KR101075558B1 - Novel Lactobacillus plantarum and compositions comprising the same - Google Patents

Abstract

The present invention provides Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) KCTC 11404BP, a composition for treating intestinal diseases, including the lactic acid bacteria, and an immune enhancing composition comprising the lactic acid bacteria.

Description

Novel Lactobacillus plantarum and compositions comprising the same

The present invention relates to a novel Lactobacillus plantarum and a composition comprising the same, and more particularly to a novel Lactobacillus plantarum and a composition comprising the same useful for the prophylactic treatment of intestinal diseases and immune diseases.

Lactobacillus, which is abundant in traditional fermented foods such as kimchi, plays a role in breaking down the fiber and complex proteins into important nutrients while living in the digestive system of the human body. Probiotics are collectively called living microorganisms that improve the environment and have a beneficial effect on the health of the host. In order to be effective as a probiotic, oral ingestion should reach the small intestine and adhere to the intestinal surface.

A typical probiotic found in traditional fermented foods such as kimchi is Lactobacillus sp. Lactobacillus microorganisms are lactic acid bacilli that are homozygous or heterozygous and are commonly found in the intestines of animals, including humans, and in fermentation of dairy products or vegetables. Lactobacillus microorganisms maintain acidic intestinal pH to inhibit the growth of harmful bacteria such as E. coli and Clostridium , improve diarrhea and constipation, as well as reduce vitamin synthesis, anticancer activity, and serum cholesterol It is known to play a role. Acidophillin produced by lactic acid bacilli is known to inhibit the growth of dysentery, Salmonella, Staphylococcus aureus, and Escherichia coli. In addition, it inhibits the growth of the causative organism of diarrhea and stops diarrhea by normalizing the intestinal flora (Michael and Philippe, Probiotics and prebiotics: Effects on diarrhea, The journal of nutrition, Volume 137, March 2007, pages 803S-811S; Roberfroid, Prebiotics and probiotics: Are they functional foods ?, American journal of clinical nutrition, Volume 71, June 2000, pages 1682S-1687S).

Research into the development of probiotics and livestock feed is actively underway using the above characteristics of Lactobacillus microorganisms. Bacterial diarrhea in livestock causes reduced weight gain and mortality. Therefore, the addition of antibiotics to feed has generally been widely practiced to prevent this and increase the production of livestock. However, due to problems such as the emergence of antibiotic resistant bacteria and residual antibiotics in livestock products, there is a tendency to regulate the use of antibiotics in feed and to emphasize the organic livestock specification law (Korean Patent Publication 1998-78358) (McEwen and Fedorka-Cray, Antimicrobial use and resistance in animals, Clinical infectious Diseases, Volume 34, June 2002, pages S93-S106).

In addition, lactic acid bacteria such as Lactobacillus microorganisms are known to have an immune enhancing effect. Recently, allergic and atopic diseases related to immunomodulatory abnormalities, which are predicted due to environmental pollution and increased intake of instant foods, are rapidly increasing. In Korea, these diseases are also increasing. Recently, as part of a microbial treatment (bacteriotheraphy) that treats diseases by oral administration of lactic acid bacteria, efforts have been made to alleviate or improve symptoms of the disease with lactic acid bacteria. Administration of Lactobacillus rhamnosus GG to infants reduced atopic incidence by half (Kalliomki et. Al. , Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial, Lancet, Volume 357, April 2001, pages 1076-1079), when Lactobacillus rhamnosus and L. reuteri are administered to a child who already has atopic eczema, there are reports of decreased eczema site and extent (Rosenfeldt et. al ., Effect of probiotic Lactobacillus strains in children with atopic dermatitis, Dermatologic and ocular diseases, Volume 111, February 2003, pages 389-395).

The mechanism of the immune enhancing effect of these lactic acid bacteria is continuously studied, and the specific mechanism is not yet clear, but it is generally orally introduced into the intestine and affects the intestinal immune system by living in the intestine. It is known. For example, lactobacillus intake through yogurt is known to increase the antimicrobial activity of lymphocytes in Peyer's patch, and some studies in experimental animals and humans have shown that lactobacillus enhances the response of IgA. In addition, lactic acid bacteria affect both innate and adaptive immunity. Innate immunity of the intestinal immune system is known to play a role in maintaining health against infection by phagocytosis and killing of pathogens. In adaptive immunity, macrophages, which break down antigens and present them to T lymphocytes, activate macrophages to increase the production of various cytokines, particularly interleukin IL-12 and IL-18, which are part of the lactic acid bacteria cell wall components. Is known to increase cytokine production by activating NF-B, STAT signaling. In addition, lactic acid bacteria are specialized antigen-presenting cells that increase the production of IL-12, IL-18, TNF- in dendritic cells that are present in the lymph nodes and mucous membranes of the digestive system, as well as MHC class II and B7-2. It is also known to increase the expression of surface molecules that activate T lymphocytes (Cross et. Al ., Anti-allergy properties of fermented foods: an important immunoregulatory mechanism of lactic acid bacteria ?, International Immunopharmacology, Volume 1, May 2001 , pages 891-901).

T lymphocytes are the cells that are the center of adaptive immunity, which can be divided into Th1 response, which is cellular immunity, and Th2 response, which is antibody immunity. Cytokines produced by Antigen Presenting Cells are different in Th1 and Th2 reactions, IL-12, IL-18, and interferon (IFN) production prevail in Th1 reaction, and PGE2, IL- in Th2 reaction 4, IL-10 production predominates. These Th1 and Th2 response should be appropriately normalized, and it is known that various immune diseases appear when the balance is broken. Th1 cells primarily fight infection, while Th2 cells are primarily involved in allergic and inflammatory responses. When they function normally, Th2 cells protect the body from dust and other unwanted substances, but if these cells show excessive activity, they increase production of IgE antibodies to proteins that have not been a threat to the body (eg pollen, food, etc.). Causes an allergic reaction. Therefore, the Th1 and Th2 responses must be balanced, and if one of them is excessive or the other lacks, the disease is caused. In addition, sustained release of cortisol leads to a decreased Th1 response and an increased Th2 response, leading to cancer, atopy, allergies, and autoimmune diseases (Elenkov and Chrousos, Stress hormones, Th1 / Th2 patterns, pro / anti-inflammatory cytokines and susceptibility to disease, Trends in Endocrinology and Metabolism, Volume 10, November 1999, pages 359-368).

