KR20140048911A - Novel lactobacillus plantarum and compositions comprising the same - Google Patents

Abstract

Provided in the present invention are Lactobacillus plantarum CJLP133 KCTC 11403BP, a bowel disease treatment composition comprising the lactobacillus, and an immunization reinforcement composition comprising the lactobacillus. [Reference numerals] (AA) 0 hour; (BB) After three hours; (CC) Log of number of cells/ml; (DD) Strains

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 useful for prophylactic treatment of intestinal diseases and immune diseases, and a composition comprising the same.

Lactic acid bacteria, which are abundant in traditional fermented foods such as kimchi, play a role in the digestive system of the human body and decompose fiber and complex proteins into important nutrients. Live microorganisms that improve the environment and have a beneficial effect on the health of the host are collectively referred to as probiotics. In order to be effective as a probiotic, since it must be taken orally to reach the small intestine and adhere to the surface of the intestine, it must have excellent acid resistance, bile acid resistance, and intestinal epithelial cell adhesion ability.

As a representative probiotic found in traditional fermented foods such as kimchi, Lactobacillus sp. is lactic acid bacteria. Microorganisms of the genus Lactobacillus are lactic acid bacillus that undergo homogeneous or heterogeneous fermentation, and are commonly found in the intestinal tract of animals including humans and in the fermentation process of dairy products or vegetables. Microorganisms of the genus Lactobacillus maintain acidic pH in the intestine , inhibiting the reproduction of harmful bacteria such as E. coli and Clostridium , improving diarrhea and constipation, as well as synthesizing vitamins, anti-cancer action, and lowering serum cholesterol. It is known to play the role of a back. Acidophillin produced by lactic acid bacillus is known to inhibit the growth of Shigella, Salmonella, Staphylococcus, E. coli. In addition, it acts to stop diarrhea by inhibiting the proliferation of diarrhea-causing bacteria and 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 to develop a probiotic and livestock feed using the above characteristics of the microorganisms of the genus Lactobacillus is actively being conducted. Bacterial diarrhea in livestock causes decreased body weight gain and death. Therefore, in order to prevent this and increase the production of livestock, it has been generally widely practiced to add antibiotics to feed. However, due to problems such as the emergence of antibiotic-resistant bacteria and residual antibiotics in livestock products, the use of antibiotics in feed is regulated and the organic livestock feeding method is being emphasized (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 microorganisms of the genus Lactobacillus are also known to have an immune enhancing effect. In recent years, allergies and atopic diseases related to immune dysregulation predicted due to environmental pollution and increased consumption of instant foods are rapidly increasing worldwide, and these diseases are also on the rise in Korea. Recently, in Europe, as part of bacteriotheraphy, which treats diseases by oral administration of lactic acid bacteria, efforts have been made to alleviate or improve symptoms of disease with lactic acid bacteria. Lactobacillus Rhamnosus GG ( Lactobacillus rhamnosus GG) atopy occurs upon administration to the infant is decreased by half (Kalliomaki et al, Probiotics in primary prevention of atopic disease:.. a randomized placebo-controlled trial, Lancet, Volume 357, April 2001, pages 1076-1079 ) If you are already a child being treated for atopic eczema progresses Lactobacillus ramno Seuss (Lactobacillus rhamnosus) and ruteri Lactobacillus (L. reuteri) has reported that the eczema area and severity decreased (Rosenfeldt et. al., Effect of probiotic Lactobacillus strains in children with atopic dermatitis, Dermatologic and ocular diseases, Volume 111, February 2003, pages 389-395).

Research on the mechanism for the immune enhancing effect of these lactic acid bacteria is ongoing, and the specific mechanism is not yet clear, but it is generally introduced orally and lives in the intestine, which affects the intestinal immune system. It is known to be. For example, intake of lactobacilli through yogurt is known to increase the antimicrobial activity of lymphocytes in Peyer's patch, and some studies conducted in experimental animals and humans indicate that lactobacilli enhance the response of IgA. In addition, lactic acid bacteria affect both innate and adaptive immunity. In innate immunity of the intestinal immune system, it is known to play a role in maintaining health against infection by groping and killing pathogens. In adaptive immunity, the production of various cytokines, especially interleukin IL-12 and IL-18, is increased by activating macrophages that degrade antigens and present them to T lymphocytes. It is known that the production of cytokines increases by activating NF-κB and STAT signaling in In addition, lactic acid bacteria, as specialized antigen presenting cells, increase the production of IL-12, IL-18, and TNF-α in dendritic cells, which are abundant in lymph nodes and mucous membranes of the digestive system, as well as MHC class II and B7- has been shown to increase the expression of surface molecule to activate T-lymphocytes, such as 2 (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 cells that become the center of adaptive immunity, and adaptive immunity can be divided into a Th1 response, a cellular immunity, and a Th2 response, an antibody immunity. In each reaction of Th1 and Th2, cytokines produced by Antigen Presenting Cells are different. In Th1 reaction, production of IL-12, IL-18, and interferon (IFN) predominate, and in Th2 reaction, PGE2, IL- 4, IL-10 production is dominant. These Th1 reactions and Th2 reactions must be properly balanced, and when the balance is broken, it is known that various immune diseases appear. Th1 cells primarily fight infection, while Th2 cells are primarily involved in allergic and inflammatory reactions. When they function normally, Th2 cells protect the body from dust and other unwanted substances, but if these cells are excessively active, the production of IgE antibodies is increased, leading to proteins (e.g., pollen, food, etc.) that were not threatened. Causes an allergic reaction. Therefore, the Th1 response and the Th2 response must be balanced, and if one of them is excessive or the other is insufficient, disease is induced. In addition, if cortisol is continuously released due to continuous stress, the Th1 response decreases and the Th2 response increases, leading to cancer, atopy, allergy, 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 In vivo test lactic acid bacteria are said to increase the production of IFN-γ Th1 cytokines from T-lymphocytes and suppress the Th2 cytokine is IL-4, IL-5 produced (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 another experiment, when lactic acid bacteria were orally administered to ovalbumin-primed mice by administering a Th2-responsive animal model of ovalbumin, IFN-γ increased in splenocytes, IL-4, IL. It is known that -5 and IgE decreased, and changes in cytokines and IgE were the same as those of oral administration experiments even when splenocytes extracted from mice that were biased by Th2 response by administration of ovalbumin were cultured with lactic acid bacteria. However, since only T cells did not increase the lactic acid bacteria and incubated during IFN-γ produced significant antigen-presenting cells, such as IFN-γ production of T lymphocytes, the macrophage, dendritic cells are thought to be essential (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). Meanwhile, IL-12 and IL-18 are important cytokines for differentiating Th0 lymphocytes into Th1 lymphocytes, and are produced from macrophages or dendritic cells. It is known that the production of IFN-α and the like increases. As such, lactic acid bacteria increase the production of IL-12, IL-18, and IFN-α in macrophages, thereby promoting differentiation into Th1 cells and inducing their IFN-γ production.Thus, when Th2 is dominant, the Th1/Th2 balance It serves to match the (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). Therefore, lactic acid bacteria are known to be helpful in preventing or treating cancer, atopy, allergies, and autoimmune diseases caused by Th1/Th2 imbalance due to excessive Th2 response.

