WO2019103198A1

WO2019103198A1 - Lactobacillus fermentum kbl 375 strain and use thereof

Info

Publication number: WO2019103198A1
Application number: PCT/KR2017/013498
Authority: WO
Inventors: 고광표; 김운기; 이준철; 이진우; 장유진; 고우리; 이기욱
Original assignee: 주식회사 고바이오랩
Priority date: 2017-11-24
Filing date: 2017-11-24
Publication date: 2019-05-31
Also published as: KR20190060271A; KR102024883B1

Abstract

The present invention relates to a Lactobacillus fermentum KBL 375 strain, which is a novel probiotic strain derived from the human gastrointestinal tract, and a use of the strain in the improvement of gut health and the treatment or prevention of enteropathy. The Lactobacillus fermentum KBL 375 strain according to the present invention has an excellent immunoregulatory function and a remarkably enhancing effect on the tight junction of the gut wall and suppresses enteritis-induced weight loss and colon length decrease to exert a therapeutic effect on enteritis, finding useful applications as a probiotic material for improving gut health functions.

Description

Lactobacillus fermentum KBL 375 strain and its uses

The present invention relates to a lactobacillus fermentum KBL 375 strain and a use thereof, and more particularly to a lactobacillus fermentum KBL 375 strain which is a novel probiotic derived from a human gastrointestinal tract, a strain of the strain, a culture of the strain A composition for health functional food for improving intestinal health, and a pharmaceutical composition for the treatment or prevention of intestinal diseases, which contain an effective amount of at least one member selected from the group consisting of the above-mentioned disruption of said strain and the extract of said strain.

Probiotics refer to microorganisms having antimicrobial activity and enzymatic activity that aid intestinal microbial balance and products produced by the microorganisms. In addition, probiotics are defined as live cells in the form of single or complex strains that are supplied to humans or animals in the form of dried cells or fermented products to improve intestinal microflora. The characteristics that probiotics should possess are those that survive on the way to the human intestine as a habitat, non-pathogenic, non-toxic, and enteric. Furthermore, they should maintain viability and activity prior to consumption in the transfer medium, be sensitive to antibiotics used to prevent infection, and not have plasmids resistant to antibiotics. In addition, resistance to acids, enzymes and bile in the intestinal environment should be established.

These probiotics include Bacillus sp . , Lactobacillus sp . Which produces lactic acid, amylase, protease, lipase, cellulase and phosphatase, Photosynthetic bacteria that use substances (ammonia, hydrogen sulfide, amines, etc.) in the metabolic process to prevent odor induction are examples.

In particular, Bacillus and Lactobacillus species are known as very useful probiotics because of the existence of various strains producing various antimicrobial substances. These lactic acid bacteria produce an antimicrobial peptide called bacteriocin, which has an antimicrobial mechanism that is not related to the antibiotic resistance mechanism. Bacteriocin has polymorphic characteristics with remarkable differences in molecular weight, biochemical properties, antimicrobial range and mechanism to the host. Klaenhammer is defined as a protein or protein complex with direct antimicrobial activity against bacteriophage-producing bacteria and closely related species (Klaenhammer, TR. Biochimie 70: 337-379, 1998).

IBS, on the other hand, is characterized by abdominal pain and / or discomfort associated with altered bowel movements or bowel habits, and these symptoms are not explained by structural or biochemical abnormalities. The feeling of urgency, abdominal distension and incomplete bowel movement are also common symptoms of IBS. Thus, the disease is classified as a functional gastrointestinal disorder including functional bloating, non-atrial chest pain, non-ulcer dyspepsia, and chronic constipation or diarrhea (Longstreth GH et al., 2006). In particular, IBS has a profound effect on morbidity and quality of life, beyond abdominal pain and discomfort, since related symptoms act on both patient well-being and normal functional aspects (Dean BB et al., 2005).

Significant research is being conducted in the field of drug development to treat IBS. In this regard, various antidepressants have been used in the public, although their efficacy in clinical trials is moderate and its clinical utility is limited due to significant side effects. Serotonergic agents have also proved effective against overall IBS symptoms. However, due to recent safety concerns, its application has been limited in many ways, and interest in developing new therapeutic agents for IBS has been greatly amplified.

WO 96/29083 and EP 554418 disclose Lactobacillus plantarum 299v (DSM 6595) and Lactobacillus casei ssp. Lactobacillus casei ssp . rhamnosus ) 271 (DSM 6594). EP 415941 discloses a process for preparing a nutritional composition which comprises treating an oat gruel with an enzyme before mixing with lactobacilli. U.S. Patent No. 7195906 discloses a Bifidobacterium strain isolated from a human gastrointestinal tract that has been resected and cleansed to treat inflammatory diseases, particularly gastrointestinal inflammatory actions such as IBD and IBS.

However, strains showing an excellent effect for improving intestinal health including irritable bowel syndrome have not yet been found, and researches for finding strains showing the above effects have continued at a number of research institutes.