In vivo experiments have shown that lactic acid bacteria increase the production of Th1 cytokine IFN-γ and inhibit the production of Th2 cytokines IL-4 and IL-5 (Matsuzaki et. Al. , The effect of oral). feeding of Lactobacillus casei strain Shirota on immunoglobulin E production in mice, Journal of Dairy Science, Volume 81, January 1998, pages 48-53). In other experiments, IFN-γ was increased in splenocytes by oral administration of lactic acid bacteria to ovalbumin-primed mice administered with Th2 response animal model of ovalbumin. IL-4, IL -5 and IgE were decreased, and the changes of cytokines and IgE were also the same as the oral administration experiments when splenocytes harvested from mice that were biased by Th2 response by administration of ovalbumin were incubated with lactic acid bacteria. However, IFN-γ production of T lymphocytes did not significantly increase when incubated with lactic acid bacteria, and antigen-presenting cells such as macrophages and dendritic cells are necessary for IFN-γ production of T lymphocytes (Kato et. Al. , Lactic) . acid bacterium potently induces the production of interleukin-12 and interferon-gamma by mouse splenocytes, International Journal of Immunopharmacology, Volume 21, February 1999, pages 121-131). IL-12 and IL-18 are important cytokines for the differentiation of Th0 lymphocytes into Th1 lymphocytes. They are produced in macrophages or dendritic cells. It is known that production of IFN-γ and the like is increased. As such, lactic acid bacteria increase the production of IL-12, IL-18 and IFN-γ in macrophages, thereby promoting differentiation into Th1 cells and inducing their IFN-γ production, and therefore Th1 / Th2 balance in the situation where Th2 predominates. (Cross et. Al ., Anti-allergy properties of fermented foods: an important immunoregulatory mechanism of lactic acid bacteria ?, International Immunopharmacology, Volume 1, May 2001, pages 891-901). Thus, lactic acid bacteria are known to help prevent or treat cancer, atopy, allergies, and autoimmune diseases caused by Th1 / Th2 imbalances due to a Th2 response excess.

Accordingly, the present inventors have studied to develop a novel lactic acid bacteria having a very good effect of controlling the Th1 / Th2 imbalance due to the Th2 reaction excess compared to the known lactic acid bacteria, a novel Lactobacillus lactic acid bacteria strain from traditional fermented foods The present invention has been completed by separation and identification.

Accordingly, the object of the present invention is not only excellent in acid resistance, bile acid resistance, intestinal epithelial cell adhesion, which are basic properties as probiotics, but also excellent in enhancing immune immunity, in particular, in controlling Th1 / Th2 imbalance due to excessive Th2 response. It is to provide a Lactobacillus strain of lactic acid bacteria.

Another object of the present invention to provide a composition for the prevention or treatment of enteric diseases, including the novel Lactobacillus lactic acid strain.

Still another object of the present invention is to provide a composition for enhancing immunity comprising the novel Lactobacillus lactic acid strain.

In order to achieve the above object, the present invention provides Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) (depositment institution: Biotechnology Research Institute Gene Bank, Deposit Date: 2008.10.16, Accession No .: KCTC 11404BP).

The present invention also provides a composition for treating intestinal diseases comprising the Lactobacillus plantarum CJLP136.

The present invention also provides a composition for enhancing immunity comprising Lactobacillus plantarum CJLP136.

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

Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) according to the present invention is characterized by a novel strain of Lactobacillus platarum isolated and identified from traditional fermented foods. The traditional fermented foods include, but are not limited to, kimchi, vegetable fermented product, miso, soy sauce, cheonggukjang or salted fish.

Lactobacillus plantarum NBRC15891 ^T , GenBank accession number AB326351) showed the highest homology (99.8%) as a result of 16S rRNA sequencing for the identification and classification of microorganisms. It showed the highest molecular systematic relationship with Lactobacillus plantarum. Therefore, the microorganism was identified as Lactobacillus plantarum, named as Lactobacillus plantarum CJLP136, and deposited on October 16, 2008 in the Biotechnology Research Institute Gene Bank (Accession No. KCTC 11404BP). The base sequence of the 16S rRNA gene of Lactobacillus plantarum CJLP136 (GenBank accession number FJ455519) is shown in SEQ ID NO. Same as 1.

The Lactobacillus plantarum CJLP136 of the present invention is a gram-positive bacterium, a facultive anaerobe capable of growing in both aerobic and anaerobic conditions, does not form spores, has no mobility, and has a cell form. More specific morphological and physiological properties of Lactobacillus plantarum CJLP136 were analyzed according to the conventional methods in the art, and are shown in Table 1 below.

TABLE 1

In order to stably preserve Lactobacillus plantarum CJLP136 of the present invention, it is preferable to dissolve cells in a storage solution made by mixing a certain amount of glycerol components in water, store them at -70 ° C, or suspend them in sterilized 10% skim milk to freeze-dry them. Do.

In addition, the Lactobacillus plantarum CJLP136 of the present invention is a probiotic, and has a general intestinal effect and an immune enhancing effect of lactic acid bacteria.