Accordingly, the present inventors studied to develop a new lactic acid bacteria that has a very excellent effect of controlling the Th1/Th2 imbalance due to excessive Th2 reaction compared to the conventionally known lactic acid bacteria. As a result, a novel Lactobacillus lactic acid bacteria strain from traditional fermented foods Separation and identification were made to complete the present invention.

Accordingly, an object of the present invention is a novel probiotic that not only has excellent acid resistance, bile acid resistance, and intestinal epithelial cell adhesion ability, which are basic properties as probiotics, but also has an immunity enhancing effect, particularly an effect of controlling Th1/Th2 imbalance due to excessive Th2 reaction. It is to provide a lactic acid bacteria strain of the genus Lactobacillus.

Another object of the present invention is to provide a composition for preventing or treating intestinal diseases comprising the novel Lactobacillus lactic acid bacteria strain.

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

In order to achieve the above object, the present invention is Lactobacillus plantarum CJLP133 (Lactobacillus plantarum CJLP133) (Donate Institution: Biotechnology Research Institute gene bank, deposit date: 2008.10.16, accession number: KCTC 11403BP).

In addition, the present invention provides a composition for preventing or treating intestinal diseases, including the Lactobacillus plantarum CJLP133.

In addition, the present invention provides a composition for enhancing immunity comprising Lactobacillus plantarum CJLP133.

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

Lactobacillus plantarum CJLP133 according to the present invention (Lactobacillus plantarum CJLP133) is characterized by being a novel strain of Lactobacillus platarum isolated and identified from traditional fermented foods. The traditional fermented foods include kimchi, fermented vegetables, miso, soy sauce, cheonggukjang, or salted fish, but are not limited thereto.

The Lactobacillus plantarum CJLP133 of the present invention has the highest homology (99.9%) with the ^{Lactobacillus plantarum standard strain (Lactobacillus plantarum NBRC15891 T} , GenBank accession number AB326351) as a result of 16S rRNA sequencing for the identification and classification of microorganisms. Showed the highest molecular phylogenetic relationship with Lactobacillus plantarum. Therefore, the microorganism was identified as Lactobacillus plantarum , named Lactobacillus plantarum CJLP133, and deposited with the Gene Bank of the Institute of Biotechnology on October 16, 2008 (accession number KCTC 11403BP). The nucleotide sequence of the 16S rRNA gene of Lactobacillus plantarum CJLP133 is SEQ ID NO. Same as 1.

The Lactobacillus plantarum CJLP133 of the present invention is a Gram-positive bacterium, a facultive anaerobe that can grow in both aerobic and anaerobic conditions, does not form spores, has no motility, and has a form of bacilli. More specific morphology and physiological properties of Lactobacillus plantarum CJLP133 were analyzed according to a conventional method in the art, and were found to be as shown in Table 1 below.

Form, physiology, and biochemical properties result Morphology Bacillus (Rod) Motility - Spore - Catalase - Homo-hetero fermentation Tongseong hetero fermentation Growth at 15°C + Growth at 45°C - Growth in NaCl 3% + Anaerobic proliferation + _{CO 2} production using glucose - Sugar fermentation properties Glycerol - Erythritol - D-arabinose - L-arabinose - Ribose + D-xylose - L-xylose - Adonitol - Xyloside - Galactose + D-glucose + D-Fructose + D-mannose + L-sorbose - Rhamnoose - Dulcitol - Inositol - Mannitol + Sorbitol + D-mannoside - D-Glocoside - Glucosamine + Amigdalin + Arbutin - Esculin + Salicin + Cellobiose + maltose + Lactose + Melibiose + Saccharose + Trehalose + Inulin - Melizitose + D-Raffinose + Amidon - Glycogen - Xylitol - Genthiobiose + D-Turanoose - D-Lixose - D-tagatose - D-fucose - L-fucose - D-arabitol - L-arabitol - Gluconate - 2-gluconate - 5-gluconate -

+: Positive reaction

-: Negative response

Lactobacillus plantarum CJLP133 of the present invention is preferably freeze-dried by dissolving the cells in a storage solution made by mixing a certain amount of glycerol in water, or suspending it in sterilized 10% skim milk for stable storage for a long period of time. Do.

In addition, the Lactobacillus plantarum CJLP133 of the present invention is a probiotic, and has a general intestinal effect and immunity enhancing effect of lactic acid bacteria.

In the present invention,'porbiotics' is understood to mean'living microorganisms that have 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, and when fed to humans or animals in the form of single or complex strains in the form of dried cells or fermented products, they can have a beneficial effect on the intestinal flora of the host. In order to be such a probiotic microorganism, first, it is checked whether it can survive in the intestine by passing through the stomach while being less affected by gastric juice and bile, and by checking whether it can survive by settling in the intestine, it is beneficial to the intestinal flora of the host. You should be able to make an impact. 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 must be no problem with the safety of the microorganism, and in this regard, a gelatin liquefaction reaction test, a phenylalaninetalamine production test, ammonia production test, a hemolytic test, etc. are generally performed. Lactobacillus plantarum CJLP133 according to the present invention not only has excellent acid resistance, bile acid resistance to bile acids, and adhesion to intestinal epithelial cells, but also in gelatin liquefaction test, phenylalanine deamine production test, and ammonia production test. It was negative, and in the hemolytic test, it was found to be safe as α-hemolysis, which was determined to be independent of pathogenicity.

Lactobacillus plantarum CJLP133 according to the present invention is very excellent in acid resistance, bile acid resistance, and intestinal epithelial cell adhesion, and an intestinal effect is expected. Accordingly, in another aspect, the present invention provides a composition for preventing or treating intestinal diseases, including Lactobacillus plantarum CJLP133.

The composition for treating intestinal diseases including microorganisms 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 bacterial infections and inflammatory bowel diseases that are harmful to the intestine, for example, infectious diarrhea caused by pathogenic microorganisms (E. coli, Salmonella, Clostridium, etc.), gastroenteritis, inflammatory bowel disease, neurogenic enteritis syndrome, Small intestine microbial hyperplasia, intestinal acute diarrhea, etc., but are not limited thereto. Lactobacillus plantarum CJLP133 contained in the composition for treating intestinal diseases may exist as live cells 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.When administered to humans and animals, live cells are concentrated and settled on the walls of the digestive tract in the intestine, preventing harmful bacteria from settling, and abortion It lowers the pH of the intestine and prevents harmful bacteria from proliferating. In addition, the administered live cells produce bacteriocin and peroxide to inhibit the proliferation of pathogens and help the intestinal villi, responsible for the absorption of nutrients. In addition, it produces substances that aid in the absorption and use of nutrients, improves feed demand in animals, and produces substances that neutralize toxic substances 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 degree of the disease, age, sex, race, treatment or prevention purpose, etc., but generally, 10 million to 100 billion animals can be administered per day based on an adult.

