KR102024883B1 - Lactobacillus Fermentum KBL 375 and Use Thereof - Google Patents

Abstract

The present invention relates to a novel probiotic Lactobacillus Permanent KBL 375 strain derived from the human gastrointestinal tract, and to the use of the strain for improving or improving gut health and treating or preventing intestinal diseases, and according to the present invention. 375 strains are excellent in immunomodulatory function, have an excellent strengthening effect on intestinal wall tight junction, and suppress the weight loss and colon length decrease by enteritis, and show the effect of treating enteritis. It can be useful as a tick material.

Description

Lactobacillus Fermentum KBL 375 and Use Thereof}

The present invention relates to a Lactobacillus Permantum KBL 375 strain and its use, and more particularly, a novel probiotic Lactobacillus Permantum KBL 375 strain, a cell of the strain, a culture of the strain. The present invention relates to a composition for health functional food for improving intestinal health, comprising an effective amount of at least one selected from the group consisting of crushed products of the strain and extracts of the strain, and a pharmaceutical composition for the treatment or prevention of intestinal diseases.

Probiotics refer to microorganisms with antimicrobial and enzymatic activity that help intestinal microbial balance and products produced by the microorganisms. In addition, probiotics are defined as live bacteria in the form of single or complex strains that are supplied to humans or animals in the form of dry cells or fermented products to improve the intestinal flora. The characteristics that probiotics must have are the intestines of the human intestine, and they must survive while going to the intestines, non-pathogenic, non-toxic. Furthermore, they must maintain viability and activity before being consumed in the delivered food, be sensitive to antibiotics used to prevent infection, and must not have antibiotic resistant plasmids. It must also be resistant to acids, enzymes and bile in the intestinal environment.

These probiotics include Bacillus sp . ( Lactobacillus sp . ), Which produce lactic acid, and Lactobacillus sp . , Which produce lactic acid and Lactobacillus sp . substances there may be mentioned (ammonia, hydrogen sulfide, amines, etc.), photosynthesis bacteria (photosynthetic bacteria) to prevent odors caused by the metabolic process, etc. for example.

Bacillus spp. And Lactobacillus spp., In particular, are known as very useful probiotics because of the presence of strains that produce a variety of antimicrobial agents. These lactic acid bacteria produce an antimicrobial peptide called bacteriocin, which has an antimicrobial mechanism that is not related to antibiotic resistance mechanisms. Bacteriocin has polymorphic features that vary considerably in molecular weight, biochemical properties, and antimicrobial range or mechanism for the host. Klaenhammer is defined as a protein or protein complex that has direct antimicrobial activity against species proximate to the bacteriocin-producing bacteria (Klaenhammer, TR. Biochimie 70: 337-379, 1998).

Irritable bowel syndrome (IBS), on the other hand, is a condition characterized by abdominal pain and / or discomfort associated with altered bowel behavior or bowel habits, which symptoms are not explained by structural or biochemical abnormalities. Feeling of urgency, bloating, and incomplete bowel movements are also common symptoms of IBS. Thus, the disease is classified as a functional gastrointestinal disorder, including diseases such as bloating, non-cardiac chest pain, non-ulcer dyspepsia and chronic constipation or diarrhea (Longstreth G. H. et al., 2006). In particular, IBS has a profound effect on morbidity and quality of life beyond abdominal pain and discomfort because related symptoms affect both patient well-being and normal functional aspects (Dean B. B. et al., 2005).

Enormous research is being conducted in the field of drug development to treat IBS. In this regard, various antidepressants have been used in public, although their efficacy in clinical trials is moderate and their clinical utility is limited due to significant side effects. Serotonergic agents have also demonstrated efficacy in the overall IBS symptoms. However, due to recent safety issues, 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. Ramno Versus (Lactobacillus casei ssp. Rhamnosus) 271 (DSM 6594) discloses the Lactobacillus strains of two or form a colony in a field, and the like. EP 415941 discloses a process for the preparation of a nutritional composition comprising treating an oat gruel with an enzyme prior to mixing with lactobacilli. U.S. Patent 7195906 discloses a Bifidobacterium strain isolated from an excised and washed human gastrointestinal tract to treat inflammatory diseases, particularly gastrointestinal inflammatory effects such as IBD and IBS.

