HK1171473B - Novel lactobacillus plantarum and composition containing same - Google Patents

Abstract

The present invention provides Lactobacillus plantarum CJLP55 KCTC 11401BP, a composition for treating bowel disease containing the lactobacillus, and a composition for immunological enhancement containing the lactobacillus.

Description

Novel lactobacillus plantarum and composition containing same

Technical Field

The invention relates to novel lactobacillus plantarum and a composition containing the same. More particularly, it relates to a novel lactobacillus plantarum for preventing and treating intestinal diseases and immune diseases and a composition comprising the same.

Background

Kimchi and other traditional fermented foods contain abundant lactic acid bacteria which are symbiotic in the digestive system of the human body and have the effect of preparing important nutritional ingredients by decomposing cellulose and complex proteins. Such microorganisms that survive in the gastrointestinal organs of animals including humans, improve the intestinal microbial environment of the host, and thus are beneficial to the health of the host are collectively referred to as probiotics. In order for probiotics to be effective, oral ingestion to reach the small intestine, adhesion to the intestinal surface and maintenance of such adhesion are required, and thus at least excellent acid resistance, bile acid resistance, and adhesion to intestinal epithelial cells are required.

Representative probiotics found in conventional fermented foods such as kimchi include Lactobacillus sp. Microorganisms of the genus lactobacillus, which are homofermentative or heterofermentative lactobacilli, are commonly found in the intestinal tract of animals including humans and in the fermentation of dairy products or vegetables. Microorganisms of the genus lactobacillus maintain the intestinal pH acidic, thereby inhibiting the growth of harmful bacteria such as escherichia coli (e.coli) and Clostridium (Clostridium), and improving diarrhea and constipation, and also have known actions such as vitamin synthesis, anticancer, and serum cholesterol reduction. It is known that lactobacillin (acidophillin) produced by lactobacilli has an effect of inhibiting the growth of dysentery bacillus, salmonella, staphylococcus, escherichia coli, and the like. And can inhibit the reproduction of bacteria causing diarrhea, so that intestinal flora is normal, thereby having the effect of stopping diarrhea. (Michael and Philippie, Probiotics and Probiotics: Effect on diarrhea, Probiotics and Probiotics: Vol. 137, p. 3, 2007, pp. 803S-811S; Roberfroid, Probiotics and Probiotics: Do. they Are functional foods.

Studies for developing probiotics and livestock feeds using the above-mentioned characteristics of microorganisms of the lactobacillus genus are actively being conducted. Bacterial diarrhea in livestock reduces the rate of weight gain, leading to death of the animal. Therefore, in order to prevent the above-mentioned situation and improve the yield of livestock, it has been generally adopted to add antibiotics to the feed. However, due to the emergence of antibiotic-resistant bacteria and the problems of residual antibiotics in livestock products, the current trend is to restrict the use of antibiotics in feed, emphasizing the organic livestock breeding method (Korean laid-open patent 1998-78358) (McEwen and Fedorka-Cray, application of antibacterial drugs in animals and drug resistance, J.Clin infectious Diseases, Vol.34, 6.2002, pp. 93-S106) (McEwen and Fedorka-Cray, anti-microbial use and resistance in animals, clinical infectious Diseases, Volume34, June 2002, pages S93-S106).

In addition, lactic acid bacteria such as microorganisms belonging to the genus Lactobacillus also have an effect of enhancing immunity. Recently, allergic diseases and atopic diseases associated with immunoregulatory abnormality are rapidly increasing due to the influence of environmental pollution, increase in intake of instant foods, etc. worldwide, and the diseases are also increasing in korea. Recently, in europe, as a link of microbial therapy (bacteriotherhy), disease is treated by oral administration of lactic acid bacteria, and efforts are being made to achieve alleviation or improvement of symptoms with lactic acid bacteria. Reducing the probability of specific reaction diseases to half when lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) is administered to infants (II)Etc., the main prevention of probiotic bacteria in hereditary allergic diseases: a randomized placebo-controlled experiment, Langerhans, Vol.357, 4/2001, p.1076-1079) (et.al.，Probiotics in primaryprevention of atopic disease：a randomized placebo-controlled tertiary, Lancet, Volume 357, April 2001, pages 1076-. It has been reported that when Lactobacillus rhamnosus (Lactobacillus rhamnosus) and Lactobacillus reuteri (L.reuteri) are administered to infants who have developed atopic eczema, the site of eczema becomes small and the symptoms of eczema are reduced (Rosenfeldt et al, the Effect of probiotic lactic acid bacterial strains on atopic dermatitis, skin diseases and eye diseases in children, Vol.111, No. 2.2003, pp.389. 395) (Rosenfeldt et al, Effect of probiotic Lactobacillus strain in childhood with atomic inflammation, Dermatologic and ocular diseases, Volume 111, February 2003, pages 389. 395).

Studies have been made on the mechanism of the immune enhancing effect (mechanism) of such lactic acid bacteria, and although the specific mechanism is not clear, it is generally known that the mechanism affects the immune system of the intestinal tract by being taken orally and inhabiting the intestine. For example, it is known that the antibacterial activity of the lymph node (Peyer's patch) lymphocytes is enhanced by taking lactic acid bacteria in the form of yogurt, and it is known that lactic acid bacteria can enhance IgA reaction according to some studies conducted on experimental animals and humans. In addition, lactic acid bacteria have an effect on both innate and adaptive immunity. It is known that in innate immune response (nature) of the intestinal immune system, pathogenic bacteria are phagocytosed to eliminate the pathogenic bacteria, thereby preventing infection and maintaining health. Adaptive immunity (adaptive immunity) is known to increase the production of various cytokines, particularly the production of interleukins IL-12, IL-18, by activating macrophages, since some of the constituent components of the cell wall of lactic acid bacteria activate the NF-. kappa. B, STAT signaling pathway in macrophages, thus increasing cytokine production. The macrophages function to break down antigens, thereby suggesting a role for T lymphocytes. Also, lactic acid bacteria are known as specialized antigen presenting cells which not only increase the production of IL-12, IL-18, TNF- α in lymph nodes, dendritic cells (dendritic cells) which are more present on the mucosa in the digestive system, but also have the effect of increasing the expression of surface molecules which, like MHC II, and B7-2, have the effect of activating T lymphocytes (Cross et al, antiallergic properties of fermented foods, an important immunoregulatory mechanism of lactic acid bacteria, J. Immunol. 1, 2001 5 months, page 891-.