Therefore, the inventors of the present invention focused on the fact that the health promotion effect of probiotics is strain-specific rather than general characteristics of genus and species (Report of a joint FAO / WHO working group on drafting guidelines the evaluation of probiotics in food, London Ontario, Canada, 2002), by screening various strains of the human digestive tract and vagina derived was identified a novel strain outstanding have immunomodulatory effects, the intestinal health improvement of the strains in vitro and all in vivo, thereby completing the present invention.

Prior art literature

[Patent Literature]

1. WO 96/29083

2. EP 554418

3. EP 415941

4. US 7195906

5. GB 10-2012-7021713

[Non-Patent Document]

L Laval et al. Lactobacillus rhamnosus CNCM I-3690 and the commensal bacterium F aecalibacterium prausnitzii A2-165 exhibit similar protective effects to induced barrier hyper-permeability in mice. Gut Microbes, Vol 6, Issue 1, p.1

발명의 요약SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel strain which can improve intestinal health and be utilized for the treatment or prevention of intestinal diseases.

To achieve the above object, the present invention provides a lactobacillus perfume KBL 375 (Accession No. KCTC 13381BP) strain.

The present invention also provides a composition comprising an effective amount of at least one member selected from the group consisting of a bacterium of Lactobacillus perfumant KBL 375 (Accession No. KCTC 13381BP), a culture of said strain, a lysate of said strain and an extract of said strain to provide.

The present invention also provides a method for the treatment and / or prophylaxis of intestinal diseases comprising enterococci, which comprise an effective amount of at least one selected from the group consisting of strains of Lactobacillus perfumant KBL 375 (Accession No. KCTC 13381BP), the culture of the strain, the lysate of the strain, Or a pharmaceutically acceptable salt thereof.

1 shows the result of confirming the immunoregulatory function of Lactobacillus fermentum KBL 375 strain according to the present invention using THP-1 cell line.

FIG. 2 shows the result of confirming the immunoregulatory function of Lactobacillus fermentum KBL 375 strain according to the present invention using PBMC.

FIG. 3 shows the results of comparing the immunoregulatory function of the Lactobacillus perfumant KBL 375 strain according to the present invention with that of the other lactobacillus fermentum strains, KCTC 5467 strain and RAW 264.7 cell line.

FIG. 4 shows the result of comparing the tight junction synergistic effect of the Lactobacillus perfumant KBL 375 strain of the present invention with that of other Lactobacillus fermentum strains, SNUV 417 strain and Caco-2 cell line.

FIG. 5 shows the result of administering Lactobacillus perfumant KBL 375 strain of the present invention to enteritis-induced mouse models and observing changes in body weight and colon length.

6 is a graph comparing the growth rate of Lactobacillus perfumant KBL 375 strain of the present invention with that of Lactobacillus fermentum KCTC5049 and Lactobacillus fermentum KCTC5467, which are different Lactobacillus fermentum strains, from De Man, Rogosa and Sharpe (MRS) This is a result.

발명의 상세한 설명 및 바람직한 DETAILED DESCRIPTION OF THE INVENTION 구현예Example

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

In the present invention, a total of 23 microorganisms derived from human digestive tract and vagina were used to compare immunomodulatory effects. Among them, Lactobacillus perfumant KBL 375 (Accession No. KCTC 13381BP) showed particularly excellent effects on immune regulation As a result of analysis of 16s rDNA of the strain, it was confirmed that the strain was a novel strain which was not known yet.

Accordingly, the present invention relates to a strain of lactobacillus fermentum KBL 375 (Accession No. KCTC 13381BP) which is a novel probiotic in a viewpoint, characterized in that the strain comprises the 16s rDNA sequence shown in SEQ ID NO: 1 do.

&Lt; SEQ ID NO.

In the present invention, it was confirmed that Lactobacillus perfume KBL 375 strain had immunosuppressive effect as well as intimate tightening effect of intestinal wall, and when lactobacillus fermentum KBL 375 strain was administered to an enteritis-induced animal model, Weight loss and colon length reduction were significantly improved.

Therefore, the present invention relates to a method for the production of an effective amount of at least one compound selected from the group consisting of Lactobacillus perfume KBL 375 (Accession No. KCTC 13381BP) strain, a culture of the strain, a lysate of the strain and an extract of the strain &Lt; / RTI >

The composition may be a food or food additive composition, but the present invention is not limited thereto. Examples of foods that are effective for improving intestinal health or preventing intestinal diseases, for example, main ingredients, additives, food additives, Or as a functional beverage.

The term " food " means a natural product or a processed product containing one or more nutrients, preferably a state of being able to be eaten directly through a certain degree of processing, Additives, health functional foods and functional beverages.

Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, gums, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food may include special nutritive foods (e.g., crude oil, spirits, baby food, etc.), meat products, fish meat products, tofu, mackerel, noodles (Such as soy sauce, soybean paste, kochujang, mixed potatoes), sauces, confectionery (eg, snacks), candies, chocolate, gums, ice cream, milk products (eg, fermented milk, cheese, But are not limited to, pickled foods (various kinds of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable beverages, beverages, fermented beverages and the like) and natural seasonings (e.g. The food, beverage or food additive may be prepared by a conventional production method.

The above-mentioned health functional food refers to a food group to which the function of the food is added to a specific purpose by physical, biochemical, and biotechnological techniques, and to control the bio-defense rhythm of the food composition, Quot; means a food which is processed by being designed so as to sufficiently express the body controlling function related to the living body. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, the functional beverage refers to a generic term for eliminating thirst or for enjoying a taste, and includes a composition for improving or preventing symptoms of intestinal diseases as an essential ingredient at a specified ratio, And may contain various flavoring agents or natural carbohydrates as additional ingredients such as ordinary beverages.

Further, in addition to the above-described foods, the food containing the composition for food for improving or preventing symptoms of intestinal diseases according to the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, And carbonating agents used in fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, And these components can be used independently or in combination.

In the food containing the food composition of the present invention, the amount of the composition according to the present invention may be 0.001% by weight to 100% by weight, preferably 1% by weight to 99% by weight, And may be contained in a ratio of 0.001 g to 10 g, preferably 0.01 g to 1 g, based on 100 ml in the case of beverages. However, in the case of long-term intake for health and hygiene purposes or for health control purposes May be less than the above range, and since the active ingredient has no problem in terms of safety, it can be used in an amount of more than the above range, so it is not limited to the above range.

The food composition of the present invention may be prepared in the form of a composition suitable for human or animal ingestion, or the Lactobacillus perfume KBL 375 strain may be added to an independent or acceptable carrier. That is, it can be added to foods that do not contain other probiotic bacteria and foods that already contain some probiotic bacteria. For example, in the production of the food of the present invention, other microorganisms that can be used in combination with the strain of the present invention are suitable for human or animal ingestion and can inhibit pathogenic harmful bacteria upon ingestion or improve the microorganism balance in mammalian gut Those having probiotic activity, and are not particularly limited. An example of such a probiotic microorganism is Saccharomyces in MRS (Saccharomyces), Candida (Candida), blood teeth (Pichia) and sat rulrop cis yeast (yeast), Aspergillus (Aspergillus) containing (Torulopsis), Rhizopus Perth ( Rhizopus), myuko (Mucor), penicillin (Penicillium) fungi and Lactobacillus bacteria (Lactobacillus), Bifidobacterium (Bifidobacterium), current Kono Stock (Leuconostoc), Lactococcus (Lactococcus), Bacillus (Bacillus), such as, Streptococcus Bacteria belonging to the genera Streptococcus , Propionibacterium , Enterococcus , Pediococcus , and the like. Specific examples of suitable probiotic microorganisms include, but are not limited to, Saccharomyces cerevisiae , Bacillus coagulans , Bacillus licheniformis , Bacillus subtilis , Bifidobacterium bifidum), Bifidobacterium Infante Tees (Bifidobacterium infantis), Bifidobacterium rongeom (Bifidobacterium longum), Enterococcus passive help (Enterococcus faecium), Enterococcus faecalis (Enterococcus faecalis), Lactobacillus ash also filler's (Lactobacillus acidophilus, Lactobacillus alimentarius, Lactobacillus casei , Lactobacillus curvatus , Lactobacillus delbruckii , Lactobacillus johnsonii , Lactobacillus spp ., Lactobacillus farciminus , Lactobacillus gasseri , Lactobacillus helveticus, Lactobacillus rhamnosus , Lactobacillus reuteri , Lactobacillus saccharomyces, ( Lactobacillus sakei ), Lactococcus lactis lactis , Pediococcus acidilactici , and the like. Preferably, the effect of the present invention can further be enhanced by additionally incorporating a probiotic microorganism mixed bacterium having an excellent probiotic activity and an excellent immunity activity and anticancer activity in the food composition of the present invention. Examples of carriers which can be used in the food composition of the present invention include extender, high fiber additive, encapsulating agent, lipid and the like, and examples of such carriers are well known in the art. The Lactobacillus perfume KBL 375 of the present invention may be in lyophilized or encapsulated form or in the form of a culture suspension or a dry powder.

The composition of the present invention may also be provided in the form of an animal feed additive containing the strain or animal feed containing it.

The animal feed additive of the present invention may be in the form of a dry or liquid preparation, and may further contain other non-pathogenic microorganisms other than Lactobacillus perfume KBL 375. Examples of microorganisms which can be added include lactic acid bacteria such as Bacillus subtilis capable of producing protease, lipolytic enzyme and glycosyltransferase, lactose with physiological activity and organic decomposition in anaerobic conditions such as bovine Lactobacillus strains, such as Aspergillus oryzae (Slyter, LLJ Animal Sci. 1976, 43), which increase the body weight of livestock and increase milk yield and increase digestion and uptake of feed. 910-926) and yeasts such as Saccharomyces cerevisiae (Johnson, D. E et al. J. Anim. Sci., 1983, 56, 735-739; Williams, PEV et al, 1990, 211) may be used.