In the present invention, 'probiotics' is understood to mean a living microorganism that has a beneficial effect on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of animals including humans. Probiotics are living microorganisms with probiotic activity that can have a beneficial effect on the intestinal flora of the host when fed in the form of dried cells or fermented products to humans or animals in the form of single or complex strains. In order to be a probiotic microorganism, first, it is possible to pass through the stomach and survive in the intestine while being less affected by gastric juice and bile. It should be able to influence. Therefore, it must have acid resistance to gastric acid, bile acid resistance to bile acids, and adhesion to intestinal epithelial cells. Second, in order to be a probiotic microorganism, there should be no problem in the safety of the microorganism, and in this regard, a gelatin liquefaction test, a phenylalanine talamine production test, ammonia production test and hemolytic test are generally performed. Lactobacillus plantarum CJLP136 according to the present invention has excellent acid resistance, bile acid resistance to bile acids, and adhesion to intestinal epithelial cells, as well as gelatin liquefaction test, phenylalaninetalamine production test, and ammonia production test. It was negative, and hemolytic laboratory tests were confirmed to be safe as α-hemolysis determined to be nonpathogenic.

Lactobacillus plantarum CJLP136 according to the present invention is very excellent in acid resistance, bile acid resistance, and intestinal epithelial cell adhesion is expected to have a formal effect. Therefore, in another aspect, the present invention provides a composition for preventing or treating intestinal diseases, including Lactobacillus plantarum CJLP136.

The composition for treating intestinal diseases comprising the microorganism according to the present invention may be used for the prevention or treatment of intestinal diseases in mammals including humans, and preferably includes livestock such as cattle, horses, and pigs. The 'intestinal disease' includes both intestinal harmful bacterial infection and inflammatory bowel disease, for example, infectious diarrhea caused by pathogenic microorganisms (E. coli, Salmonella, Clostridium, etc.), gastroenteritis, inflammatory bowel disease, neuro enteritis syndrome, Small intestinal microflora, growth, enterotropic diarrhea, and the like. The Lactobacillus plantarum CJLP136, which is included in the composition for treating intestinal diseases, may exist as live or dead cells, but is preferably present as live cells. In general, live cells have the effect of treating and ameliorating all symptoms caused by abnormal fermentation of the intestinal flora, and when administered to humans and animals, they are concentrated and settled in the gut wall of the intestine, preventing harmful bacteria from settling. It lowers the pH of the intestine by preventing the growth of harmful bacteria. In addition, the administered probiotics produce bacteriocins and peroxides to inhibit the growth of pathogens and help the intestinal villi work to absorb nutrients. In addition, they produce substances that help the absorption and utilization of nutrients, improve feed requirements in animals, and produce substances that neutralize the toxins produced by pathogens.

The method of administering the composition for preventing or treating intestinal diseases of the present invention is not particularly limited, but is preferably administered orally. The dosage may vary depending on the type of intestinal disease, the severity of the disease, age, sex, race, treatment or prophylactic purpose, etc., but in general, 10 to 100 billion mice may be administered per adult.

In addition, the Lactobacillus plantarum CJLP136 of the present invention has a significantly superior immunopotentiating effect compared to conventional lactic acid bacteria in addition to the formal effect. Lactobacillus plantarum CJLP136 increases the production of IL-12 that induces a Th1 response in splenocytes and inhibits the production of IL-4 that induces a Th2 response. In addition, as antigen presenting cells, it stimulates immune-regulated related cells such as macrophages and dendritic cells that regulate T cell immune responses, thereby promoting the production of cytokines that induce differentiation of Th0 lymphocytes into Th1 lymphocytes, resulting in an excess of Th2 response. It has been shown to have immunomodulatory ability to control Th1 / Th2 imbalance. When explaining the immune enhancing effect of Lactobacillus plantarum CJLP136 in more detail.

Lactobacillus plantarum CJLP136 produces 11.9 to 13.2 times higher Th1 response-induced cytokine IL-12 when treated with splenocytes of mice that are biased by Th2 response by administration of ovalbumin (OVA) compared to negative control Inhibition of the production of Th2 response-induced cytokine IL-4 to 3.9-13.5%, compared to other typical lactic acid bacteria Lactobacillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC13416) It was confirmed that it was remarkably excellent. Therefore, it can be said that Lactobacillus plantarum CJLP133 has an immunomodulatory ability to inhibit Th2 response and promote Th1 response, thereby regulating Th1 / Th2 imbalance due to the excess of Th2 response.

In addition, Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) was treated with macrophage cell lines RAW264.7 and dendritic cells JAWSII, it was confirmed that as the number of lactic acid bacteria increased macrophage cell line to stimulate the immune response. Treatment of the macrophage cell line RAW264.7 and dendritic cell line JAWSII with Lactobacillus plantarum CJLP136 results in the production of Th1 differentiation-inducing cytokines IL-12, IL-18 and the inhibition of the Th1 differentiation-inducing cytokine IL-10. It has been shown to promote Th1 differentiation induction by producing relatively less than the amount of IL-12 produced. Therefore, even from these experimental results, it can be said that Lactobacillus plantarum CJLP136 has an immunomodulatory ability to suppress Th2 response and promote Th1 response, thereby controlling Th1 / Th2 imbalance due to the excess of Th2 response.

IL-4 is derived from Th2 cells and specifically plays a pivotal role in the cellular immune response and has an anti-inflammatory cytokine function that inhibits the production of the cytokine IL-12 of Th1 cells. Recently, peripheral blood and skin lesions of patients with atopic dermatitis have been found to have relatively increased Th2 cells, which mainly produce IL-4 and IL-5 (Miraglia et. Al , Immune dysregulation in atopic dermatitis, Allergy and Asthma Proceedings). , Volume 27, November-December 2006, pages 451-455. Thus, Th1 / Th2 imbalance due to the Th2 response excess leads to diseases such as atopy. In addition, as discussed above, an excess or lack of one of the Th1 and Th2 responses causes disease, and when the Th1 response decreases and the Th2 response increases, cancer, atopy, allergy, and autoimmune diseases are caused. (Elenkov and Chrousos, Stress hormones, Th1 / Th2 patterns, pro / anti-inflammatory cytokines and susceptibility to disease, Trends in Endocrinology and Metabolism, Volume 10, November 1999, pages 359-368). Thus, Lactobacillus plantarum CJLP136 according to the present invention by controlling the cytokines produced by Th1 cells, Th2 cells, macrophages, and dendritic cells involved in immunomodulation, thereby controlling the imbalance of Th1 / Th2 due to the Th2 response excess, Not only can it be expected to work effectively against diseases such as atopy and allergy, but also to prevent or treat cancer and autoimmune diseases.