T lymphocytes are the core cells of adaptive immunity (adaptive immunity), which can be divided into the Th1 response of cellular immunity and the Th2 response of antibody immunity. Cytokines produced by Antigen Presenting cells (Antigen Presenting cells) were different from each other in the respective Th1 and Th2 responses, and IL-12, IL-18 and Interferon (IFN) were dominant in the Th1 response, while PGE2, IL-4 and IL-10 were dominant in the Th2 response. In order to achieve a proper balance between the Th1 response and the Th2 response, various immune diseases may occur when the balance is disrupted. Th1 cells are mainly combated with infectious disorders, whereas Th2 cells are mainly associated with allergic diseases and inflammatory reactions. When they function normally, Th2 cells protect the body from dust and other unwanted substances, but if these cells show excessive activity, IgE antibody production is increased, and allergic reactions are induced by proteins (e.g., pollen, food, etc.) that are not originally threatening the human body. Also, the Th1 response and Th2 response must be balanced, and if one is excessive or the other is insufficient, disease is induced. Furthermore, due to the constant pressure, and the continuous isolation of cortisol, there is a decrease in Th1 responses and an increase in Th2 responses, which can induce cancer, atopic diseases, allergic diseases, and autoimmune diseases (Elenkov and Chrosus, Stress hormones, model Th1/Th2, pro/anti-inflammatory cytokines and susceptibility to disease, endocrinological and metabolic Trends, Vol.10, 11.1999, p.359. (Elenkov and Chuuss., Stress hormones, Th1/Th2 tablets, pro/anti-inflammatory cytokines and cytokine diseases, Trends in Endocrinology and Metabolism, Volume 10, November 1999, tablets 359-).

It is known from In vivo (In vivo) experiments that lactic acid bacteria can increase IFN-. gamma.production as a Th1 cytokine In T lymphocytes and can suppress The production of IL-4 and IL-5 as Th2 cytokines (Matsuzaki et al, action of oral Lactobacillus casei Shirota on immunoglobulin E production In mice, Journal of Dairy Science, Vol.81, p.1998, pp.48-53) (Matsuzaki et al, The effect of oral feeding of Lactobacillus casei strain Shiroata on immunological glucose E production In mice, Journal of Dairy Science, Volume 81, January 1998, pages 48-53). In other experiments, when lactic acid bacteria were orally administered to mice (ovalbumin-printed mice) biased to the Th2 response, which was used as an animal model of the Th2 response, the level of INF- γ in splenocytes (spleenocytes) was increased and IL-4, IL-5 and IgE were decreased, respectively, and ovalbumin-administered mice were used as the animal model of the Th2 response. Further, it is known that when spleen cells extracted from mice biased to Th2 reaction after administration of ovalbumin were cultured together with lactic acid bacteria, the change in cytokines and IgE was the same as in the oral administration experiment. However, since the production of IFN-. gamma.is not significantly increased when only T lymphocytes are cultured with Lactic acid bacteria, it is considered that antigen-presenting cells such as macrophages and dendritic cells are essential for the production of IFN-. gamma.from T lymphocytes (Kato et al, Lactic acid bacteria efficiently induce the production of interleukin 12and interferon-. gamma.by spleen cells, J.Immunol.21, 1999, 2.121-131) (Kato et al, Lactic acid bacteria potential peptides of products of interleukin-12and interferon-gamma. beta. -cells, International Journal of immunological science, Volume 21, February 1999, pages 121-131). On the other hand, IL-12 and IL-18 are important cytokines for differentiating Th0 lymphocytes into Th1 lymphocytes, and are produced in macrophages or dendritic cells, and it is known that when spleen cells or macrophages are cultured, the production of IL-12, IL-18, IFN-. alpha.and the like increases with the increase in concentration by treatment with lactic acid bacteria. It is known that lactic acid bacteria increase the production of IL-12, IL-18, IFN-. alpha.and the like in macrophages, thus promoting differentiation to Th1 cells and inducing IFN-. gamma.production, and thus can play a role in regulating the balance Th1/Th2 in the case where Th2 is dominant (Cross et al, antiallergic property of fermented foods: an important immunological mechanism of lactic acid bacteria, International Immunopharmacology, Vol.1, 5.2001, page 891-. In addition, lactic acid bacteria are known to be useful for preventing and treating cancer, atopic disease, allergic disease, and autoimmune disease induced by the imbalance of Th1/Th2 due to excess Th2 reaction.

Disclosure of Invention

Technical problem to be solved by the invention

The inventor of the invention researches and develops a novel lactic acid bacterium which has greatly better effect than the existing lactic acid bacterium in the aspect of regulating Th1/Th2 imbalance caused by excessive reaction of Th 2. As a result of the research, a novel strain of Lactobacillus was isolated and identified from conventional fermented foods, thereby completing the present invention.

The purpose of the present invention is to provide a novel lactobacillus strain which has excellent acid resistance, bile acid resistance, and adhesion to intestinal epithelial cells, which are basic properties of probiotics, and also has excellent immune enhancing effects, and particularly has excellent effects of regulating the Th1/Th2 imbalance caused by excess Th2 reaction.

Another object of the present invention is to provide a composition for preventing or treating intestinal diseases, comprising the novel lactobacillus strain.

Still another object of the present invention is to provide a composition for enhancing immunity, which contains the above novel lactobacillus strain.

Technical scheme

In order to achieve the above object, the present invention provides Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) (Collection center: institute of Life engineering Gene Bank, Collection date: 2008.10.16, Collection number: KCTC11401 BP).

Also, the present invention provides a composition for preventing and treating intestinal diseases, comprising the lactobacillus plantarum CJLP55 described above.

Also, the present invention provides a composition for enhancing immunity, which contains lactobacillus plantarum CJLP 55.

The present invention will be described in further detail below.

The Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) related to the present invention is characterized as a novel strain of Lactobacillus plantarum isolated and identified from conventional fermented foods. The conventional fermented food includes kimchi, vegetable fermented product, soybean paste, soy sauce, koji sauce or salted seafood, but is not limited thereto.

In order to identify and classify microorganisms, the lactobacillus plantarum CJLP55 of the present invention was subjected to base sequence analysis on 16S rRNA, and the results were: and a Lactobacillus plantarum standard strain (Lactobacillus plantarum NBRC 15891)^TGenBank accession No. AB326351) showed the highest homology (99.9%) showing the highest phylogenetic relationship with lactobacillus plantarum. The microorganism was identified as Lactobacillus plantarum (Lactobacillus plantarum), named Lactobacillus plantarum CJLP55, and deposited in the institute of bioscience Gene Bank (accession No. KCTC11401BP) at 10/16 of 2008. The base sequence of the 16S rRNA gene of the Lactobacillus plantarum CJLP55 is shown in a base sequence table SEQ ID NO.1 in the attachment of the specification.