The animal feed additive of the present invention may further comprise at least one enzyme preparation in addition to the above-mentioned Lactobacillus perfume KBL 375. The added enzyme preparation can be either dry or liquid. Enzyme agents include lipase such as lipase, phytase which decomposes phytic acid to make phosphate and inositol phosphate, Amylase which is an enzyme which hydrolyzes an α-1,4-glycoside bond contained in glycogen and the like, phosphatase which is an enzyme which hydrolyzes an organic phosphate ester, cellulose which is an enzyme which hydrolyzes an organic phosphate ester, Carboxymethylcellulase which degrades xylose, xylase which degrades xylose, maltase which hydrolyzes maltose into glucose with glucose and maltase which hydrolyzes maltose into glucose Sugar-producing enzymes such as invertase and the like which hydrolyze saccharose to produce a glucose-fructose mixture can be used.

The lactobacillus fermentum KBL 375 strain of the present invention can be used as an animal feed additive in various feedstuffs such as peanut, pea, beet, pulp, grain by-products, animal powder, fish meal, And they can be used without limitation, either unprocessed or processed. The processing is not necessarily limited to this. For example, a process in which a feed material is filled with a constant outlet under pressure in the state of being filled with feed, and in the case of a protein, extrusion is used in which denaturation and availability are increased . Extrusion is advantageous in that it modifies the protein through heat treatment and destroys the antioxidant factor. In the case of soy protein, it is possible to improve protein digestibility through extrusion and to inactivate antitrophic factors such as trypsin inhibitor, which is one of protease inhibitors present in soybean, And the nutritional value of soy protein can be increased.

In another aspect, the present invention provides an effective amount of at least one selected from the group consisting of Lactobacillus fermentum KBL 375 (Deposit No. KCTC 13381BP) strain, a culture of the strain, a lysate of the strain and an extract of the strain And to a pharmaceutical composition for the treatment or prevention of intestinal diseases.

The pharmaceutical composition of the present invention may be provided as a composition that can be combined with cells of a live cell, a form of a dry strain, a culture of the strain, a lysate of the strain, or a pharmaceutically acceptable carrier or medium thereof. The carrier or medium employed may be selected from the group consisting of a solvent, a dispersing agent, a coating, an absorption promoting agent, a controlled release agent (i. E., Sustained release), and one or more inert excipients (starch, polyol, granule, microfine cellulose, microcrystalline cellulose (Including, for example, cell peers, Celphere beads (registered trademark)), diluents, lubricants, binders, disintegrants, and the like. If desired, the tablet formulations of the disclosed compositions may be coated by standard aqueous or non-aqueous techniques. Examples of excipients, fillers, disintegrants, lubricants, antimicrobial agents, and coatings include, but are not limited to, pharmaceutically acceptable carriers and excipients for use as pharmaceutically acceptable inert carriers and examples of such additional ingredients It is not.

In the present invention, the enteric disease is selected from the group consisting of abdominal distension, abdominal discomfort, infectious diarrhea caused by pathogenic microorganisms, gastroenteritis, inflammatory bowel disease, neurodegenerative inflammatory syndrome, irritable bowel syndrome, small intestinal microorganisms, And includes diseases caused by normal barrier function impairment of the intestines.

Inflammatory Bowel Disease (IBD) refers to a group of diseases including Crohn's disease, intestinal lesions associated with Behcet's disease, ulcerative colitis, haemorrhoidal rectal ulcer, and ileocystitis, including Crohn's disease and ulcerative colitis. Ulcerative colitis affects only the large intestine. Inflammation and ulcers in ulcerative colitis are limited to the mucosal and submucosal layers, the two innermost layers of the four layers of the colon. In Crohn's disease, inflammation and ulcers can spread through all layers of the barrier in both the small intestine and the large intestine.

Irritable Bowel Syndrome (IBS) is a chronic disease that accompanies abdominal discomfort and abdominal pain such as abdominal bloating, which is repeated for a long time without any underlying cause, accompanied by diarrhea and constipation, and it causes mental factors or stress It is also deteriorated by the social environment of

Refers to reversing, alleviating, inhibiting, or preventing the disease or condition to which the term applies, or one or more symptoms of the disease or disorder, unless otherwise stated And the term " treatment " used in the present invention refers to an action to be performed when " treating " is defined as described above. The therapeutic or therapeutic treatment of intestinal disorders in mammals thus may include one or more of the following:

(1) inhibiting the growth of enteric disease, i.e., inhibiting its development,

(2) prevent the spread of intestinal diseases,

(3) relieve bowel disease.