In addition, the Lactobacillus plantarum CJLP133 of the present invention has a remarkably superior immunity enhancing effect compared to the conventional lactic acid bacteria in addition to the intestinal effect. Lactobacillus plantarum CJLP133 increases the production of IL-12, which induces Th1 response in splenocytes, and inhibits the production of IL-4, which induces Th2 response. In addition, as antigen-presenting cells, it stimulates macrophages that regulate the immune response of T cells and immune regulation-related cells such as dendritic cells to promote the production of cytokines that induce the differentiation of Th0 lymphocytes into Th1 lymphocytes. It was confirmed that there is an immunomodulatory ability to control the Th1/Th2 imbalance. The immune enhancing effect of Lactobacillus plantarum CJLP133 will be described in more detail as follows.

Compared to the negative control group, Lactobacillus plantarum CJLP133 produced 7.3-9.5 times higher levels of IL-12, a cytokine that induces Th1 responses when treated with ovalbumin (OVA) to treat the splenocytes of mice biased by Th2 response. In addition, the production of IL-4, a Th2 response-inducing cytokine, was suppressed to a level of 3.2-12.1%, which is another typical lactobacillus Lactobacillus. rhamnosus GG (KCTC 5033), Lactobacillus It was found to be remarkably superior compared to casei (KCTC 3109) and Lactobacillus sakei CJLS118 (KCTC 13416). Therefore, it can be said that Lactobacillus plantarum CJLP133 inhibits the Th2 response and promotes the Th1 response, thereby controlling the Th1/Th2 imbalance due to the excess of the Th2 response.

In addition, Lactobacillus plantarum CJLP133 ( Lactobacillus plantarum CJLP133) was treated with the macrophage cell line RAW264.7 and the dendritic cell JAWSII, and it was confirmed that as the number of lactic acid bacteria increased, the macrophage cell line was stimulated to enhance the immune response. As a result of treating the macrophage cell line RAW264.7 and the dendritic cell line JAWSII with Lactobacillus plantarum CJLP133, it promotes the production of Th1 differentiation-inducing cytokines IL-12 and IL-18, and IL-10, a cytokine that inhibits the induction of Th1 differentiation. It was found to promote the induction of Th1 differentiation by producing relatively small amounts of IL-12. Therefore, even from these experimental results, it can be said that Lactobacillus plantarum CJLP133 suppresses Th2 response and promotes Th1 response, thereby controlling the Th1/Th2 imbalance due to excessive Th2 response.

IL-4 is derived from Th2 cells, specifically plays a pivotal role in cellular immune responses, and has an anti-inflammatory cytokine function that inhibits the production of IL-12, a cytokine in Th1 cells. Recently peripheral blood and skin lesions of atopic dermatitis patients, there is the fact that Th2 cells are relatively increased known to produce mainly including IL-4, IL-5 ( Miraglia et. Al, Immune dysregulation in atopic dermatitis, Allergy and Asthma Proceedings , Volume 27, November-December 2006, pages 451-455). Therefore, the imbalance of Th1/Th2 due to excessive Th2 response causes diseases such as atopy. In addition, as mentioned earlier, when one of the Th1 and Th2 responses is excessive or the other is insufficient, disease is caused, and when the Th1 response is lowered and the Th2 response is increased, cancer, atopy, allergies, 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). Therefore, Lactobacillus plantarum CJLP133 according to the present invention regulates the imbalance of Th1/Th2 due to excessive Th2 response by controlling the cytokines produced by Th1 cells, Th2 cells, macrophages, and dendritic cells related to immune regulation, Not only can it be expected to act effectively against diseases such as atopy and allergies, but it is also expected to be effective in preventing or treating cancer and autoimmune diseases.

Accordingly, in another aspect, the present invention provides a composition for enhancing immunity comprising Lactobacillus plantarum CJLP133. In the composition for enhancing immunity according to the present invention, Lactobacillus plantarum CJLP133 is a lactic acid bacterium and has an immunity enhancing effect due to the immunity enhancing effect of the general lactic acid bacteria as described in the prior art. In particular, the composition for enhancing immunity according to the present invention has the effect of controlling the imbalance of Th1/Th2 due to excessive Th2 reaction as Lactobacillus plantarum CJLP133 has an effect of promoting Th1 reaction as demonstrated in the following Examples. , It is effective in preventing or treating diseases caused by an imbalance of Th1/Th2 due to excessive Th2 response. Therefore, the composition for enhancing immunity in 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 and hay fever.

The method of administering the composition for enhancing immunity of the present invention is not particularly limited, but is preferably administered orally. The dosage may vary depending on the type of disease, the degree of the disease, age, sex, race, treatment or prevention purpose, etc. for which immunity is required, but in general, 10 million to 100 billion animals can be administered per day for adults. have.

Since the composition for preventing or treating bowel disease and the composition for enhancing immunity including Lactobacillus plantarum CJLP133 according to the present invention contains lactic acid bacteria that have proven safety, medicines, health functional foods, cosmetics, feed without fear of side effects, etc. , Or may be used as a feed additive.

When the composition according to the present invention is used as a pharmaceutical, it can be formulated into a conventional pharmaceutical formulation known in the art. The pharmaceuticals may preferably be formulated in an oral dosage form, and for example, may be formulated in a dosage form for oral administration such as a solution, a suspension, a powder, a granule, a tablet, a capsule, a pill, or an extract.

When formulated into each of the above formulations, it can be prepared by adding a pharmaceutically acceptable carrier or additive necessary for the manufacture of each formulation. Typically, 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 can be selected and used as the carrier. As an additive, flavoring, vitamins, and antioxidants You can select and use one or more of them.

The carriers and additives may be 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 preferable. In addition, as a disintegrant, calcium carboxymethylcellulose, sodium starch glycolate, potassium polyacrylate, or crospovidone, as a sweetener, sucrose, fructose, sorbitol, or aspartame, as a stabilizer, sodium carboxymethylcellulose, beta-cyclodextrin, Pewter, or xanthan gum, as the preservative, is preferably methyl paraoxybenzoate, propyl paraoxybenzoate, or potassium sorbate.

In addition, in addition to the above ingredients, in order to enhance the taste as well-known additives, natural flavors such as plum flavor, lemon flavor, pineapple flavor, herbal flavor, natural fruit juice, natural colors such as chlorophyllin, flavonoids, fructose, honey, sugar alcohol, sugar A sweetening component such as, or an acidifying agent such as citric acid or sodium citrate may be mixed and used.