The lactobacillus plantarum CJLP55 of the present invention is a gram-positive bacterium, is a facultative anaerobe (viable anaerobe) that can grow under both aerobic and anaerobic conditions, does not form spores, does not have motility, and has a bacillus cell morphology. The results of analyzing more specific morphology and physiological properties of Lactobacillus plantarum CJLP55 using a method conventional in the art are shown in Table 1 below.

TABLE 1

Morphological, physiological and biochemical characteristics	Results
		Morphology (Morphology)	Bacillus (Rod)
Motility (mobility)	-
		Spore (Spore)	-
Catalase enzyme	-
		Homotypic and heterotypic fermentations	Facultative heterotypic fermentation
Propagation at 15 ℃	+
		Propagation at 45 ℃	-
Propagation in 3% NaCl	+
		Anaerobic reproduction	+
CO production from glucose2	-
		Sugar fermentation characteristics
Glycerol	-
		Erythritol and its preparation method	-

D-arabinose	-
		L-arabinose	-
Ribose	+
		D-xylose	-
L-xylose	-
		Pentahydric alcohol	-
Xyloside	-
		Galactose	+
D-glucose	+
		D-fructose	+
D-mannose	-
		L-sorbose	-
Rhamnose	+
		Galactitol	-
Inositol	-
		Mannitol	+
Sorbitol	+
		D-mannoside	+
D-glucoside	-
		Glucosamine	+
Amygdalin	+
		Arbutin	+
Esculin	+
		Salicin	+
Cellobiose	+
		Maltose	+
Lactose	+
		Melibiose	+
Sucrose	+
		Trehalose	+
Inulin	+
		Melezitose (melizitose)	+
D-raffinose	+
		Methadone (amidon)	-
Glycogen	-
		Xylitol, its preparation method and use	-
Gentiobiose	+
		D-turanose	-
D-lyxose	-
		D-tagatose	-
D-fucose	-
		L-fucose	-

D-arabinitol	-
		L-arabinitol	-
Gluconate	-
		2-gluconate	-
5-gluconate	-

+: positive reaction

-: negative reaction

In order to stably store the lactobacillus plantarum CJLP55 of the present invention for a long period of time, it is preferable to disperse the cells in a storage solution mixed with a certain amount of aqueous solution of glycerol and store them at-70 ℃, or to suspend them in sterilized 10% skim milk and freeze-dry them.

In addition, the lactobacillus plantarum CJLP55 of the invention has the intestinal regulation effect and the immunity enhancement effect commonly possessed by lactic acid bacteria as probiotics.

Probiotics in the context of the present invention are understood to be: microorganisms which survive in the gastrointestinal organs of animals including humans and improve the intestinal microbial environment of the host, thereby being beneficial to the health of the host. A probiotic is a live microorganism with probiotic activity, in the form of a single or complex strain, capable of exerting a beneficial effect on the intestinal flora of the host when used in dry cell form or in the form of a fermentation product in humans or animals. In order to confirm whether the microorganism is a probiotic microorganism, it is necessary to confirm whether the microorganism is less affected by gastric juice and bile and passes through the stomach and is present in the intestine, and whether the microorganism is retained in the intestine and is present in the intestine, and it is necessary to favorably influence the intestinal flora of the host. Therefore, it must have acid resistance to gastric acid, bile acid resistance, and adhesion to intestinal epithelial cells. Secondly, in order to confirm whether or not a probiotic microorganism is present, it is necessary that the microorganism has no problem in terms of safety, and in this connection, a gelatin liquefaction reaction test, a phenylalanine deamination production test, an ammonia production test, a hemolytic test, and the like are generally performed. The lactobacillus plantarum CJLP55 of the present invention was found to be safe because it was negative in the gelatin liquefaction reaction assay, phenylalanine deamination assay, and ammonia production assay, and was confirmed to be alpha-hemolytic independent of pathogenicity in the hemolytic assay, in addition to having excellent acid resistance, bile acid resistance, and adhesion to intestinal epithelial cells.

The lactobacillus plantarum CJLP55 of the present invention is very excellent in acid resistance, bile acid resistance, and adhesion of intestinal epithelial cells, and thus it is predicted to have an intestinal conditioning effect. Thus, in another aspect, the present invention provides a composition for preventing or treating intestinal diseases, comprising lactobacillus plantarum CJLP 55.

The composition for treating intestinal diseases containing the microorganism of the present invention can be used for preventing or treating intestinal diseases in mammals including humans, preferably domestic animals including cattle, horses, pigs, and the like. The "intestinal disease" includes infection with harmful intestinal bacteria, inflammatory intestinal diseases, and the like, and includes, for example, infectious diarrhea caused by pathogenic microorganisms (e.g., escherichia coli, salmonella, clostridium, and the like), gastroenteritis, inflammatory intestinal diseases, neurogenic enteritis syndrome, small intestinal bacterial overgrowth, acute diarrhea, and the like, but is not limited thereto. The lactobacillus plantarum CJLP55 contained in the composition for treating intestinal diseases may be present as viable bacteria or dead bacteria, but is preferably present as viable bacteria. Generally, live bacteria have an effect of treating and ameliorating various symptoms caused by abnormal fermentation of intestinal flora, and when used in humans and animals, live bacteria can be densely retained on the intestinal wall of the digestive tract, thereby preventing harmful bacteria from being retained, and lactic acid is produced to lower the intestinal pH and inhibit the growth of harmful bacteria. In addition, the used live bacteria produce bacteriocin (bacteriocin) and peroxide, thereby inhibiting the reproduction of pathogenic bacteria and helping the movement of intestinal villi which are responsible for absorbing nutrient components. In addition, substances are produced which aid absorption and utilization of nutrients, improve feed conversion rates for animals, and also produce a substance which neutralizes toxic substances produced by pathogenic bacteria.

The mode of administration of the composition for preventing or treating intestinal diseases of the present invention is not particularly limited, but oral administration is preferred. The dose varies depending on the type of intestinal disease, the degree of the disease, age, sex, race, therapeutic or preventive purpose, etc., but generally 1 million to 1000 million can be administered per day on an adult basis.

In addition to having an intestinal regulation effect, lactobacillus plantarum CJLP55 of the present invention has a significantly superior effect of enhancing immunity compared to the existing lactobacillus. Lactobacillus plantarum CJLP55 is capable of increasing the production of IL-12 inducing a Th1 response and inhibiting the production of IL-4 inducing a Th2 response in splenocytes (spleenocytes). The lactobacillus plantarum CJLP55 stimulates cells involved in immune regulation, such as macrophages and dendritic cells, which are antigen presenting cells regulating T cell immune responses, thereby promoting the production of cytokines that induce the differentiation of Th0 lymphocytes into Th1 lymphocytes, and further, it was confirmed that it has immunoregulatory ability and can regulate Th1/Th2 imbalance caused by excess Th2 responses. The specific description of the immunological enhancement effect of Lactobacillus plantarum CJLP55 is as follows.