(4) preventing recurrence of bowel disease, and

(5) palliating symptoms of bowel disease

The composition for the prevention or treatment of intestinal diseases according to the present invention may comprise a pharmaceutically effective amount of Lactobacillus perfumant KBL 375 (Accession No. KCTC 13381BP) alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents .

In the present invention, the term " effective amount (or an effective amount) " means an amount which is sufficiently high to deliver a desired effect but which is sufficiently small to sufficiently prevent serious side effects within the medical judgment range. The amount of the microorganism to be administered into the body by the composition of the present invention can be appropriately adjusted in consideration of the route of administration and the subject to be administered.

The composition of the present invention may be administered to the subject once or more daily. Unit dose refers to units physically discrete suitable for unit administration for human subjects and other mammals and each unit contains a suitable pharmaceutical carrier and contains a predetermined amount of the microorganism of the invention which exhibits a therapeutic effect . The dosage unit for oral administration to an adult patient preferably contains 0.001 g or more of the microorganism of the present invention, and the oral dose of the composition of the present invention is 0.001 to 10 g, preferably 0.01 to 5 g, at one time. The pharmaceutically effective amount of the microorganism of the present invention is 0.01 to 10 g / day. However, the dosage varies depending on the severity of the enteric disease of the patient and the microorganism and auxiliary active ingredient used. In addition, the total daily dose may be divided into several doses and administered sequentially as necessary. Thus, the dosage ranges do not limit the scope of the invention in any way.

The term " pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to humans.

The compositions of the present invention may be formulated using methods known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatine capsules, sterile injectable solutions, sterile powders. In addition, the composition for preventing or treating intestinal diseases according to the present invention may be administered through various routes including oral, transdermal, subcutaneous, intravenous, or muscular, and the dose of the active ingredient may vary depending on the administration route, The composition for preventing or treating gastrointestinal diseases according to the present invention may be appropriately selected according to various factors such as body weight and the severity of the patient and the composition for preventing or treating gastrointestinal diseases according to the present invention may be used in combination with a known compound having an effect of preventing, Lt; / RTI >

The pharmaceutical composition of the present invention can be provided particularly as an enteric coated preparation as an oral unit dosage form. As used herein, " enteric coating " includes all known pharmaceutically acceptable coatings which are not degraded by stomach acid to maintain the coating, but which are sufficiently degraded in the small intestine so that the active ingredient can be released into the small intestine do. The " enteric coating " of the present invention remains intact for at least 2 hours when contacting an artificial juice, such as a HCl solution at pH 1, at 36 ° C to 38 ° C, and is preferably subsequently maintained at a pH of 6.8, such as KH 2 PO 4 buffer Refers to a coating that decomposes within 30 minutes of the juice.

The enteric coating of the present invention is coated on one core in an amount of about 16 to 30, preferably 16 to 20 or 25 mg or less. When the thickness of the enteric coating of the present invention is 5 to 100 mu m, preferably 20 to 80 mu m, satisfactory results are obtained as an enteric coating. Materials for enteric coatings are appropriately selected from known polymeric materials. Suitable polymeric materials are described in numerous publications (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd ed., 1986, pp. 365-373; H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359 Hager Handbuchter pharmazeutischen Praxis, 4th Edition, Vol.7, pp. 739-742, and 766-778, (Springer Verlag, 1971); and Remington's Pharmaceutical Sciences, 13th Edition, pp. 1689-1661 (Mack Publ. , 1970), and include cellulose ester derivatives, cellulose ethers, methyl acrylate copolymers of acrylic resins, and copolymers of maleic acid and phthalic acid derivatives.

The enteric coating of the present invention can be prepared using conventional enteric coating methods in which an enteric coating solution is sprayed onto the core. Suitable solvents for use in enteric coating processes include alcohols such as ethanol, ketones such as acetone, halogenated hydrocarbon solvents such as dichloromethane (CH2Cl2), and mixed solvents of these solvents may also be used. A softener such as di (di) n-butyl phthalate or triacetin is added to the coating solution in a ratio of from about 0.05 to about 0.3 (coating material to softener) per coat. It is appropriate to carry out the spraying process continuously and it is possible to adjust the spraying amount in consideration of the conditions of the coating. The spraying pressure can be varied and generally satisfactory results are obtained with spray pressures of from about 1 to about 1.5 bar.

In another aspect, the present invention provides the use of the strain or composition for use in the prevention or treatment of intestinal diseases and the use of the strain or composition for the preparation of a therapeutic agent for intestinal diseases.

In the present invention, the term " prevention " relates to averting, delaying, impeding or hindering the disease.

In the present invention, the term " treatment " relates to caring for a subject afflicted with a disease in order to ameliorate, cure, or reduce the symptoms of the disease or to reduce or stop the progression of the disease.