Therefore, in another aspect, the present invention provides a composition for enhancing immunity comprising Lactobacillus plantarum CJLP136. The immune enhancing composition according to the present invention has an immune enhancing effect due to the immune enhancing effect of Lactobacillus plantarum CJLP136 is a lactic acid bacterium as described in the prior art. In particular, the immune enhancing composition according to the present invention has the effect of promoting the Th1 response as Lactobacillus plantarum CJLP136 has the effect of promoting Th1 / Th2 imbalance due to the excessive Th2 response as demonstrated in the following examples Therefore, it is effective in preventing or treating a disease caused by an imbalance of Th1 / Th2 due to an excessive Th2 response. Therefore, the immune enhancing composition of the present invention can be effectively used for the prevention or treatment of atopy, allergy, cancer and autoimmune diseases. The autoimmune diseases include, but are not limited to, asthma, high fever, and the like.

The method of administering the immune enhancing composition of the present invention is not particularly limited, but is preferably administered orally. Dosages may vary depending on the type of disease, the severity, age, sex, race, treatment or prophylaxis of the disease requiring immunity, but generally between 10 and 100 billion animals per day Can be.

The composition for preventing or treating intestinal diseases and the composition for enhancing immunity, including Lactobacillus plantarum CJLP136, according to the present invention includes lactic acid bacteria, which have been proven to be safe, and thus, pharmaceuticals, foods, cosmetics, feed, or It can be used as a feed additive.

When the composition according to the present invention is used as a medicine, it may be formulated into a conventional pharmaceutical formulation known in the art. The medicament may preferably be formulated in an oral dosage form and may be formulated in a dosage form for oral administration such as, for example, liquids, suspensions, powders, granules, tablets, capsules, pills, or extracts.

When formulated into each of the above formulations, it may be prepared by the addition of a pharmaceutically acceptable carrier or additive necessary for the preparation of each formulation. Representatively, when formulated into a dosage form for oral administration, one or more of a diluent, a lubricant, a binder, a disintegrant, a sweetener, a stabilizer, and a preservative may be used as the carrier, and as an additive, flavors, vitamins, and antioxidants may be used. One or more types can be selected and used out of them.

The carrier and the additive may be any pharmaceutically acceptable, and specifically, lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol as a diluent, magnesium stearate or talc as a lubricant, and polyvinylpyrrolidone as a binder. Or hydroxypropyl cellulose is preferred. In addition, as a disintegrant, calcium carboxymethyl cellulose, sodium starch glycolate, potassium polyacrylic acid, or crospovidone, sweetener as white sugar, fructose, sorbitol, or aspartame, stabilizer as carboxymethyl cellulose sodium, beta-cyclodextrin, As lead, or xanthan gum, and preservative, methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate is preferable.

In addition to the above ingredients, in order to enhance the taste as well-known additives, natural flavors such as plum, lemon, pineapple, herbal flavor, natural pigments such as fruit juice, chlorophyllin, flavonoids, fructose, honey, sugar alcohol, sugar Sweetening ingredients such as, or may be used by mixing an acidulant such as citric acid, sodium citrate.

Such formulation methods and the carriers and additives required for formulation are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The composition according to the present invention can be used as food. The food includes not only the health functional food, but also general foods that humans consume in general and daily. When used as a nutraceutical, it may be formulated into a formulation of a conventional nutraceutical known in the art together with a pharmaceutically acceptable carrier or additive. The health functional food may be prepared, for example, as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, liquids, extracts, teas, jelly, or beverages. As a food acceptable carrier or additive, any carrier or additive known to be available in the art for the preparation of the formulation to be prepared may be used.

Since the composition according to the present invention has a prophylactic or therapeutic effect of atopy, it may be used as a cosmetic. When the composition according to the present invention is used as a cosmetic can be prepared in a variety of cosmetics of conventional formulations known in the cosmetic art. When formulated into each of the above formulations, it may be prepared by adding an acceptable carrier or additive for the preparation of cosmetics necessary for the preparation of each formulation.

The composition according to the present invention can be used as a feed additive or feed.

When used as a feed additive, the composition may be 20 to 90% high concentrate or prepared in powder or granule form. The feed additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherols and rosemary Extracts, vitamin C, green tea extracts, licorice extracts, chitosan, tannic acid, phytic acid, such as any one or more may be further included. When used as a feed, the composition may be formulated in a conventional feed form and may include common feed ingredients.

The feed additives and feeds are cereals, for example milled or crushed wheat, oats, barley, corn and rice; Vegetable protein feed, such as feed consisting mainly of rapeseed, soybean, and sunflower; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; Sugar and dairy products, for example, may further include a dry ingredient consisting of various powdered milk and whey powder, and may further include a nutritional supplement, digestion and absorption enhancer, growth promoter and the like.

The feed additive may be administered alone or in combination with other feed additives in an edible carrier. In addition, the feed additive may be easily administered to the animal as a top dressing or directly mixed into the animal feed or in an oral formulation separate from the feed. When administered separately from the animal feed, the feed additive can be prepared in immediate release or sustained release formulation, in combination with a pharmaceutically acceptable edible carrier, as is well known in the art. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the feed additive may be a tablet, capsule, powder, troche or sugar-containing tablet or top dressing in microdisperse form. If a liquid carrier is used, the feed additive may be a gelatin soft capsule or a formulation of syrup or suspension, emulsion, or solution.