When mouse spleen cells (spleenocytes) biased to Th2 response by addition of Ovalbumin (OVA) were treated with lactobacillus plantarum CJLP55, cytokine IL-12 inducing Th1 response was produced up to 12.4-12.7 times and production of cytokine IL-4 inducing Th2 response was suppressed to 6.1-9.8%, compared to the negative control group, which was confirmed to be significantly superior to other typical lactic acid bacteria such as lactobacillus rhamnosus GG (KCTC5033), lactobacillus casei (KCTC3109), lactobacillus sakei CJLS118(KCTC 13416). Lactobacillus plantarum CJLP55 can inhibit Th2 response and promote Th1 response, and thus has immunoregulatory ability to regulate Th1/Th2 imbalance caused by excess Th2 response.

The results of treatment of macrophage strain RAW264.7 and dendritic cell JAWSII with Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) confirmed that the macrophage strain was stimulated to enhance immune response as the number of lactic acid bacteria increased. The results of treatment of macrophage strain RAW264.7 and dendritic cell strain JAWSII with lactobacillus plantarum CJLP55 demonstrated that lactobacillus plantarum CJLP55 promoted induction of differentiation of Th1 because it was able to promote production of cytokines IL-12 and IL-18 that induced Th1 differentiation and inhibited production of cytokine IL-10 that induced Th1 differentiation relatively less than IL-12. The results of this experiment revealed that Lactobacillus plantarum CJLP55 has an immunoregulatory ability to suppress Th2 response and promote Th1 response, thereby regulating Th1/Th2 imbalance caused by excess Th2 response.

IL-4 is derived from Th2 cells, and particularly plays a central role in cellular immune response, and inhibits the production of cytokine IL-12 of Th1 cells, thereby having the function of anti-inflammatory cytokine. Recently, it was found that Th2 cells, which produce mainly IL-4, IL-5, etc., are relatively increased in peripheral blood or skin lesions of patients with atopic dermatitis (Miraglia et al, immunoregulatory abnormalities in atopic dermatitis, conference on Allergy and Asthma, Vol.27, 11-12 2006, p.451-455) (Miraglia et al, Immune Allergy in atomic distribution, Allergy and Ashma procedures, Volume 27, November-December 2006, pages 451-455). Further, imbalance of Th1/Th2 caused by excessive Th2 reaction may induce diseases such as atopic diseases. Also, as described above, it is known that diseases are caused when either one of the Th1 response and the Th2 response is excessive or the other is insufficient, and cancers, atopic diseases, allergic diseases, and autoimmune diseases are caused when the Th1 response is decreased and the Th2 response is increased (Elenkov and Chrosus, Stress hormones, Th1/Th2 pattern, pro-inflammatory/anti-inflammatory cytokines and disease susceptibility, endocrinological and metabolic Trends, Vol.10, 11 months 1999, p. 359) (Elenkov and Chrosus, Stress hormons, Th1/Th2 patters, pro/anti-inflammatory cytokines and survival diagnosis, Trends in Endocrinology and Metabolism, Volume 10, November 1999, pages 359 368). The lactobacillus plantarum CJLP55 of the present invention regulates Th1/Th2 imbalance caused by excess Th2 response by regulating cytokines that produce Th1 cells, Th2 cells, macrophages, and dendritic cells involved in immune regulation, and thus is expected to have an effective effect not only on diseases such as atopic diseases and allergic diseases, but also on prevention and treatment of cancer and autoimmune diseases.

In another aspect of the present invention, there is provided a composition for enhancing immunity comprising lactobacillus plantarum CJLP 55. The lactobacillus plantarum CJLP55 in the composition for enhancing immunity according to the present invention has an effect of enhancing immunity as it is lactobacillus having the effect of enhancing immunity as described in the above prior art. In particular, as exemplified in the examples below, lactobacillus plantarum CJLP55 in the above composition for enhancing immunity of the present invention has the effect of promoting Th1 response and the effect of regulating Th1/Th2 imbalance due to excessive Th2 response, and thus has the effect of preventing or treating diseases induced by Th1/Th2 imbalance due to excessive Th2 response. Therefore, the composition for enhancing immunity of the present invention can be effectively used for preventing or treating atopic diseases, allergic diseases, cancers, and autoimmune diseases. The above autoimmune diseases include asthma, hay fever, etc., but are not limited thereto.

The administration mode of the composition for enhancing immunity of the present invention is not particularly limited, and oral administration is preferable. The dose varies depending on the kind of disease, the degree of disease, the age, sex, race, therapeutic or preventive purpose, etc. required for enhancing immunity, but generally 1 million to 1000 million can be administered per day on an adult basis.

Since the composition for preventing or treating intestinal diseases and the composition for enhancing immunity containing lactobacillus plantarum CJLP55 of the present invention described above contain safe and acceptable lactic acid bacteria, they can be used as drugs, foods, cosmetics, feeds or feed additives without concern for side effects.

When the above-mentioned composition of the present invention is used as a pharmaceutical, a conventional pharmaceutical preparation known in the art can be prepared. The above-mentioned pharmaceutical product is preferably in the form of an oral administration preparation. For example, it can be formulated into oral preparations such as liquid, suspension, powder, granule, tablet, capsule, pill, or extract.

When the composition is prepared into the various dosage forms, carriers or additives required for preparing the various dosage forms and allowed in pharmaceutics can be added. When the pharmaceutical composition is prepared into a typical dosage form for oral administration, one or more selected from diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives can be used as the carrier, and one or more selected from flavors, vitamins and antioxidants can be used as the additive.

The carrier and the additive may be any pharmaceutically acceptable carrier, and preferably, the diluent includes lactose, corn starch, soybean oil, microcrystalline cellulose or mannitol, the lubricant includes magnesium stearate or talc, and the binder includes polyvinylpyrrolidone or hydroxypropyl cellulose. Further, preferably, calcium carboxymethylcellulose, sodium starch glycolate, potassium polacrilin or crospovidone are used as a disintegrant, white sugar, fructose, sorbitol or aspartame are used as a sweetener, sodium carboxymethylcellulose, β -cyclodextrin, white wax or xanthan gum are used as a stabilizer, and methyl paraben, propyl paraben or potassium sorbate are used as a preservative.