Such formulation methods and 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 foods include not only health functional foods, but also general foods that are widely and commonly consumed daily by humans. When used as a health functional food, it may be formulated in a formulation of a conventional health functional food known in the art together with a food physiologically acceptable carrier or additive. The health functional food may be prepared, for example, as a powder, granule, tablet, capsule, suspension, emulsion, syrup, liquid, extract, tea, jelly, or beverage. As the food pharmaceutically acceptable carrier or additive, any carrier or additive known to be usable in the art may be used in the manufacture of a formulation to be prepared.

Since the composition according to the present invention has a preventive 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, it can be prepared into various cosmetic products 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 to the manufacture of cosmetics required for the manufacture of each formulation.

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

When used as a feed additive, the composition may be a high concentration of 20 to 90% or may be prepared in the form of a powder or granules. The feed additives include organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherol, rosemary. Any one or more of natural antioxidants such as extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid may be further included. When used as a feed, the composition may be formulated in a conventional feed form, and may include a common feed component together.

The feed additives and feeds include grains such as crushed or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds, such as feeds based on rapeseed, soybeans, and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include a dry component composed of sugar and dairy products, for example, various milk powders and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters, and the like.

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

The feed may contain any protein-containing organic grain flour commonly used to satisfy the dietary needs of animals. These protein-containing grains typically consist primarily of corn, soybean meal, or corn/soybean meal mix.

In addition, the feed additive and feed may contain adjuvants, such as preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, and the like. The feed additive may be used by infiltrating, spraying, or mixing and adding to animal feed.

The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry and fish. As the mammal, pigs, cows, sheep, goats, experimental rodents, and experimental rodents, as well as pets (eg, dogs, cats), etc., can be used, and as the poultry, chickens, turkeys, ducks, geese, pheasants, And quail, and may be used for trout as the fish, but is not limited thereto.

As described above, the novel lactic acid bacteria Lactobacillus plantarum CJLP133 according to the present invention, as a probiotic, has excellent acid resistance, bile acid resistance, and intestinal epithelial cell adhesion, so that it has an intestinal effect, and has an effect of promoting Th1 reaction. It has the effect of controlling the imbalance of Th1/Th2 due to excessive Th2 reaction. Therefore, the novel lactic acid bacteria Lactobacillus plantarum CJLP133 according to the present invention can be used as a composition for treating intestinal diseases and a composition for enhancing immunity, and in particular, prevention of diseases caused by imbalance of Th1/Th2 due to excessive Th2 reaction or It is effective in treatment.

1 is a graph showing the acid resistance test results of Lactobacillus plantarum CJLP133.
Figure 2 is a graph showing the bile acid resistance test results of Lactobacillus plantarum CJLP133.
3 is a graph showing the test results of intestinal epithelial cell adhesion ability of Lactobacillus plantarum CJLP133.
FIG. 4 shows the results of measuring the concentration of IL-12, a Th1 response-inducing cytokine, after treatment of the Lactobacillus plantarum CJLP133 strain to the splenocytes of mice biased by the Th2 reaction by administering ovalbumin, and comparing the results with other lactic acid bacteria. It is a graph.
Figure 5 shows the results of measuring the concentration of IL-4, a Th2 response-inducing cytokine, after treatment of the Lactobacillus plantarum CJLP133 strain to the splenocytes of mice biased by the Th2 reaction by administering ovalbumin, compared with other lactic acid bacteria. It is a graph.
6 is a graph showing the results obtained by treating the Lactobacillus plantarum CJLP133 strain with the macrophage cell line RAW264.7, and then measuring the concentrations of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.
7 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the dendritic cell line JAWSII, and then measuring the concentrations of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.
FIG. 8 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the macrophage cell line RAW264.7, and then measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR compared with other lactic acid bacteria.
9 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the dendritic cell line JAWSII, and then measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR, compared with other lactic acid bacteria.
Figure 10a is a graph showing the results of measuring the skin thickness of the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
FIG. 10B is a photograph of a result of observing the degree of accumulation of lymphocytes in inflammatory tissues in the extracted skin after administration of lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.
FIG. 11A is a graph showing the results of measuring the number of cells of eosinophil in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
FIG. 11B is a photograph of a result of observing the degree of infiltration of eosinophil in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.
12A is a graph showing the results of measuring the number of mast cells in the skin extracted after lactic acid bacteria were administered to NC/Nga mice inducing atopic dermatitis.
FIG. 12B is a photograph of a result of observing the degree of infiltration of mast cells in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.
13A is a graph showing the results of measuring the number of nerve fibers in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
13B is a photograph of a result of observing the degree of penetration of nerve fibers in the extracted skin with an optical microscope after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
FIG. 14 is a photograph of axillary lymph nodes (A) and spleens (B) that were excised after lactic acid bacteria were administered to NC/Nga mice inducing atopic dermatitis.
15 is a graph showing the results of counting the total number of cells in the axillary lymph nodes (a) and spleen (b) extracted after administration of lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
Figure 16 is a graph showing the results of counting the number of T-cells in the axillary lymph nodes (a) and spleen (b) extracted after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
FIG. 17 is a graph showing the results of counting the number of B-cells in the axillary lymph nodes (a) and spleen (b) extracted after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.
Figure 18 shows the amount of IL-12 after cultivation by adding house dust mite extract to the single cell suspension obtained from the extracted axillary and lymph nodes (a) and spleen (b) after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis. It is a graph showing the results measured by the ELISA method.
Figure 19 shows the amount of IFN-γ after cultivation by adding house dust mite extract to the single cell suspension obtained from the extracted axillary and lymph nodes (a) and spleen (b) after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis. It is a graph showing the results measured by the ELISA method.

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

Example 1: microbes Lactobacillus Planta Room CJLP133 Isolation and identification of strains

The Lactobacillus plantarum CJLP133 strain isolated from kimchi was spread on MRS solid medium (Difco, USA) containing 1.5% agar and cultured at 37°C for 24 hours, and then the colonies confirmed to have been purely separated were platinum. It was taken as a (loop) and cultured at 37° C. for 18-24 hours in MRS liquid medium (Difco, USA).

Then, the morphology and physiological characteristics of the CJLP133 strain were determined by Kim et al . (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) were used. As a result, the morphology and physiological characteristics of the identified CJLP133 strain are summarized in Table 1 above.

In addition, the base sequence of the 16S rRNA gene for identification and classification of lactic acid bacteria was analyzed. The nucleotide sequence determination and analysis of the 16S rRNA gene was conducted by Kim et al . (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). The method of was used. As a result, the nucleotide sequence of the 16S rRNA gene of CJLP133 confirmed as a result was described in the sequence list (SEQ ID NO: 1).

Lactobacillus plantarum CJLP133 strain was 16S rRNA sequencing analysis results Lactobacillus plantarum standard strain ( Lactobacillus plantarum NBRC15891 ^T , GenBank accession number AB326351) showed the highest homology (99.9%) and was identified as Lactobacillus plantarum, named Lactobacillus plantarum CJLP133, and was named as Lactobacillus plantarum CJLP133. It was deposited on October 16, 2014 (accession number KCTC 11403BP).