The feed may comprise any protein-containing organic flour commonly used to meet the dietary needs of the animal. Such protein-containing flours typically consist primarily of corn, soy flour, or corn / bean flour mixtures.

In addition, the feed additives and feed may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifiers, solution promoters and the like. The feed additive may be used in addition to the feed of the animal by acupuncture, spraying or mixing.

The feed or feed additive of the present invention is applicable to many animal diets, including mammals, poultry and fish. The mammals can be used not only for pigs, cows, sheep, goats, experimental rodents, and experimental rodents, but also for pets (eg, dogs, cats), and the like as chickens, turkeys, ducks, geese, and pheasants. And can also be used for quail, etc., may be used in trout, such as fish, but is not limited thereto.

As described above, the novel lactic acid bacterium Lactobacillus plantarum CJLP136 according to the present invention is probiotic and has an excellent effect on acid resistance, bile acid resistance, and intestinal epithelial cell adhesion, and has an effect of promoting a Th1 response. It is effective in controlling the imbalance of Th1 / Th2 due to the Th2 reaction excess. Therefore, the novel lactic acid bacterium Lactobacillus plantarum CJLP136 according to the present invention can be used as a composition for treating intestinal diseases, and as an immune enhancing composition, and particularly for the prevention of diseases caused by the imbalance of Th1 / Th2 due to the excessive Th2 response. Effective for treatment

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for the understanding of the present invention, and the scope of the present invention is not limited by them in any sense.

Example 1 Isolation and Identification of Microbial Lactobacillus Plantarum CJLP136 Strains

Lactobacillus plantarum CJLP136 strain isolated from Kimchi was plated in MRS solid medium (Difco, USA) containing 1.5% agar and incubated for 24 hours at 37 ° C. Taken as a loop and incubated 18-24 hours at 37 ℃ in MRS liquid medium (Difco, USA).

The morphological and physiological characteristics of the CJLP136 strain were then determined by Kim et al ., Leuconostoc inhae sp.nov ., A lactic acid bacterium isolated from kimchi, International Journal of Systematic and Evolutional Microbiology, Volume 53, July 2003 , pages 1123-1126) and API50CH and API50CHL kits (manufactured by Biomerio). As a result, the morphological and physiological characteristics of the identified CJLP136 strain are summarized in Table 1 above.

In addition, the nucleotide sequence of 16S rRNA gene for lactic acid bacteria identification and classification was analyzed. Nucleotide sequence determination and analysis of 16S rRNA genes is described by Kim et. Al ., Leuconostoc kimchii sp.nov ., A new species from kimchi.International Journal of Systematic and Evolutional Microbiology, Volume 50, September 2000, pages 1915-1919 ) Method was used. The resulting nucleotide sequence of 16S rRNA gene of CJLP133 was listed in the sequence list (SEQ ID NO: 1).

Lactobacillus plantarum CJLP136 strain showed the highest homology (99.8%) with Lactobacillus plantarum NBRC15891 ^T , GenBank accession number AB326351, according to 16S rRNA sequencing. plantarum), Lactobacillus plantarum CJLP136, and was deposited on October 16, 2008 with the Biotechnology Research Institute Gene Bank (Accession Number KCTC 11404BP).

Example 2: Acid resistance test in artificial gastric juice of Lactobacillus plantarum CJLP136 strain and bile resistance test in artificial bile

Acid resistance experiments in artificial gastric fluids include Kobayashi et al ., Studies on biological characteristics of Lactobacillus : II.Tolerance of the multiple antibiotic resistance strain, L. casei PSR3002, to artificial digestive fluids.Japan Journal of Microbiology.Volume 29 , July 1974, pages 691-697), using artificial gastric fluid prepared by modifying the experiment. Specifically, artificial gastric fluid was adjusted to pH 2.5 with 1 N HCl and pepsin to 1000 units / ml. Prepared by addition and then sterilized.

The Lactobacillus plantarum CJLP136 isolated and identified in Example 1 was cultured in MRS liquid medium at 37 ° C. for 18 hours by centrifuging the cells to precipitate the lactic acid strain and washed twice with sterile saline solution (0.85% NaCl). After that, the cell suspension was inoculated in the control medium and artificial gastric juice at a level of about 10 ⁷ cfu / ml, respectively, and cultured at 37 ° C., and the number of viable cells was measured 0 and 3 hours after inoculation. Total bacterial count was measured by diluting 10-fold with phosphate buffer (pH 6.8) containing KH ₂ PO 4, Na ₂ HPO, L-cysteine, HCl, Tween 80, and the like.

Bile resistance experiments in artificial bile have been described by Casey et. Al. , Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract, Letters in Applied Microbiology.Volume 39, 2004, pages 431-438 Oxygen bile was added to the MRS liquid medium used in the acid resistance evaluation, followed by the same method as the acid resistance evaluation method, and the viable bacterial counts were measured 0, 12, and 24 hours after lactic acid inoculation. .

Similar comparison experiments were performed for Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC13416), and Lactobaillus rhamnosus GG (KCTC 5033), which are typical lactic acid bacteria in both the acid resistance evaluation and the bile acid resistance evaluation.

The results are shown in FIGS. 1 and 2. 1 is a graph showing the results of an acid resistance test of Lactobacillus plantarum CJLP136. Figure 2 is a graph showing the results of the bile resistance test of Lactobacillus plantarum CJLP136.

According to the results of FIGS. 1 and 2, Lactobacillus plantarum CJLP136 was found to have more than the equivalent acid resistance and bile acid resistance compared to all other lactic acid bacteria tested. This indicates that the novel strain according to the present invention can reach the intestine without the influence of gastric juice in the body and can survive without the influence of bile in the intestine.