In addition, in order to enhance the taste, natural flavors such as plum blossom, lemon, pineapple, and vanilla may be mixed as known additives in addition to the above components; natural pigments such as natural fruit juice, chlorophyllin, and flavonoid; sweet components such as fructose, Mel, sugar alcohol, and white sugar; or sour agents such as citric acid and sodium citrate.

The formulation method and the carriers and additives required for formulation are described in detail in Remington's Pharmaceutical Sciences (19 th edition, 1995).

The composition of the present invention described above can be used as a food. The food is not only a health functional food, but also a general food which is taken by people every day. In the case of using as a health functional food, a dosage form of a conventional health functional food known in the art can be prepared together with a sitologically acceptable carrier or additive, and the health functional food can be prepared, for example, as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, a liquid, an extract, tea, jelly, a beverage, or the like. The aforementioned dietetically acceptable carriers or additives may be selected from any known carriers or additives acceptable in the art according to the preparation form to be prepared.

The composition of the present invention can be used in cosmetics because it has an effect of preventing or treating atopic diseases. When the above-described composition of the present invention is used for cosmetics, various cosmetics of conventional formulations well known in the art of cosmetics technology can be prepared. When the cosmetic is prepared into various dosage forms, carriers or additives which are allowed to be used in preparing cosmetics and are required for preparing various dosage forms can be added to prepare the cosmetic.

The composition of the present invention described above can also be used as a feed additive or feed.

When used as a feed additive, the composition may be prepared into a highly concentrated solution or powder of 20 to 90% or in the form of granules. The feed additive may also comprise organic acid selected from citric acid, fumaric acid, adipic acid, lactic acid, malic acid, etc.; or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polymeric phosphate); or one or more natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid, etc. When used as a feed, the composition may be prepared in a conventional feed form, and may include conventional feed ingredients.

The feed additive and feed may also include cereals such as crushed or broken wheat, oat, barley, corn and rice; vegetable protein feeds, for example, feeds containing rape, soybean and sunflower seeds as main ingredients; animal protein feed such as blood meal, meat meal, bone meal and seafood meal; examples of the sugar and dairy products include dry components such as various milk powders and whey powders, and may include nutritional supplements, digestive absorption enhancers, growth promoters, and the like.

The feed additive can be used for feeding animals alone or in combination with other feed additives in an edible carrier. The feed additive can be easily mixed with the animal feed as a top dressing (topdressing) or prepared into an oral dosage form separately from the feed. When the feed additive is prepared into an oral feeding formulation without being fed together with an animal feed, the feed additive can be used in combination with an edible carrier which is known in the art and allowed in pharmaceutics, and can be prepared into a formulation which can be released immediately or slowly. These edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil, and propylene glycol. In the case of a solid carrier, the feed additive may be in the form of a tablet, capsule, powder, dragee or sugar-containing tablet or a top dressing (topdressing) in a microdispersed form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, syrup, suspension, emulsion, or solution.

Any commonly used protein-containing organic flour that meets the food requirements of animals may be included in the feed. Such protein-containing flours are typically composed primarily of corn, soy flour, or corn/soy flour mixtures.

The feed additive and feed may further contain adjuvants such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters, and the like. The feed additive can be added into animal feed by soaking, spraying or mixing.

The feed or feed additive of the present invention can be applied to the diet of many animals including mammals, poultry, and fish. The mammals include not only pigs, cows, sheep, goats, laboratory rodents and laboratory rodents, but also pets (e.g., dogs and cats). The poultry may be chicken, turkey, duck, goose, pheasant, quail, etc., and the fish may be trout, etc., but is not limited thereto.

Advantageous effects

As described above, the novel lactobacillus plantarum CJLP55 of the present invention has excellent acid resistance, bile acid resistance, and intestinal epithelial cell adhesion as a probiotic, thereby having an intestinal regulation effect, and has an effect of promoting Th1 reaction, and an effect of regulating Th1/Th2 imbalance caused by excess Th2 reaction. In addition, the novel lactobacillus plantarum CJLP55 of the present invention may be used in a composition for preventing or treating intestinal diseases and a composition for enhancing immunity, and particularly, has an effect of preventing or treating diseases induced by Th1/Th2 imbalance due to excess Th2 response.

Drawings

Fig. 1 is a graph showing the results of an acid resistance experiment of lactobacillus plantarum CJLP 55.

Fig. 2 is a graph showing the results of a bile acid tolerance experiment of lactobacillus plantarum CJLP 55.

Fig. 3 is a graph showing the results of an intestinal epithelial cell adhesion experiment of lactobacillus plantarum CJLP 55.

FIG. 4 is a graph comparing the results of treating spleen cells of mice, which are biased to a Th2 response by the addition of ovalbumin, with Lactobacillus plantarum CJLP55 strain, and then measuring the concentration of IL-12, a cytokine inducing a Th1 response, with other lactic acid bacteria.

FIG. 5 is a graph comparing the results of treating spleen cells of mice, which are biased to a Th2 response by the addition of ovalbumin, with Lactobacillus plantarum CJLP55 strain, and then measuring the concentration of the cytokine IL-4 inducing a Th2 response with other lactic acid bacteria.

FIG. 6 is a graph comparing the results of enzyme-linked immunosorbent assay (ELISA) for IL-12 and IL-10 concentrations after treating macrophage strain RAW264.7 with Lactobacillus plantarum CJLP55 strain with other lactic acid bacteria.

FIG. 7 is a graph showing the results of ELISA measurement of IL-12 and IL-10 concentrations in the dendritic cell strain JAWSII treated with Lactobacillus plantarum CJLP55 strain, compared with other lactic acid bacteria.

FIG. 8 is a graph comparing the results of treating macrophage strain RAW264.7 with Lactobacillus plantarum CJLP55 strain and then measuring the expression of IL-12p40 and IL-18mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) method with other lactic acid bacteria.

FIG. 9 is a graph comparing the results of RT-PCR measurement of the expression of IL-12p40 and IL-18mRNA with other lactic acid bacteria after treating dendritic cell strain JAWSII with Lactobacillus plantarum CJLP55 strain.

Detailed Description

The present invention will be described more specifically with reference to the following examples. However, these examples are only for the purpose of facilitating understanding of the present invention, and the scope of the present invention is not limited in any way by these examples.

Example 1: isolation and characterization of the microbial Lactobacillus plantarum CJLP55 Strain

Lactobacillus plantarum CJLP55 strain isolated from kimchi was spread on MRS solid medium (Difco, USA) containing 1.5% agar (agar), cultured at 37 ℃ for 24 hours, and then colonies that had been confirmed to be pure isolates were picked up with an inoculating loop (loop) and cultured in MRS liquid medium (Difco, USA) at 37 ℃ for 18-24 hours.