Example 2: Lactobacillus Planta Room Acid resistance of CJLP133 strain in artificial gastric juice Experiments and tests on bile resistance in artificial bile

Acid resistance experiments in artificial gastric juice were conducted by Kobayashi et al . (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) was performed using artificial gastric juice prepared by modifying the experiment. Specifically, the artificial gastric juice was adjusted to pH 2.5 with 1N HCl in MRS liquid medium and pepsin to 1000 units/ml. After addition, it was prepared by sterilization.

Lactobacillus plantarum CJLP133 isolated and identified in Example 1 above was centrifuged in MRS liquid medium at 37°C for 18 hours to precipitate the lactic acid strain and washed twice with sterile saline (0.85% NaCl). Then, the cell suspension was inoculated into the control medium and the artificial gastric juice at a ^{level of about 10 7} cfu/ml, respectively, and the number of viable bacteria was measured 0 and 3 hours after the inoculation while incubating at 37°C. The total number of bacteria was _{measured by diluting 10 times with a phosphate buffer (pH 6.8) containing KH 2} PO4, Na ₂ HPO, L-cysteine, HCl, Tween 80, and the like.

Within the biliary experiments in artificial bile Casey, etc. (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) After 0.3% of ox bile was added to the MRS liquid medium used in the acid resistance evaluation, the number of viable bacteria was measured 0 hours, 12 hours, and 24 hours after inoculation of lactic acid bacteria in the same manner as the acid resistance evaluation method. .

Lactobacillus casei (KCTC 3109), Lactobacillus , which are typical lactic acid bacteria in both the acid resistance evaluation and the bile acid resistance evaluation The same comparative experiment was performed for sakei CJLS118 (KCTC 13416), and Lactobaillus rhamnosu s GG (KCTC 5033).

The results are shown in FIGS. 1 and 2. 1 is a graph showing the acid resistance test results of Lactobacillus plantarum CJLP133. 2 is a graph showing the results of an experiment on bile resistance of Lactobacillus plantarum CJLP133.

According to the results of FIGS. 1 and 2, it was found that Lactobacillus plantarum CJLP133 had an acid resistance equal to or higher than that of all other lactic acid bacteria and bile acid resistance compared to the other lactic acid bacteria tested. This indicates that the novel strain according to the present invention can reach the intestine without being affected by gastric juice in the body, and can survive in the intestine without being affected by bile.

Example 3: Lactobacillus Planta Room Attaching function of the intestinal epithelial cells of a strain CJLP133 Experiment

Chapter was the HT-29 as an animal cells for the epithelial cell adhesion function tests used under pre-sale from Korea Cell Line Bank (KCLB), experimental methods, such as Kim (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, etc. (Hirano et. al., the effect of Lactobacillus rhamnosus on enterohemorrhagic Escherichia coli infection of human intestinal cells in In vitro , Microbiology and Immunology, Volume 47, 2003, pages 405-109 ) was used.

HT-29 cells were heat-inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin G (100 IU/mL), and RPMI 1640 supplemented with streptomycin (100 mg/mL) (Gibco, USA). ) Incubated at 37°C in the presence of 5% CO _{2 using a medium.} For the adhesion test and adhesion inhibition test, HT-29 cells were ^{dispensed into 24 well-plates at a number of 1.0×10 5} cells/mL per well, and the medium was exchanged every other day until a completely monolayer was formed. It was cultured and used in the experiment. HT-29 cells forming a complete monolayer were washed 5 times with a PBS buffer solution at 25°C, and 0.5 mL of RPMI 1640 medium without antibiotics was added.

Lactobacillus plantarum CJLP133 was ^{suspended in RPMI to a concentration of about 1.0×10 9} cfu/mL, respectively, and then inoculated into the well plate and cultured at 37° C. for 2 hours in the presence of _{5% CO 2.} After the cultivation was completed, in order to confirm the removal of non-adherent lactic acid bacteria and the adhesion ability due to washing, washing was performed three times using a PBS buffer solution while stirring at a speed of 200 rpm for 3 minutes. After washing is complete, 0.2% trypsin-EDTA is dispensed to remove the attached cells, plated on MRS-agar by continuous dilution using peptone water, and incubated at 37°C for 24 hours to obtain the number of bacteria. It was measured.

Separately, to confirm some adhesion, a cover glass completely sterilized by immersing it in 70% alcohol for about a day was attached to the bottom of a Petri dish, and then HT-29 cells were cultured and the same amount of lactic acid bacteria was added to the above. Lactobacillus adhered to HT-29 cells without being washed off by washing was dried, followed by Gram staining, observed with an optical microscope, and the number of bacteria was measured. Lactobacillus The same comparative experiment was performed for sakei CJLS118 (KCTC 13416), and Lactobaillus rhamnosus GG (KCTC 5033).

The results are shown in FIG. 3. 3 is a graph showing the test results of intestinal epithelial cell adhesion ability of Lactobacillus plantarum CJLP133

According to the results of Figure 3, Lactobacillus plantarum CJLP133 is a commercially well known Lactobacillus probiotic strain. Rhamnosus GG (KCTC 5033) and Lactobacillus sakei CJLS118 (KCTC 13416) showed superior adhesion to intestinal epithelial cells after 24 hours, especially Lactobacillus Compared to sakei CJLS118 (KCTC 13416), intestinal epithelial cell adhesion was significantly higher. These results indicate that the novel strain according to the present invention can improve the intestinal environment by adhering to intestinal epithelial cells.

Example 4: Lactobacillus Planta Room CJLP133 Safety evaluation of strains

In order to evaluate the safety of the strain isolated in Example 1, the hemolysis phenomenon test, gelatin liquefaction test, the production of harmful metabolites (ammonia), and phenynealaninetal were carried out according to the safety evaluation test method proposed by the Korean Bioventure Association group standard. An amine test was performed.

The results are shown in Table 2 below.

Safety evaluation results of Lactobacillus plantarum CJLP133 Strain Item Gelatin liquefaction reaction test Phenylalaninetalamine Hemolytic test result Ammonia generation CJLP133 voice voice α-hemolysis, safe voice

According to the above results, Lactobacillus plantarum CJLP133 ( Lactobacillus plantarum CJLP133) was negative for gelatin liquefaction reaction, production of harmful metabolites (ammonia), and production of phenolalaninetalamine. In hemolysis test, it was confirmed as α-hemolysis, which was judged to be independent of pathogenicity. Therefore, it was confirmed that Lactobacillus plantarum CJLP133 is a safe strain that can be administered to the human body.

Example 5: mouse Splenocytes After processing IL - 12 generation Acceleration evaluation

Lactobacillus Planta Room CJLP133 ( Lactobacillus plantarum CJLP133) strain,

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 the like Fujiwara (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).