Example 3: Adhesion test on enteroepithelial cells of Lactobacillus plantarum CJLP136 strain

As an animal cell for intestinal epithelial cell adhesion test, HT-29 was distributed and used by Korea Cell Line Bank (KCLB), and the experimental method was Kim et. Al. , Probiotic properties of Lactobacillus and Bifidobacterium strains isolated from porcine gastrointestinal tract, Applied Microbiology and Biotechnology, Volume 74, April 2007, pages 1103-1111) and Hirano et al. ( Hirano et. al. , The effect of Lactobacillus rhamnosus on enterohemorrhagic Escherichia coli infection of human intestinal cells in vitro , Microbiology and Immunology, Volume 47, 2003, pages 405-109 ) .

HT-29 cells were RPMI 1640 (Gibco, USA) supplemented with 10% fetal calf serum (FBS), 1% L-glutamine, penicillin G (100 IU / mL), and streptomycin (100 mg / mL). ) Was incubated at 37 ° C. in the presence of 5% CO ₂ . For adhesion and anti-adhesion experiments, HT-29 cells were aliquoted into 24 well-plates at a number of 1.0 x 10 ⁵ cells / mL per well, changing media every other day, until a complete monolayer was formed. The culture was used for the experiment. HT-29 cells forming a complete monolayer were washed five times with PBS buffer at 25 ° C. and 0.5 mL of RPMI 1640 medium without antibiotics was added.

Lactobacillus plantarum CJLP136 was suspended in RPMI to a concentration of about 1.0 × 10 ⁹ cfu / mL, respectively, and then inoculated into the well plates and incubated at 37 ° C. for 2 hours in the presence of 5% CO ₂ . After the incubation was completed, washing was performed three times using PBS buffer while stirring at a speed of 200 rpm for 3 minutes to confirm the ability to remove and adhere to the lactic acid bacteria not attached. After washing, dispense 0.2% trypsin-EDTA to remove the adhered cells, plate them on MRS-agar by continuous dilution method using peptone water, and incubate for 24 hours at 37 ℃. Measured.

Separately, in order to confirm some adhesion, the sterilized cover glass was soaked in 70% alcohol for one day, and then attached to the bottom of the Petri dish, followed by culturing HT-29 cells and adding the same amount of lactic acid bacteria. Lactobacillus attached to HT-29 cells without being washed off by washing was dried and observed with an optical microscope by Gram staining, and the number of bacteria was measured. Similar experiments were performed for Lactobacillus sakei CJLS118, and Lactobaillus rhamnosus GG (KCTC 5033).

The results are shown in Fig. Figure 3 is a graph showing the results of intestinal epithelial cell adhesion capacity of Lactobacillus plantarum CJLP136

According to the results of FIG. 3, Lactobacillus plantarum CJLP136 showed superior intestinal epithelial cell adhesion after 24 hours compared to Lactobacillus rhamnosus GG (KCTC 5033) and Lactobacillus sakei CJLS118, which are commercially known as probiotic strains. Intestinal epithelial cell adhesion was significantly higher than that of Lactobacillus sakei CJLS118. These results indicate that the novel strain according to the present invention can be attached to intestinal epithelial cells to improve the intestinal environment.

Example 4: Safety Evaluation of Lactobacillus Planta Room CJLP136 Strains

In order to evaluate the safety of the strain isolated in Example 1, hemolytic phenomenon test, gelatin liquefaction test, harmful metabolite (ammonia) production confirmation, phenalanalantal in accordance with the safety evaluation test method presented in the Korea Bio Venture Association group standard An amine test was performed.

The results are shown in Table 2 below.

TABLE 2

According to the above results, Lactobacillus plantarum CJLP136 was negative for gelatin liquefaction, noxious metabolite (ammonia) production, phenalanlanthalamine production, and hemolysis test was found to be pathogenic. confirmed by α-hemolysis. Thus, Lactobacillus plantarum CJLP136 has been identified as a safe strain that can be administered to humans.

Example 5 Evaluation of Promoting IL-12 Production after Mouse Spleen Cell Treatment

Lactobacillus plantarum CJLP136 strain,

To evaluate the ability of Th12 response-inducing cytokine to stimulate IL-12 production when treated with spleen cells of mice that are biased by Th2 response by administering ovalbumin, Fujiwara et al. (Fujiwara et. Al. A double-blind trial of Lactobacillus paracasei strain KW3110 administration for immunomodulation in patients with pollen allergy, Allergology International, 2005, volume 54, pages 143-149) and Fujiwara et al. (Fujiwara et. Al., The anti-allergic effects of lactic acid bacteria are strain dependent and mediated by effects on Both Th1 / Th2 cytokine expression and balance, International Archives of Allergy and Immunology, 2004, Volume 135, pages 205-215).

Immunization was obtained by purchasing five 6-week-old female Balb / c mice, mixing well with 1.538 mL of alum hydroxide (Sigma) 13 mg / mL solution, 10 mg of ovalbumin and 0.4615 mL of PBS, and reacting at room temperature for 20 minutes. The mixture was injected intraperitoneally with 0.2 mL (1 mg OVA + 2 mg alum) per mouse and boosted by injecting the same amount again into the abdominal cavity on the 6th day. Mice were sacrificed on day 13 and spleens were extracted and 100 μl (4 × 10 ⁶ cells / mL) of the splenocytes obtained therefrom, 50 μl of the dead bacterium and 50 μl of ovalbumin (4 mg / mL) were tested for cell culture wells. Plates were added to the cell culture well plates and incubated in a 10% CO ₂ incubator for 7 days in DMEM-10 medium. After incubation for 7 days, the supernatant was taken and assayed with an IL-12 ELISA kit (Biosource) to measure the concentration of IL-12.

The dead bacteria of the test subject bacteria were obtained as follows.