Then, morphological and physiological characteristics of CJLP55 strain were identified using Kim et al (Kim et al, Leucosotonic inhae sp. nov, a lactic acid bacterium isolated from kimchi, J. International Systematic evolution Microbiology, Vol. 53, 7.2003, page 1123 & gt 1126) (Kim et al, Leucosotonic inhae sp. nov, a lactic acid bacterial isolated from kimchi, International Journal of Systematic and evolution Microbiology, Volume53, July 2003, pages 1123 & gt) and API50CH and API50CHL kit (product of biomeriella). The morphology and physiological properties of the finally confirmed CJLP55 strain are collated in table 1 above.

In addition, the nucleotide sequence of the 16S rRNA gene was analyzed for the identification and classification of lactic acid bacteria. The base sequence of 16S rRNA gene was determined and analyzed by the method of Kim et al (Kim et al, novel species of kimchi lactic acid bacteria, novel species derived from kimchi, Journal of International systems and evolutionary Microbiology, Vol. 50, 9.2000, pp. 1915-1919) (Kim et al, Leuconostoc kimchi sp. nov., a new species from kimchi. International Journal of Systematic and empirical Microbiology, Volume50, September 2000, pages 1915-1919). The last identified 16S rRNA gene base sequence of CJLP55 was described in the sequence List (SEQ ID NO: 1).

As a result of analysis of the base sequence of 16S rRNA, Lactobacillus plantarum CJLP55 strain and Lactobacillus plantarum Standard strain (Lactobacillus plantarum NBRC 15891)^TGenBank accession number AB326351) showed the highest homology (99.9%) and was thus identified as Lactobacillus plantarum (Lactobacillus plantarum) and named Lactobacillus plantarum CJLP55, which was deposited in the gene bank of the institute of life engineering (accession number KCTC11401BP) at 10/16 of 2008.

Example 2: acid resistance experiment of Lactobacillus plantarum CJLP55 strain in artificial gastric juice and bile resistance experiment in artificial bile

The acid resistance test in artificial gastric juice was carried out by using an artificial gastric juice prepared after modifying the test of Kobayashi et al (Kobayashi et al, study of biological characteristics of lactic acid bacteria: Multi-antibiotic resistant strains, Lactobacillus casei PSR3002, tolerance to artificial digestive juices, Japan Journal of microbiology, Vol.29, 1974, 7.691-697) (Kobayashi et al, Studies on biological characteristics of Lactobacillus 691, II.Tolerance of the multiple antibiotic resistance response strain, L.casei PSR3002, to specific diagnostic fluids, Japan Journal of biological fluids 29, July 1974, pages 691-6987). Specifically, the artificial gastric juice is prepared by adjusting the pH of MRS liquid medium to 2.5 with 1N HCl, adding pepsin at 1000 units/mL, and sterilizing.

Lactobacillus plantarum CJLP55 isolated and identified in example 1 above was centrifuged in MRS broth at 37 ℃ for 18 hours to precipitate the lactic acid bacterial strain, which was then washed 2 times with sterile saline (0.85% NaCl), and the bacterial suspension was washed at about 10%⁷cfu/mL were inoculated in the control medium and the artificial gastric juice, respectively, cultured at 37 ℃ and viable cell counts were measured at 0 and 3 hours after inoculation. The total bacteria count is KH₂PO₄、Na₂The assay was carried out after 10-fold dilution of phosphate buffer solutions (pH6.8) of HPO, L-cysteine, HCl, Tween 80, etc.

Bile resistance experiments in artificial bile were carried out according to the method of Casey et al (Casey et al, Isolation and characterization of Salmonella resistant Lactobacillus plantarum from porcine gastrointestinal tract, Letters in Applied Microbiology, vol.39, 2004, p.431-. After 0.3% of bovine bile was added to the MRS liquid medium used in the acid resistance evaluation, the viable cell count was measured 0 hours, 12 hours, and 24 hours after inoculation of lactic acid bacteria by the same method as the acid resistance evaluation method.

In the acid resistance evaluation and bile acid resistance evaluation, the same comparative experiment was performed for typical lactobacillus casei (KCTC3109), lactobacillus sakei CJLS118(KCTC13416), and lactobacillus rhamnosus GG (KCTC 5033).

The results are shown in fig. 1 and 2. Fig. 1 is a graph showing the results of an acid resistance experiment of lactobacillus plantarum CJLP 55. Fig. 2 is a graph showing the results of a bile acid tolerance experiment of lactobacillus plantarum CJLP 55.

According to the results of fig. 1 and 2, lactobacillus plantarum CJLP55 was shown to have acid resistance and bile acid resistance equal to or higher than those of other lactic acid bacteria in the comparative experiment. This indicates that the novel strain of the present invention can reach the intestine without being affected by gastric juice in vivo, and can survive without being affected by bile in the intestine.

Example 3: adhesion experiment of Lactobacillus plantarum CJLP55 strain to intestinal epithelial cells

HT-29, which is an animal cell used for The intestinal epithelial adhesion test, is derived from Korean Cell Line Bank (KCLB), and gold and The like (gold and The like, Probiotic properties of Lactobacillus and Bifidobacterium isolated from pig gastrointestinal tract, Applied Microbiology and Biotechnology, Vol.74, 4.2007, p.1103-, 2003, pages 405-.

Using RPMI1640(Gibco, USA) medium supplemented with heat-inactivated 10% Fetal Bovine Serum (FBS), 1% L-glutamate, penicillin G (100IU/mL), and streptomycin (100mg/mL) in the presence of 5% CO₂Culturing HT-29 cells at 37 ℃. For adhesion and inhibition experiments, HT-29 cells were plated at 1.0X 10/well⁵The cells/mL were added to 24-well plates and the culture was changed every other dayMedium, cultured until a monolayer is completely formed (monolayer), and used for experiments. HT-29 cells that had completely formed monolayers were washed 5 times with 25 ℃ PBS buffer solution, and then 0.5mL of RPMI1640 medium without antibiotics was added.

Lactobacillus plantarum CJLP55 was added at about 1.0X 10, respectively⁹After suspending in RPMI at a concentration of cfu/mL, the suspension was plated onto the well plate and then incubated in the presence of 5% CO₂Under the conditions, the cells were cultured at 37 ℃ for 2 hours. After the completion of the incubation, the cells were stirred at 200rpm for 3 minutes while being washed 3 times with PBS buffer solution in order to remove the lactic acid bacteria not adhering thereto and to confirm the adhesion of the cells to the washing. After washing, 0.2% trypsin-EDTA was added to remove the adhered cells, and then plated on MRS-agar (agar) by a serial dilution method using peptone (peptone) water, and after culturing at 37 ℃ for 24 hours, the number of bacteria was measured.

