt; its protein.
A Lactobacillus gasseri strain according to any one of claims 1 to 5, a lysate of said strain, a culture of said strain, a concentrate of said culture, a dried product of said culture and a fermentation product of said strain Or a pharmaceutically acceptable salt thereof, as an active ingredient.
The method of claim 6, wherein the inflammatory disease is inflammatory bowel disease, allergy, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;
A Lactobacillus gasseri strain according to any one of claims 1 to 5, a lysate of said strain, a culture of said strain, a concentrate of said culture, a dried product of said culture and a fermentation product of said strain Wherein the composition comprises at least one selected from the group consisting of the active ingredient and the active ingredient.
9. The method of claim 8, wherein the inflammatory disease is inflammatory bowel disease, allergy, dermatitis, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, Sjogren's syndrome, multiple sclerosis, and acute and chronic inflammatory diseases such as psoriatic arthritis, rheumatoid arthritis, osteoarthritis, rheumatoid arthritis, shoulder periitis, tendinitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, Lt; RTI ID = 0.0 &gt; of: &lt; / RTI &gt;
A Lactobacillus gasseri strain having probiotic activity deposited under Accession No. KCCM 11777P of any one of claims 1 to 5, a lysate of said strain, a culture of said strain, a culture of said culture Wherein the active ingredient is at least one selected from the group consisting of an extract, a concentrate, a dried product of the culture, and a fermented product of the strain.

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