The test cells were inoculated in MRS liquid medium (Difco) and incubated for 24 hours at 37 ℃, and the cells obtained by centrifugation for 1 minute at 13,000 rpm was washed twice in physiological saline and only the cells were taken. The recovered cells were heated for 10 minutes at 100 ° C. in sterile distilled water of the same volume as the original culture volume for animal cell line inoculation test, centrifuged at 13,000 rpm for 1 minute, then recovered and diluted in DMEM medium in an appropriate amount based on the cell line culture volume. The cells were allowed to have a concentration of 50 μg / ml and 5 μg / ml. Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), and Lactobacillus sakei CJLS118 (KCTC 13416) were used as Lactobaillus plantarum CJLP136 as test targets, and the results were compared.

IL-12 assay using the IL-12 ELISA kit was carried out in accordance with the instructions provided in the IL-12 ELISA kit and the calibration of the IL-12 control sample provided in the kit by measuring the OD value measured in the ELISA reader According to the formula, the amount of IL-12 produced was converted. Thus, the obtained measurement result is shown in FIG.

Figure 4 shows Lactobacillus plantarum CJLP136 strain treated with ovalbumin to splenocytes of mice that were biased by Th2 response, and then measured the concentration of IL-12, a Th1 response-induced cytokine, compared with other lactic acid bacteria. It is a graph.

According to the results of Figure 4, Lactobacillus plantarum CJLP136 was shown to significantly promote the production of Th1 response-inducing cytokine IL-12 compared to other lactic acid bacteria. Therefore, it was confirmed that Lactobacillus plantarum CJLP136 according to the present invention effectively induces the Th1 response in mice biased by the Th2 response.

Example 6: Evaluation of Inhibition of IL-4 Production After Mouse Spleen Cell Treatment

The Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) strain was treated with ovalbumin to spleen cells of mice deflected by Th2 response to confirm the inhibitory effect of Th2 response-induced cytokine IL-4 production. The method of Example 5 is different from using the IL-4 kit (Biosource) instead of the IL-12 kit, and the rest of the conditions were the same experiment. Thus measured results are shown in FIG.

5 shows Lactobacillus plantarum CJLP136 strain treated with ovalbumin to splenocytes of mice biased by Th2 response, and then measured the concentration of IL-4, a Th2 response-induced cytokine, compared with other lactic acid bacteria. It is a graph.

According to the results shown in FIG. 5, it was confirmed that Lactobacillus plantarum CJLP136 had an inhibitory effect on Th2 response in mouse splenocytes biased by Th2 response by inhibiting the Th2 response-inducing cytokine, IL-4 cytokine.

Example 7: Expression of Th1 lymphocyte differentiation-inducing cytokines IL-12p40 and IL-18 and inhibition of Th1 lymphocyte differentiation-induced cytokine cytokine IL-10 expression in macrophages and dendritic cells

Antigen presenting cells (APCs), such as macrophages and dendritic cells, produce IL-12 and IL-18 to induce differentiation of Th0 lymphocytes into Th1 lymphocytes, and on the other hand, to produce IL-10 Inhibit differentiation induction. In order to evaluate the effect of the lactic acid bacteria according to the present invention on the production of IL-12, IL-10 and IL-18 of macrophages and dendritic cells, the following experiment was performed.

The test subject was treated with macrophages cell line RAW264.7 at a concentration of 510 ⁷ / mL, and cultured at 37 ° C. and 10% CO ₂ for 48 hours, and then the medium was taken to measure the concentration of IL-12p40 and IL-10 by ELISA. Measured. In addition, the inoculation and incubation of the test bacteria in the dendritic cell line JAWSII in the same manner, and then the medium was taken to measure the production of IL-12p40 and IL-10.

Lactobacillus plantarum CJLP136 was used as the test target bacteria, lipopolysaccharide was used as a positive control, Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC 13416). The same experiment was performed and the results were compared.

The concentration measurement by the ELISA method is IL-12p40 kit (BD Biosciences, USA) to measure the concentration of IL-12 and IL-10 kit (BD Biosciences, USA) to measure the concentration of IL-10 Using according to the manufacturer's instructions. Thus, the respective results measured are shown in FIGS. 6 and 7.

6 is a graph showing Lactobacillus plantarum CJLP136 strain treated with macrophage cell line RAW264.7, and the concentration of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

7 is a graph showing Lactobacillus plantarum CJLP136 strain treated with dendritic cell line JAWSII, and the concentration of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

6 and 7 show that Lactobacillus plantarum CJLP136 produces the Th1 differentiation-inducing cytokine IL-12 cytokine and the production of Th1 differentiation inhibiting cytokine IL-10 is significantly less than IL-12. It can be seen that it significantly increases the production of IL-12 compared to other lactic acid bacteria.

In addition, in order to confirm the production of IL-12 and IL-18 at the gene level, the test subject was treated with macrophages cell line RAW264.7 at a concentration of 510 ⁷ / mL, and cultured at 37 ° C. and 10% CO _2. After 6 hours was performed to extract the total RNA and the production of IL-12 and IL-18 mRNA was measured by RT-PCR method. JAWSII, a dendritic cell line, was inoculated and cultured in the same manner, and RNA was extracted to measure the production of IL-12 and IL-18 mRNA by RT-PCR.

Thus, each result measured is shown in FIGS. 8 and 9.

8 is a graph showing the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating Lactobacillus plantarum CJLP136 strain in macrophage cell line RAW264.7.

9 is a graph showing the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating Lactobacillus plantarum CJLP136 strain to dendritic cell line JAWSII, compared with other lactic acid bacteria.

8 and 9 show that Lactobacillus plantarum CJLP136 promotes the production of mRNAs that direct the production of the Th1 differentiation-inducing cytokines IL-12 and IL-18, in particular IL-12 mRNA. It was confirmed that it is remarkably superior to other lactic acid bacteria in the expression promotion of.