In addition, in order to confirm partial attachment, the experiment was performed by immersing the plate in 70% ethanol for 1 day, attaching a completely sterilized cover glass to the bottom surface of a petri dish, culturing HT-29 cells, and then adding lactic acid bacteria in an amount equivalent to that described above. The lactic acid bacterial strain attached to HT-29 cells without being washed off was dried, gram-stained, observed under an optical microscope, and the number of bacteria was measured. The same comparative experiments were also performed on Lactobacillus sakei CJLS118 and Lactobacillus rhamnosus GG (KCTC 5033).

The results are shown in FIG. 3. Fig. 3 is a graph showing the results of an intestinal epithelial cell adhesion experiment of lactobacillus plantarum CJLP 55.

According to the results of fig. 3, lactobacillus plantarum CJLP55 showed superior intestinal epithelial cell adhesion after 24 hours, compared to the commercially well-known probiotics lactobacillus rhamnosus GG (KCTC5033) and lactobacillus sakei CJLS118, and the intestinal epithelial cell adhesion of lactobacillus plantarum CJLP55 was significantly higher than lactobacillus sakei CJLS 118. Such results show that the novel strain of the present invention can attach to intestinal epithelial cells and improve the intestinal environment.

Example 4: safety evaluation of Lactobacillus plantarum CJLP55 Strain

In order to evaluate the safety of the strain isolated in example 1, hemolysis, liquefaction, confirmation of harmful metabolite (ammonia) formation, and deamination of phenylalanine were performed according to the safety evaluation test method proposed in the incorporated standards of the korean society of biological industries.

The results are shown in table 2 below.

TABLE 2 evaluation results of safety of Lactobacillus plantarum CJLP55

Bacterial strains	Item
						Gelatin liquefaction reaction detection	Deamination of phenylalanine	Results of hemolytic experiments	Ammonia production
CJLP55	Negative of	Negative of	α-Hemolysis and safety	Negative of

From the above results, it was confirmed that Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) was negative for the gelatin liquefaction reaction, the production of harmful metabolite (ammonia) and the deamination of phenylalanine, and was judged as alpha-hemolysis unrelated to pathogenicity in the hemolysis detection. It was also confirmed that lactobacillus plantarum CJLP55 is a safe strain that can be used in human body.

Example 5: evaluation of IL-12 production promoting ability after mouse splenocyte treatment

In order to evaluate the promotion of the production of the cytokine IL-12 which induces the Th1 response when spleen cells of mice which are biased toward the Th2 response by addition of ovalbumin are treated with Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) strains, reference is made to Fuiwara et al (Fujiwara et al, Lactobacillus paracasei KW3110 double blind test conducted for immunomodulation of pollen allergic patients, International allergy, 2005, Vol. 54, p. 143) double blind test, amino-acid-blue trial for immunomodulation in patients, functional-blue study, axial-blue study, Volume 54, pages 143 allergy 149, and Fujiwara et al (Th-resistant factors of Th-dependent allergy, strain 2, Th-dependent immune response of Lactobacillus strain, Th-dependent allergy, Vol. 1, page 205-.

Immunization (Immunizatio)n) the method is as follows: 5 female Balb/c mice of 6 weeks old were purchased, 13mg/mL of 1.538mL of a hydroxylate (Sigma) solution, 10mg of ovalbumin and 0.4615mL of PBS were mixed uniformly, reacted at room temperature for 20 minutes, and the resulting mixture was injected into the abdominal cavity of each mouse at a dose of 0.2mL (1mg of OVA +2mg of alum (alum)) and re-injected intraperitoneally at the same dose on day 6 for boosting (boosting). Mice were sacrificed on day 13, spleens were removed, and 100. mu.L (4X 10) of spleen-derived material was collected⁶cells/mL) spleen cells (spleenocytes) and 50. mu.L of dead bacteria of the test subject bacteria, and 50. mu.L (4mg/mL) of ovalbumin were applied to a cell culture well plate (cell culture well plate) in DMEM-10 medium at 10% CO₂Culturing in an incubator for 7 days. After 7 days of culture, the supernatant was analyzed with IL-12ELISA kit (Biosource) to determine the IL-12 concentration.

The dead bacteria of the test object bacteria were obtained by the following method.

The test object bacteria were inoculated into MRS liquid medium (Difco), cultured at 37 ℃ for 24 hours, and then the cells obtained by centrifugation at 13,000rpm for 1 minute were washed 2 times with physiological saline, and only the cells were collected. For the inoculation test of the animal cell line, the collected cells were placed in sterilized distilled water of the same volume as the original culture medium, heated at 100 ℃ for 10 minutes, centrifuged at 13,000rpm for 1 minute, collected, and diluted in an appropriate amount into DMEM medium to obtain cells at concentrations of 50. mu.g/mL and 5. mu.g/mL based on the volume of the culture medium of the cell line. The same experiment was performed for Lactobacillus rhamnosus GG (KCTC5033), Lactobacillus casei (KCTC3109) and Lactobacillus sakei CJLS118(KCTC13416) using Lactobacillus plantarum CJLP55 as the test object bacteria, and the results were compared.

The IL-12 analysis using the IL-12ELISA kit was performed in accordance with the information provided by the IL-12ELISA kit, and the O.D. value measured in the ELISA reader was measured, and the IL-12 production amount was calculated from the calibration equation (calibration equation) for the IL-12 control sample provided in the kit. The measurement results obtained are shown in fig. 4.

FIG. 4 is a graph showing the results of treating spleen cells of mice, which are biased to a Th2 response by the addition of ovalbumin, with Lactobacillus plantarum CJLP55 strain, and then measuring the concentration of the cytokine IL-12 inducing a Th1 response, compared with other lactic acid bacteria.

According to the results of fig. 4, lactobacillus plantarum CJLP55 was shown to have a significant promoting effect on the production of cytokine IL-12 inducing Th1 response, compared to other lactic acid bacteria. In addition, it was confirmed that the lactobacillus plantarum CJLP55 of the present invention efficiently induced a Th1 response in mice biased to a Th2 response.

Example 6: evaluation of IL-4 production inhibitory Activity after mouse splenocyte treatment

When splenocytes of mice, which were biased to the Th2 reaction by ovalbumin addition, were treated with Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55), the method of example 5 was used to confirm the effect of inhibiting the production of IL-4, a cytokine inducing the Th2 reaction, except that IL-12 kit was replaced with IL-4 kit (Biosource), and the experiment was carried out under the same conditions. The measurement results are shown in FIG. 5.