For immunization, 5 6-week-old female Balb/c mice were purchased, mixed well with 1.538 mL of alum hydroxide (Sigma) 13 mg/mL solution, 10 mg of ovalbumin and 0.4615 mL of PBS, and reacted for 20 minutes at room temperature. The mixed solution was injected into the abdominal cavity at 0.2 mL (1 mg OVA + 2 mg alum) per mouse, and the same amount was injected into the abdominal cavity on the 6th day for boosting. Mice were sacrificed on the 13th day, and the spleen was removed, and 100 µl (4x10 ⁶ cells/mL) of splenocytes obtained therefrom, 50 µl of dead cells and 50 µl of ovalbumin (4 µg/ml) of the test bacteria were added to the cell culture wells. It was added to a plate (cell culture well plate) and cultured in a 10% CO ₂ incubator for 7 days in DMEM-10 medium. After incubation for 7 days, the supernatant was taken and assayed (Biosource) with an IL-12 ELISA kit to measure the concentration of IL-12.

Dead cells of the test subject bacteria were obtained as follows.

The test subject bacteria were inoculated in MRS liquid medium (Difco) and cultured at 37° C. for 24 hours, and the cells obtained by centrifugation at 13,000 rpm for 1 minute were washed twice in physiological saline, and only the cells were taken. For the animal cell line inoculation test, the recovered cells were heated at 100°C for 10 minutes in sterilized distilled water equal to the volume of the original culture solution, centrifuged at 13,000 rpm for 1 minute, and then collected and diluted in DMEM medium, based on the volume of the cell line culture solution. The cells were made to have concentrations of 50 µg/ml and 5 µg/ml. Lactobaillus plantarum CJLP133 was used as the test subject bacteria, and Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei The same experiment was performed for CJLS118 (KCTC 13416) and the results were compared.

The IL-12 assay using the IL-12 ELISA kit was tested according to the instructions provided in the IL-12 ELISA kit, and the OD value measured in the ELISA reader was measured, and the calibration for the IL-12 control sample provided in the kit. The amount of IL-12 produced was converted according to the formula. Thus, the obtained measurement results are shown in FIG. 4.

FIG. 4 shows the results of measuring the concentration of IL-12, a Th1 response-inducing cytokine, after treatment of the Lactobacillus plantarum CJLP133 strain to the splenocytes of mice biased by the Th2 reaction by administering ovalbumin, and comparing the results with other lactic acid bacteria. It is a graph.

According to the results of FIG. 4, it was found that Lactobacillus plantarum CJLP133 significantly promoted the production of IL-12, a Th1 response-inducing cytokine, compared to other lactic acid bacteria. Therefore, it was confirmed that the Lactobacillus plantarum CJLP133 according to the present invention effectively induces the Th1 response in mice biased by the Th2 response.

Example 6: mouse Splenocytes After processing IL - 4 generation Deterrent evaluation

Lactobacillus Planta Room CJLP133 ( Lactobacillus plantarum CJLP133) strain, in order to confirm the effect of inhibiting the production of IL-4, a cytokine that induces Th2 response, when treated with ovalbumin to the splenocytes of mice biased by the Th2 response, IL-12 in the method of Example 5 above. The only difference was that the IL-4 kit (Biosource) was used instead of the kit, and the other conditions were the same. The measurement results are shown in FIG. 5.

Figure 5 shows the results of measuring the concentration of IL-4, a Th2 response-inducing cytokine, after treatment of the Lactobacillus plantarum CJLP133 strain to the splenocytes of mice biased by the Th2 reaction by administering ovalbumin, compared with other lactic acid bacteria. It is a graph.

According to the results shown in FIG. 5, it was confirmed that Lactobacillus plantarum CJLP133 suppressed the Th2 response-inducing cytokine IL-4 cytokine, thereby having an inhibitory effect on the Th2 response in mouse splenocytes biased by the Th2 response.

Example 7: macrophage differentiation and induce Th1 lymphocytes in dendritic cell cytokine IL-12p40 and IL-18 expression and Th1 lymphocytes, differentiation inhibiting cytokine-cytokine is IL-10 expression in the verification experiment

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, produce IL-10 to produce Th1 lymphocytes. Inhibits differentiation induction. The following experiment was performed 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 in macrophages and dendritic cells.

The test target bacteria were treated in the macrophage cell line RAW264.7 at a ^{concentration of 5×10 7} /mL, cultured at 37°C and 10% CO ₂ for 48 hours, and then the medium was taken and the IL-12p40 and IL-10 were prepared by ELISA. The concentration was measured. In addition, the dendritic cell line JAWSII was inoculated and cultured in the same manner, and then the medium was taken to measure the production amount of IL-12p40 and IL-10.

Lactobacillus plantarum CJLP133 was used as the test target bacteria, lipopolysaccharide was used as a positive control, and Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus The same experiment was performed for sakei CJLS118 (KCTC 13416) and the results were compared.

The concentration measurement by the ELISA method includes an IL-12p40 kit (BD Biosciences, USA) capable of measuring the concentration of IL-12 and an IL-10 kit (BD Biosciences, USA) capable of measuring the concentration of IL-10. Was performed according to the manufacturer's instructions. Thus, each of the measured results is shown in FIGS. 6 and 7.

6 is a graph showing the results obtained by treating the Lactobacillus plantarum CJLP133 strain with the macrophage cell line RAW264.7, and then measuring the concentrations of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

7 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the dendritic cell line JAWSII, and then measuring the concentrations of IL-12 and IL-10 by ELISA compared with other lactic acid bacteria.

According to the results of FIGS. 6 and 7, Lactobacillus plantarum CJLP133 generates IL-12 cytokine, a Th1 differentiation-inducing cytokine, and production of IL-10, a cytokine that inhibits Th1 differentiation, is significantly less than IL-12. It can be seen that, compared to other lactic acid bacteria, it can be seen that it significantly increases the production of IL-12.

In addition, in order to confirm the production of IL-12 and IL-18 at the gene level, the test target bacteria ^{were treated with a macrophage cell line, RAW264.7, at a concentration of 5×10 7} /mL, and 37°C, 10% CO _{2 After} culturing for 6 hours, total RNA was extracted and the amount of IL-12 and IL-18 mRNA produced was measured by RT-PCR method. The dendritic cell line JAWSII was inoculated and cultured in the same manner, and then RNA was extracted and the amount of IL-12 and IL-18 mRNA produced was measured by RT-PCR method.

Thus, each of the measured results is shown in FIGS. 8 and 9.

FIG. 8 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the macrophage cell line RAW264.7, and then measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR compared with other lactic acid bacteria.

9 is a graph showing the results of treating the Lactobacillus plantarum CJLP133 strain with the dendritic cell line JAWSII, and then measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR, compared with other lactic acid bacteria.