Example 8 Preparation of Probiotics Comprising Lactobacillus plantarum CJLP136

Probiotics Lactobacillus plantarum CJLP136 (Lactobacillus plantarum CJLP136) identified in Example 1 was mass-produced in order to be applied as a raw material of medicines, foods, feeds, feed additives, or cosmetics and freeze-dried to probiotics.

For the production of bacteria, the cells were recovered by incubation at 37 ° C. for about 18 hours while adjusting the pH to 6.0 using 25% NaOH solution in MRS liquid medium (Difco) and centrifugation. The recovered cells were pulverized by using a dextrin 5% and skim milk 10% as a cryoprotectant to freeze at -40 ℃ and dried cells at 37 ℃ by a mixer. The powdered live bacteria were packed in an aluminum pouch sealed by mixing with an appropriate amount of excipients such as glucose, lactose, skim milk, etc. in order to store and store in accordance with the target number of bacteria.

Probiotics prepared as described above are used as feed probiotics by mixing with grains used as raw materials in feeds, or as carriers or additives and used as probiotics for pharmaceuticals, foods such as tablets and capsules, or as raw materials used in cosmetics. By mixing a certain amount can be utilized in a variety of fields such as pharmaceuticals, food, feed, cosmetics according to the conventional method in the art.

1 is a graph showing the results of an acid resistance test of Lactobacillus plantarum CJLP136.

Figure 2 is a graph showing the results of the bile acid resistance test of Lactobacillus plantarum CJLP136.

Figure 3 is a graph showing the results of intestinal epithelial cell adhesion capacity of Lactobacillus plantarum CJLP136

Figure 4 shows Lactobacillus plantarum CJLP136 strain treated with ovalbumin to splenocytes of mice that were biased by Th2 response, and then measured the concentration of IL-12, a Th1 response-induced cytokine, compared with other lactic acid bacteria. It is a graph.

5 shows Lactobacillus plantarum CJLP136 strain treated with ovalbumin to splenocytes of mice biased by Th2 response, and then measured the concentration of IL-4, a Th2 response-induced cytokine, compared with other lactic acid bacteria. It is a graph.

6 is a graph showing Lactobacillus plantarum CJLP136 strain treated with macrophage cell line RAW264.7, and the concentration of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

7 is a graph showing Lactobacillus plantarum CJLP136 strain treated with dendritic cell line JAWSII, and the concentration of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

8 is a graph showing the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating Lactobacillus plantarum CJLP136 strain in macrophage cell line RAW264.7, compared with other lactic acid bacteria.

9 is a graph showing the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating Lactobacillus plantarum CJLP136 strain to dendritic cell line JAWSII and comparing with other lactic acid bacteria.

<110> CJ Cheiljedang Corporation <120> Novel Lactobacillus plantarum and compositions comprising the          same <130> pn081724 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1462 <212> DNA <213> Lactobacillus plantarum <400> 1 agtcgaacga actctggtat tgattggtgc ttgcatcatg atttacattt gagtgagtgg 60 cgaactggtg agtaacacgt gggaaacctg cccagaagcg ggggataaca cctggaaaca 120 gatgctaata ccgcataaca acttggaccg catggtccga gtttgaaaga tggcttcggc 180 tatcactttt ggatggtccc gcggcgtatt agctagatgg tgaggtaacg gctcaccatg 240 gcaatgatac gtagccgacc tgagagggta atcggccaca ttgggactga gacacggccc 300 aaactcctac gggaggcagc agtagggaat cttccacaat ggacgaaagt ctgatggagc 360 aacgccgcgt gagtgaagaa gggtttcggc tcgtaaaact ctgttgttaa agaagaacat 420 atctgagagt aactgttcag gtattgacgg tatttaacca gaaagccacg gctaactacg 480 tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag 540 caagcgcagg cggtttttta agtctgatgt gaaagccttc ggctcaaccg aagaagtgca 600 tcggaaactg ggaaacttga gtgcagaaga ggacagtgga actccatgtg tagcggtgaa 660 atgcgtagat atatggaaga acaccagtgg cgaaggcggc tgtctggtct gtaactgacg 720 ctgaggctcg aaagtatggg tagcaaacag gattagatac cctggtagtc cataccgtaa 780 acgatgaatg ctaagtgttg gagggtttcc gcccttcagt gctgcagcta acgcattaag 840 cattccgcct ggggagtacg gccgcaaggc tgaaactcaa aggaattgac gggggcccgc 900 acaagcggtg gagcatgtgg tttaattcga agctacgcga agaaccttac caggtcttga 960 catactatgc aaatctaaga gattagacgt tcccttcggg gacatggata caggtggtgc 1020 atggttgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc 1080 ttattatcag ttgccagcat taagttgggc actctggtga gactgccggt gacaaaccgg 1140 aggaaggtgg ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct 1200 acaatggatg gtacaacgag ttgcgaactc gcgagagtaa gctaatctct taaagccatt 1260 ctcagttcgg attgtaggct gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg 1320 gatcagcatg ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg 1380 agagtttgta acacccaaag tcggtggggt aaccttttag gaaccagccg cctaaggtgg 1440 gacagatgat tagggtgaag tc 1462  

Claims (9)

Lactobacillus plantarum CJLP136 KCTC 11404BP. Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) A composition for preventing or treating intestinal diseases, including KCTC 11404BP. Lactobacillus plantarum CJLP136 ( Lactobacillus plantarum CJLP136) A composition for enhancing immunity comprising KCTC 11404BP. 4. The composition according to claim 3, which is for the prevention or treatment of immune diseases caused by Th1 / Th2 imbalance due to the Th2 response excess. The composition of claim 4, wherein the immune disease is selected from the group consisting of allergic diseases, atopy, cancer, and autoimmune diseases. The composition according to any one of claims 2 to 5, which is a medicament. The composition according to any one of claims 2 to 5, which is a food. The composition according to any one of claims 2 to 5, which is a feed or feed additive. The composition according to any one of claims 2 to 5, which is a cosmetic.

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