FIG. 5 is a graph showing the results of treating spleen cells of mice, which are biased to a Th2 response by the addition of ovalbumin, with Lactobacillus plantarum CJLP55 strain, and then measuring the concentration of the cytokine IL-4 inducing a Th2 response, compared with other lactic acid bacteria.

From the results shown in FIG. 5, it was confirmed that Lactobacillus plantarum CJLP55 has the effect of inhibiting the Th2 response in mouse splenocytes biased toward the Th2 response by inhibiting the cytokine IL-4 which induces the Th2 response.

Example 7: experiments to confirm the expression of cytokines IL-12p40 and IL-18 that induce Th1 lymphocyte differentiation in macrophages and dendritic cells and the expression of cytokine IL-10 that inhibits Th1 lymphocyte differentiation

Antigen Presenting Cells (APC), such as macrophages and dendritic cells, produce IL-12 and IL-18, thereby inducing differentiation of Th0 lymphocytes into Th1 lymphocytes, and IL-10, thereby inhibiting induction of differentiation into Th1 lymphocytes. In order to evaluate the effect of the lactic acid bacteria of the present invention on IL-12, IL-10 and IL-18 production by macrophages and dendritic cells, the following experiments were conducted.

Subjecting the test bacteria to a condition of 5X 10⁷RAW264.7 as a macrophage cell line was treated at a concentration of 10% CO at 37 ℃%₂After culturing for 48 hours, the culture medium was taken and the concentrations of IL-12p40 and IL-10 were measured by ELISA. In addition, dendritic cell strain JAWSII was inoculated with test bacteria and cultured in the same manner, and then the culture medium was taken to measure the amounts of IL-12p40 and IL-10 produced.

The same experiment was performed for lactobacillus rhamnosus GG (KCTC5033), lactobacillus casei (KCTC3109), and lactobacillus sakei CJLS118(KCTC13416), and the results thereof were compared, using lactobacillus plantarum CJLP55 as the test object bacteria and lipopolysaccharide as the positive control group.

The concentration measurement according to the ELISA method described above was carried out using IL-12p40 kit (BD Biosciences, USA) capable of measuring the concentration of IL-12 and IL-10 kit (BD Biosciences, USA) capable of measuring the concentration of IL-10, and was carried out according to the manufacturer's guidelines. Fig. 6 and 7 show the measurement results, respectively.

FIG. 6 is a graph showing the results of treating macrophage strain RAW264.7 with Lactobacillus plantarum CJLP55 strain and then measuring the concentrations of IL-12 and IL-10 by ELISA in comparison with other lactic acid bacteria.

FIG. 7 is a graph showing the results of ELISA measurement of IL-12 and IL-10 concentrations in the dendritic cell strain JAWSII treated with Lactobacillus plantarum CJLP55 strain, compared with other lactic acid bacteria.

From the results of fig. 6 and 7, it was confirmed that lactobacillus plantarum CJLP55 produced cytokine IL-12 inducing Th1 differentiation, and that production of cytokine IL-10 inhibiting Th1 differentiation was significantly less than IL-12, and that production of IL-12 was significantly increased compared to other lactic acid bacteria.

In order to confirm the production of IL-12 and IL-18 at the gene level (level), the test bacteria were treated at 5X 10⁷RAW264.7 as a macrophage cell line was treated at a concentration of 10% CO at 37 ℃%₂After culturing for 6 hours, total RNA was extracted, and the amounts of IL-12 and IL-18mRNA produced were measured by RT-PCR. The test-target bacteria were inoculated and cultured in the same manner as in JAWSII, which is a dendritic cell strain, and then RNA was extracted, and the amounts of IL-12 and IL-18mRNA produced were measured by RT-PCR.

The results of the measurement are shown in fig. 8 and 9, respectively.

FIG. 8 is a graph showing the results of RT-PCR measurement of the expression of IL-12p40 and IL-18mRNA in Lactobacillus plantarum CJLP55 strain treated with macrophage strain RAW264.7, compared with other lactic acid bacteria.

FIG. 9 is a graph showing the results of RT-PCR measurement of the expression of IL-12p40 and IL-18mRNA in a dendritic cell strain JAWSII treated with Lactobacillus plantarum CJLP55 strain, compared with other lactic acid bacteria.

From the results shown in FIGS. 8 and 9, it was revealed that Lactobacillus plantarum CJLP55 promotes the production of mRNA indicating the production of cytokines IL-12 and IL-18 inducing Th1 differentiation.

Example 8: preparation of probiotic agent containing Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55)

In order to apply the probiotic Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) identified in the above example 1 to raw materials of medicines, foods, feeds, feed additives or cosmetics and to mass-produce, it was freeze-dried to make a probiotic.

For producing the cells, MRS liquid medium (Difco) was adjusted to pH 6.0 using 25% NaOH solution, and cultured at 37 ℃ for about 18 hours, and then the cells were recovered by centrifugation. Using 5% dextrin and 10% skimmed milk as cryoprotectant, freezing recovered thallus at-40 deg.C, grinding dried thallus at 37 deg.C with a stirrer, crushing, mixing with crushed viable bacteria to obtain target bacteria count, mixing with appropriate amount of excipient such as glucose, lactose, skimmed milk, etc., placing in sealed aluminum bag, and packaging.

The probiotic prepared in this way can be mixed with cereal flour as a feed raw material to be used as a feed probiotic, or can be mixed with a carrier or an additive to be used as a medicine such as a tablet or a capsule, or used as a food probiotic, or can be mixed with a certain amount of raw materials for cosmetics, so that the probiotic can be applied to various fields such as medicines, foods, feeds, cosmetics and the like according to a conventional method in the technical field.

Claims (9)

1. Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) KCTC11401 BP.

2. A composition for preventing or treating intestinal diseases comprises Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) KCTC11401 BP.

3. Composition for enhancing immunity contains Lactobacillus plantarum CJLP55(Lactobacillus plantarum CJLP55) KCTC11401 BP.

4. The composition according to claim 3, wherein the composition is used for preventing or treating immune diseases induced by Th1/Th2 imbalance caused by excess Th2 response.

5. The composition of claim 4, wherein the immune disease is selected from the group consisting of allergic diseases, atopic diseases, cancer, and autoimmune diseases.

6. Composition according to any one of claims 2 to 5, characterized in that it is a pharmaceutical product.

7. Composition according to any one of claims 2 to 5, characterized in that it is a food product.

8. Composition according to any one of claims 2 to 5, characterized in that it is a feed or feed additive.

9. Composition according to any one of claims 2 to 5, characterized in that it is a cosmetic product.