In another aspect, the present invention provides a method for preventing or treating enteric diseases, comprising the step of administering a pharmaceutically effective amount of the above strain or composition to a subject in need of prevention or treatment of bowel disease or improvement of intestinal health.

Since the pharmaceutical composition and the administration method used in the intestinal disease prevention or treatment method have been described above, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

On the other hand, the individual to which the composition for preventing or treating bowel disease can be administered includes all animals. For example, it may be an animal such as a dog, a cat, or a mouse.

[Example]

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Screening of Probiotic Strains Having Immune Regulation

A THP-1 cell line, a leukemic mononuclear cell, and a peripheral blood mononuclear cell (PBMC) were screened for a probiotic strain having an immunoregulatory function. A strain derived from a human gut or vagina was added to each of the above two cell lines to a ratio of 1: 100, and inflammation of the cytokine IL-6, which is a main index of the inflammatory response, (IL-10 / IL-6) of the cytokine IL-10, which is the main indicator of the regulation of IL-10. For screening, two kinds of Lactobacillus gasseri , one Lactobacillus reuteri , five Lactobacillus rhamnosus , two Lactobacillus fermentum , and two Lactobacillus paracase , (Lactobacillus paracasei) 4 species, Lactobacillus salivarius (Lactobacillus salivarius) 4 species, Lactobacillus Planta Room (Lactobacilus plantarum ), two Lactobacillus acidophilus strains and two Lactococcus lactis strains were used as a total of 23 strains

As a result of screening, Lactobacillus fermentum KBL 375 (KCTC 13381BP) showed an IL-10 / IL-6 value of 4.94 in the THP-1 cell line and an IL-10 / IL-6 value of 7.79 in the peripheral blood mononuclear cell Effect. Table 1 below shows the results of comparing IL-10 / IL-6 values between two Lactobacillus fermentum strains among the 23 probiotic strains used in the present screening.

As can be seen in Table 1, the Lactobacillus fermentum KBL 375 strain showed a very high IL-10 / IL-6 value, and the strain was predicted to be a highly immunomodulatory strain. Lactobacillus fermentum KBL 375 And further experiments were conducted using strains.

Example 2. In vitro test of lactobacillus fermentum KBL 375 strain

In order to examine the immunoregulatory effect of the Lactobacillus perfumant KBL 375 (accession number KCTC 13381BP) strain, when Lactobacillus perfumant KBL 375 strain was treated with two cell lines of THP-1 and RAW 264.7, , And the expression of marker genes of T cell differentiation when treated with Lactobacillus fermentum KBL 375 strain in PBMC.

First, the THP-1 cell line was dispensed into each well of a 24-well plate (1 × 10 ⁵ cells) and matured into macrophages. Lactobacillus fermentum KBL 375 strain was added 3 hours after the culture medium was replaced Each well was dispensed at a rate of 1 × 10 ⁷ cells on the basis of the viable cell count. As a control, LPS was treated at a concentration of 1 μg / ml instead of Lactobacillus fermentum KBL 375 strain under the same conditions to induce an inflammatory reaction. After 24 hours, the amount of each cytokine was measured according to the manufacturer's method using a BD cytometric Bead Array (CBA) human inflammation kit (Cat No. 551811).

1, IL-6, TNF, IL1-b, and IL-8 were more prominent in the THP-1 cell line than in the LPS-treated group, which was treated with Lactobacillus fermentum KBL 375 strain And it was observed to be significantly lower. Therefore, it was confirmed that Lactobacillus fermentum KBL 375 strain significantly inhibited the production of inflammatory cytokines in THP-1 cells.

Next, PBMC cells were dispensed into each well of a 24-well plate at 5 × 10 ⁵ cells, and then Lactobacillus perfumant KBL 375 was added at a concentration of 5 × 10 ⁶ cells per viable cell count. Escherichia coli O157 (H7 EC4115) isolated from the intestine was added to the wells at a concentration of 5 × 10 ⁶ cells per well or LPS was added at a concentration of 500 ng / ml. The PBMC cell group prepared under the above conditions was cultured for 5 days, and then the cells were collected. To determine the amount of gene expression, RNA was first extracted using an easy-spin ™ (total RNA) extraction kit (Intron) and cDNA was synthesized with a High Capacity RNA-to-cDNA Kit (Thermofisher). The synthesized cDNA was subjected to real-time PCR using a Rotor-Gene® Q (Qiagen) kit and a Rotor-Gene SYBR Green PCR kit (Qiagen) bet, Th2 marker gene, GATA3, Th17 marker gene RORrt, and Immune regulatory T cell (Treg cell) marker gene, FOXP3 mRNA expression. In order to measure the expression level of the B2M gene, a 5'-CCA GCA GAG AAT GGA AAG TC-3 '(reverse) 5'-GAT The degree of expression was measured using a primer having a sequence of GCT TCT TAC ATG TCT CG-3 '. The sequences of the primers for confirming each gene expression were forward 5'-CCC CAA GGA ATT GAC AGT TG-3 '(reverse) 5'-GGG AAA CTA AAG CTC ACA AAC-3' , 5'-CTG CAA TGC CTG TGG GCT C-3 '(reverse) 5'-GAC TGC AGG GAC TCT CGC T-3' for amplification of GATA3, 5'- AAG ACT CAT CGC CAA AGC AT-3 '(reverse) 5'-TCC ACA TGC TGG CTA CAC A-3', 5'-TCA AGC ACT GCC AGG CG-3 'reverse for FOXP3 amplification 5'-CAG GAG CCC TTG TCG GAT-3 'was used.