According to the results of FIGS. 8 and 9, it can be seen that Lactobacillus plantarum CJLP133 promotes the production of mRNAs indicative of the production of IL-12 and IL-18, which are Th1 differentiation-inducing cytokines, and in particular IL-12 mRNA It was found to be remarkably superior to other lactic acid bacteria in promoting the expression of.

Example 8: Lactobacillus Planta Room In vivo test for atopic skin disease of CJLP133 strain

1) Breeding of experimental animals and Experimental group Classification

A 4-week-old female NC/Nga mouse was purchased and stabilized for 1 week, and oral administration of the lactic acid bacteria strain was started. The breeding environment was maintained at 24±2℃, the light and dark cycle was maintained at 12 hours intervals, and powdered feed without antibiotics was used. Lactobacillus was evenly mixed with powdered feed so that mice were ingested for 10 weeks (1×1010 cfu/mouse), and atopy was induced by applying Biostar AD (Biostir, Japan) ointment for 5 weeks from 6 weeks after administration of lactic acid bacteria. The experimental group was divided into a non-induction group that does not induce atopy, a control group that induces atopy, and an experimental group that induces atopy and administered a lactic acid bacteria strain, and each group was classified into 8 mice (Table 3). ). The lactic acid bacteria is a typical lactic acid bacteria of Lactobacillus by a conventional sakei CJLS118(KCTC 13416), Lactobacillus rhamnosu s GG (KCTC 5033), strain CJLP55 (KCTC 11401BP), CJLP56 (KCTC 11402BP), CJLP136 (KCTC 11404BP) and strain CJLP133 (KCTC 11403BP) according to the present invention developed by the applicant of the present invention were targeted.

group Administered lactobacilli Induce atopy Non-induction - × Control - ○ GG GG(KCTC 5033) ○ LP55 CJLP55(KCTC 11401BP) ○ LP56 CJLP56(KCTC 11402BP) ○ LP133 CJLP133(KCTC 11403BP) ○ LP136 CJLP136(KCTC 11404BP) ○ LS118 CJLS118(KCTC 13416) ○ LA12 CJLA12 ○

2) induce atopy

The NC/Nga mouse's back and upper auricles were removed as much as possible with an epilator, and the hairs were completely removed using a hair removal cream. A 4% SDS aqueous solution was sprayed on the application site to remove the fat component of the skin, dried completely for about 1 hour, and then 100 mg of Biostir AD ointment (Biostir, Japan) was evenly applied to the back and auricles using a flat stick. . Biostir AD ointment was applied twice a week, a total of 10 times for 5 weeks.

3) tissue staining

In atopic dermatitis, the thickness of the skin increases according to repeated inflammatory reactions, and major immune cells such as lymphocytes, monocytes, eosinophils, and mast cells infiltrate a lot into the tissue, causing an inflammatory reaction. It is known to cause itching. Therefore, the skin of atopic-induced mice was excised, and the number of immune cells and neurons were counted and observed.

After induction of atopy for 5 weeks, the mouse was rapidly killed, the back skin was removed, the tissue was fixed with an Accustain formalin-free fixative solution, and a paraffin block was prepared. After cutting into 5 μm thickness, hematoxylin/eosin staining was performed to observe changes in skin (epidermis + dermis) thickness, and the degree of accumulation of lymphocytes in the inflamed tissue was observed in the range of 2×2 mm under an optical microscope . In addition, toluidine blue staining for mast cell identification, Congo red staining for eosinophil identification, and observation at a range of 2×2mm under an optical microscope from the epidermis The number of mast cells or eosinophil cells present in the skin between muscle tissues was counted. In order to confirm the invasion of nerve fibers in skin tissue by immunohistochemical staining, an anti-protein gene product (PGP9.5) antibody that recognizes nerve fibers was reacted to the previously immobilized tissue, followed by biotin-conjugated goat anti-rabbit antibody and peroxidase. The -conjugated streptavidin was reacted in order and colored by a peroxidase enzyme reaction.

The results are shown in FIGS. 10 to 13.

Figure 10a is a graph showing the results of measuring the skin thickness of the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

FIG. 10B is a photograph of a result of observing the degree of accumulation of lymphocytes in inflammatory tissues in the extracted skin after administration of lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.

FIG. 11A is a graph showing the results of measuring the number of cells of eosinophil in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

FIG. 11B is a photograph of a result of observing the degree of infiltration of eosinophil in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.

12A is a graph showing the results of measuring the number of mast cells in the skin extracted after lactic acid bacteria were administered to NC/Nga mice inducing atopic dermatitis.

FIG. 12B is a photograph of a result of observing the degree of infiltration of mast cells in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.

13A is a graph showing the results of measuring the number of nerve fibers in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

13B is a photograph of a result of observing the degree of penetration of nerve fibers in the extracted skin after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis with an optical microscope.

According to the above results, it was confirmed that the skin thickness of about 100 μm appeared in most of the experimental groups of NC/Nga mice that caused atopy, while maintaining the thickness of 50 μM, which is half the level in the CJLP133-treated group (FIG. 10A), and that of lymphocytes and monocytes. When invasion was observed, a number of immune cells were stained purple in the control and GG groups, but a remarkably small number of immune cells were observed in the CJLP133 administration group (FIG. 10B).

As a result of confirming the invasion of eosinophil and mast cells in the inflamed tissue, the number of eosinophil and mast cells increased compared to the non-induction group when atopy was induced (control group). However, in the group administered with CJLP133, the number of eosinophil and mast cells increased. It was found that the number of eosinophil and mast cells was significantly less than that of the lactic acid bacteria administration group (FIGS. 11A and 12A ). In the actual staining picture, a large number of blue-blue eosinophil and red-brown mast cells were observed in the control group and the GG-administered group, respectively, whereas a small number of eosinophil and mast cells were observed in the CJLP133-administered group (FIGS. 11B and 12B).

As a result of confirming the penetration of nerve fibers by the immunohistochemistry method, the penetration of nerve fibers was not observed in the non-inducing group, but a number of brown nerve fibers appeared in the control group (Fig. 13a), and nerves that penetrated the skin in all groups administered with lactic acid bacteria The number of fibers decreased, and in particular, the number of nerve fibers was significantly reduced in the group administered with CJLP55, CJLP133, and CJLP136 (FIG. 13B).

4) Axillary lymph node And Splenocytes Composition confirmation

The axillary lymph node (ALN) is an immune organ that plays an important role in an animal model of chronic atopic disease, and it is reported that the size of the axillary lymph node is increased in some severe chronic atopic patients, and atopic animal model induced by house dust mites NC/Nga mice In many studies, the axillary lymph node was designated as the direct target lymph node and observed. Therefore, in this study, after induction of atopy for 5 weeks, the axillary lymph nodes as well as the spleen, which is the main immune organ, were excised to observe changes in size and cell composition.