As a result, as shown in FIG. 2, the Lactobacillus perfume KBL375 strain significantly decreased the expression levels of T-bet, GATA3 and RORrt genes, which are effector cell markers of T cells, compared with E. coli, while Foxp3 mRNA was significantly increased.

In addition, RAW 264.7 cell lines were each subdivided into each well of a 24-well plate at a rate of 1 × 10 ⁵ cells. LPS was treated at a concentration of 2 ng / ml to induce an inflammatory reaction, and KCTC 5467, a representative subspecies of Lactobacillus fermentum Or Lactobacillus fermentum KBL 375 was added to each well at a rate of 1 × 10 ⁶ cells per well and then cultured for 24 hours to determine the concentration of TNF and IL-10 in an ELISA kit (Mouse TNF ELISA Set II No. 558534, BD OptEIATM), and Mouse IL-10 ELISA Set (Cat No. 555252, OptEIATM). Experimental groups not treated with Lactobacillus fermentum strains under the same experimental conditions were used as controls and the measured values were compared. As a result, as shown in Fig. 3, the IL-10 concentration of the anti-inflammatory cytokine was significantly increased in the cells treated with Lactobacillus perfumant KBL 375 strain as compared with that of the Lactobacillus perfumant KCTC 5467 strain of the control group and the comparative experiment group I could confirm. The ability of IL-10 / TNF-α, which is a ratio of inflammation-promoting cytokine TNF-α concentration, to immunoregulatory ability was evaluated.

As a result, in the cells treated with Lactobacillus perfumant KBL 375 strain, IL-10 / TNF-α value was significantly higher than that of the control and comparison experimental group Lactobacillus fermentum KCTC 5467, 375 strains have higher immunoregulatory and anti-inflammatory effects than the known lactobacillus fermentum strains.

Example 3. Tight junction tightening effect of Lactobacillus fermentum KBL 375 strain

The transepithelial / transendothelial electrical resistance (TEER) assay was performed to confirm the effect of Lactobacillus fermentum KBL 375 (KCTC 13381BP) on the intestinal wall adhesion tightening effect. For comparison, Lactobacillus fermentum SNUV 417 strain derived from human vagina was used as a comparative experimental group.

The Caco-2 (ATCC® HTB-37 ™) cell line for the TEER assay contains 20% FBS, 1% non-essential amino acids solution, 1% HEPES, 1.5% sodium bicarbonate solution, 50 μg / ml gentamicin, penicillin-streptomycin at 37 ° C and 5% CO ₂ .

Caco-2 cells were dispensed at a rate of 3 × 10 ⁴ cells / well in a 12-well plate, and the medium was changed every 2 days. Then, the medium was changed to a medium without FBS and antibiotics on the 6th day. On the 7th day, the plate was kept at room temperature for at least 20 minutes. The temperature of the plate was adjusted to room temperature, and the TEER was measured using a VOM resistance meter (World Precision Instruments, Sarasota, FL, USA). Then, the Lactobacillus fermentum KBL 375 strain of the present invention and the Lactobacillus fermentum SNUV 417 strain of the comparative experiment group were all washed with PBS and then released into MEM. FACS was used to perform live / dead cell counting and the strain was treated with Caco-2 cells so as to have a cell count of 3 × 10 ⁷ cells / well. The TEER value was measured at 12h and 24h after the strain was treated. In this case, the plate was kept at room temperature for at least 20 minutes to adjust the temperature of the plate to room temperature. After the measurement, TEER (Ω cm ² ) = (resistance (Ω) - background resistance (Ω)) × membrane area (cm ² )

Change in TEER (%) = TEER (Ω cm ² ) / initial TEER (Ω.cm ² ) × 100

As a result, as shown in FIG. 4, it was confirmed that the cells treated with Lactobacillus fermentum KBL 375 strain were significantly increased compared with the control and comparative experimental group, Lactobacillus perfumant SNUV 417 strain. This indicates that the Lactobacillus perfume KBL 375 strain has an effect of enhancing tight junctions in the intestinal wall and can be used for the treatment of irritable bowel syndrome.