After 5 weeks of atopy induction, the mice were suddenly killed, the axillary lymph node and the spleen were removed to compare the size, and then red blood cells were removed and a single cell suspension of each tissue was obtained. Dispense into 1×10 ⁶ cells per FACS tube, stain with anti-Thy1.2-FITC, anti-CD19-FITC, anti-F4/80-FITC, anti-CD11c-FITC antibody, respectively, and FACS analysis to analyze T lymphocytes and The composition of B lymphocytes was confirmed. The results are shown in FIGS. 14 to 17.

FIG. 14 is a photograph of axillary lymph nodes (A) and spleens (B) that were excised after lactic acid bacteria were administered to NC/Nga mice inducing atopic dermatitis.

FIG. 15 is a graph showing the results of counting the total number of cells in the axillary lymph nodes (a) and spleen (b) extracted after administration of lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

Figure 16 is a graph showing the results of counting the number of T-cells in the axillary lymph nodes (a) and spleen (b) extracted after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

FIG. 17 is a graph showing the results of counting the number of B-cells in the axillary lymph nodes (a) and spleen (b) extracted after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis.

According to the above results, the size of axillary lymph nodes increased in the control group that caused atopic disease, and was observed to be similar in size to the control group in the GG-treated group, but in the CJLP55, CJLP56, CJLP133, CJLP136, and CJLS118 groups, the size of the axillary lymph nodes was the control It was confirmed to be smaller than (Figure 14A). In the case of the spleen, there was little difference between the experimental groups (FIG. 14B). As a result of measuring the number of cells by separating cells from the axillary lymph nodes, it was confirmed that the number of cells increased by 4.5 times or more in the control group compared to the non-inducing group, and the number of cells was significantly decreased in the CJLP55, CJLP133, and CJLP136-administered groups compared to the control group (Fig. 15A). ). In the case of the spleen, there was no significant change in the number of cells as in the size (FIG. 15B).

According to the results of FACS analysis by staining T lymphocytes and B lymphocytes from axillary lymph nodes and spleen, respectively, in the case of T lymphocytes and B lymphocytes from axillary lymph nodes, the number of cells increased by 5 times in the control group that caused atopy, CJLP55, CJLP56. , CJLP133, CJLP136 showed a statistically significant decrease in cell number compared to the control group in all of the groups, in particular, CJLP133 administration group was found to be significantly reduced compared to other lactic acid bacteria administration group (Figs. 16a and 17a). The number of T lymphocytes and B lymphocytes in the spleen did not significantly change (Figs. 16B and 17B).

5) Axillary lymph node Cells and Splenic Cytokine Generating ability

IL-12, mainly produced from macrophages, induces ThO lymphocytes to differentiate into Th1 lymphocytes, and IFN-γ produced from Th1 lymphocytes not only activates macrophages, but also inhibits the differentiation and activity of Th2 lymphocytes, so IL-12 and IFN- The change in the amount of γ produced was measured.

In the above 4) axillary lymph node and splenocyte composition confirmation experiment, a single cell suspension of each tissue obtained from axillary lymph node and splenocytes was dispensed into a 24-well plate by 5×10 ⁶ cells each, and house dust mite (Dermatophagoides farinae body, Dfb) extract Was added to a final concentration of 10 μg/ml. After incubation at 37° C. for 48 hours, the supernatant was taken and the amount of IFN-γ and IL-12 produced was measured by ELISA. The results are shown in FIGS. 18 and 19.

Figure 18 shows the amount of IL-12 after cultivation by adding house dust mite extract to the single cell suspension obtained from the extracted axillary and lymph nodes (a) and spleen (b) after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis. It is a graph showing the results measured by the ELISA method.

Figure 19 shows the amount of IFN-γ after cultivation by adding house dust mite extract to the single cell suspension obtained from the extracted axillary and lymph nodes (a) and spleen (b) after administering lactic acid bacteria to NC/Nga mice inducing atopic dermatitis. It is a graph showing the results measured by the ELISA method.

According to the above results, it was found that the amount of IL-12 production was significantly increased in both the axillary lymph nodes and spleen in the CJLP133 administration group compared to the control group (FIG. 18), and the production of IFN-γ was also significantly increased (FIG. 19). In addition, it was found that the increase in the amount of IFN-γ and IL-12 production was significantly higher than that of other known lactic acid bacteria.

In summarizing the experimental results of 1) to 5) above, CJLP55, CJLP56, CJLP133, and CJLP136 administration groups all show an effect of improving the symptoms of atopic dermatitis.In particular, the CJLP133 administration group has excellent effects, so the size of the axillary lymph nodes and the axillary lymph nodes are excellent. It was confirmed that the effect on atopic dermatitis was remarkably superior to that of conventional lactic acid bacteria in both cellular and molecular mechanisms, such as the number of cells in, immune cell infiltration and neuronal cell penetration into skin inflammatory tissues, and Th1 / Th2 cytokine balance.

Example 9: Lactobacillus Planta Room CJLP133 ( Lactobacillus plantarum CJLP133)

In order to apply the probiotics Lactobacillus plantarum CJLP133 (Lactobacillus plantarum CJLP133) identified in Example 1 as a raw material for pharmaceuticals, foods, feeds, feed additives, or cosmetics, mass production and freeze-drying were made to probiotics.

For the production of bacteria, the cells were incubated at 37° C. for about 18 hours while adjusting the pH to 6.0 using a 25% NaOH solution in MRS liquid medium (Difco), followed by centrifugation to recover the cells. The recovered cells were frozen at -40°C using 5% dextrin and 10% skim milk as a cryoprotectant, and then dried at 37°C with a mixer to pulverize. The powdered live cells were packaged in a sealed aluminum pouch after mixing with an appropriate amount of excipients such as glucose, lactose, and skim milk for storage to match the target number of bacteria.

The probiotic prepared in this way is used as a probiotic for feed by mixing with grain powder used as a raw material for feed, or as a probiotic for pharmaceuticals such as tablets and capsules, health functional foods by mixing carriers or additives, or raw materials used in cosmetics. By mixing a certain amount, it can be used in various fields such as pharmaceuticals, foods, feeds, cosmetics, etc. according to a conventional method in the art.

Korea Research Institute of Bioscience and Biotechnology KCTC11403BP 20081016

<110> CJ Cheiljedang Corporation <120> Novel Lactobacillus plantarum and compositions comprising the same <130> pn084437 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1486 <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 cgagcgcagg 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 tcgtaacaag tatgccgttg cccccc 1486

Claims (4)

Lactobacillus plantarum CJLP133 ( Lactobacillus plantarum CJLP133) KCTC 11403BP. A composition for a health functional food comprising Lactobacillus plantarum CJLP133 KCTC 11403BP. A composition for feed or feed additives comprising Lactobacillus plantarum CJLP133 KCTC 11403BP. A composition for cosmetics comprising Lactobacillus plantarum CJLP133 KCTC 11403BP.

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