Example 4 Lactobacillus buffer momentum enteritis relaxation effect verification of KBL strain 375 (in vivo test)

Lactobacillus fermentum KBL 375 (KCTC 13381BP) strain is in vivo And the effect of improving the bowel function was also confirmed. For this purpose, C57BL / 6 mice were divided into 10 clusters of each, and 2% DSS was dissolved in tap water, and drinking water for 9 days to induce enteritis. At the same time, 200 μl of PBS was orally administered to the control mice daily, and Lactobacillus fermentum KBL 375 strain was diluted to 2 × 10 ¹⁰ CFU / ml in the test mice and then orally administered in 200 μl each day. Thereafter, body weight changes of control and experimental mice were measured daily for 9 days during which DSS was induced and enteritis was induced. On the 9th day after feeding DSS, mice were autopsied and colon length was measured.

As a result, as shown in FIG. 5, on day 9 when DSS was supplied and enteritis was induced, the body weight of the mice was reduced to 87% of the initial body weight in the control group. However, the Lactobacillus fermentum KBL 375 strain , The weight of the mouse was 97% of the initial body weight, and the weight change was very slight. As a result of measuring the length of the colon, the length of the colon was found to be less than 80% of that of the control group on day 9 in the mice in which DSS was supplied and the enteritis was induced. In the experimental group of Lactobacillus perfumant KBL 375, The length was 90% of the control group, and it was confirmed that Lactobacillus perfumant KBL 375 can exert a beneficial effect on intestinal health.

Example 5. Productivity Assay of Lactobacillus fermentum KBL 375 strain

To confirm the productivity of Lactobacillus fermentum KBL 375 (KCTC 13381BP), two representative subspecies ( L. fermentum KCTC5049, L. fermentum KCTC5467) of Lactobacillus fermentum were registered in the BRC. As shown in FIG. 6, when Lactobacillus fermentum strains were cultured in De Man, Rogosa and Sharpe (MRS) medium, respectively, Lactobacillus perfumant KBL 375 strain had a faster growth rate than the other two Lactobacillus fermentum strains I could confirm. The specific growth rate of Lactobacillus fermentum KBL375 was 0.756, which was 1.1 to 1.3 times faster than that of other Lactobacillus fermentum (Table 2). Lactobacillus fermentum KBL 375 and Lactobacillus fermentum KCTC5467 were required to have a doubling time of 0.91 hours, 1.16 hours and 1.02 hours, respectively. (Table 2). Therefore, it was confirmed that Lactobacillus fermentum KBL 375 was superior to other Lactobacillus fermentum strains reported above by more than 10%.

Specific growth rate and doubling time of Lactobacillus fermentum KBL375, Lactobacillus fermentum KCTC5049 and Lactobacillus fermentum KCTC5467

StrainsStrains	Specific growth rate (h^-1)Specific growth rate (h ^-1 )	Doubling time (h)Doubling time (h)	CharacterCharacter
L. fermentum KBL375 L. fermentum KBL375	0.7560.756	0.9160.916	Isolated from feces adultIsolated from feces adult
L. fermentum KCTC5049 L. fermentum KCTC5049	0.6770.677	1.1631.163	Isolated from salivaIsolated from saliva
L. fermentum KCTC5467 L. fermentum KCTC5467	0.5960.596	1.0241.024	Isolated from intestineIsolated from intestine

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

The lactobacillus fermentum KBL 375 strain according to the present invention is excellent in immunomodulating function, excellent in reinforcing effect on intestinal wall adhesion, and is effective in treating enteritis by inhibiting weight loss and reduction of colon length due to enteritis , And as a probiotic material for improving intestinal health function. In addition, Lactobacillus fermentum KBL 375 strain according to the present invention has an advantage of being industrially advantageous because of its excellent productivity because of its rapid growth rate compared with similar strains during culturing.

I attached an electronic file.

[Amended by Rule 26, 10.05.2018]

Claims

Lactobacillus fermentum KBL 375 (Accession No. KCTC 13381BP) strain.
The strain of claim 1, wherein the strain has the 16s rDNA sequence shown in SEQ ID NO: 1.
A composition comprising an effective amount of at least one member selected from the group consisting of the bacterium of claim 1, the culture of the bacterium, the bacterium of the bacterium, and the extract of the bacterium.
4. The composition of claim 3, wherein the composition is a food or food additive composition.
The composition according to claim 4, wherein the food is a health functional food for improving bowel health.
A pharmaceutical composition for the treatment or prevention of intestinal diseases, which comprises an effective amount of at least one selected from the group consisting of the strains of the strain of claim 1, the culture of the strain, the lysate of the strain and the extract of the strain.
The method according to claim 6, wherein the bowel disease is selected from the group consisting of abdominal distension, abdominal discomfort, infectious diarrhea caused by pathogenic microorganisms, gastroenteritis, inflammatory bowel disease, neurodegenerative bowel syndrome, irritable bowel syndrome, small intestine microbial growth and intestinal diarrhea &Lt; / RTI >