However, no strain has yet been found to have an excellent effect on improving intestinal health, including irritable bowel syndrome, and studies to find strains showing such effects continue in many research institutes.

Accordingly, the present inventors have focused on the fact that the health promoting effect of probiotics is strain specific rather than general features of genus and species (Report of a joint FAO / WHO working group on drafting guidelines for 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 This invention was completed by confirming all in vivo.

1. WO 96/29083 2.EP 554418 3.EP 415941 4.US 7195906 5.KR 10-2012-7021713

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

It is an object of the present invention to provide novel strains that can be used for improving bowel health and for treating or preventing bowel disease.

In order to achieve the above object, the present invention provides a Lactobacillus permanentum KBL 375 (Accession No. KCTC 13381BP) strain.

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

The present invention also provides an intestinal disease containing an effective amount of at least one selected from the group consisting of Lactobacillus latent KBL 375 (Accession No. KCTC 13381BP) strain, culture of the strain, lysate of the strain and extract of the strain. It provides a pharmaceutical composition for the treatment or prevention of.

Lactobacillus Permanent KBL 375 strain according to the present invention is excellent in immunomodulatory function, excellent strengthening effect of close contact of the intestinal wall, and suppresses the weight loss and colon length reduction caused by enteritis to show the treatment of enteritis It can be usefully used as a probiotic material for improving intestinal health function. In addition, the Lactobacillus latent KBL 375 strain according to the present invention has the advantage that the growth rate is faster than the similar strain in the culturing process excellent productivity and suitable for industrial use.

1 is a result of confirming the immunomodulatory function of the Lactobacillus fermentum KBL 375 strain according to the present invention using a THP-1 cell line.
Figure 2 is a result of confirming the immunomodulatory function of the Lactobacillus fermentum KBL 375 strain according to the present invention using PBMC.
Figure 3 is a result of comparing the immunomodulatory function of the Lactobacillus Permantum KBL 375 strain according to the present invention in another Lactobacillus Permantum strain KCTC 5467 strain and RAW 264.7 cell line.
Figure 4 is a result of comparing the adhesion-strengthening strengthening effect of the Lactobacillus fermentum KBL 375 strain of the present invention in another Lactobacillus fermenton strain SNUV 417 strain and Caco-2 cell line.
5 is a result of administering the Lactobacillus latement KBL 375 strain of the present invention to a mouse model induced enteritis and observed the weight change and the change in colon length.
FIG. 6 is a comparison of the growth rate of the Lactobacillus latent KBL 375 strain of the present invention in other Lactobacillus latent strains, Lactobacillus latent KCTC5049 and Lactobacillus latent KCTC5467, and De Man, Rogosa and Sharpe (MRS) media. One result.

Unless defined otherwise, 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 strains of microorganisms derived from the human digestive tract and the vagina were used to compare the immunomodulatory effects, and among them, the Lactobacillus permanentment KBL 375 (Accession No. KCTC 13381BP) strain was observed to have a particularly excellent effect on immune regulation. As a result of analyzing 16s rDNA of the strain, it was confirmed that the strain is a novel strain that has not been known in the prior art.

Therefore, the present invention relates to a novel probiotic Lactobacillus latent KBL 375 (Accession No. KCTC 13381BP) strain in a consistent point, characterized in that the strain comprises a 16s rDNA sequence represented by SEQ ID NO: 1 below. do.

<SEQ ID NO 1>

In the present invention, it was also confirmed that the Lactobacillus Percentum KBL 375 strain had an immunomodulatory effect as well as the tight junctional strengthening effect of the intestinal wall, and when entertaining the Lactobacillus Percentum KBL 375 strain in the enteritis-induced animal model, It was found that weight loss and colon length decrease were significantly improved.

Therefore, the present invention contains an effective amount of at least one selected from the group consisting of Lactobacillus latent KBL 375 (Accession Number KCTC 13381BP) strain, culture of the strain, lysate of the strain and extract of the strain. It relates to a composition to be.

The composition may be a food or a composition for food addition, but is not limited thereto, and foods that are effective in improving intestinal health or preventing intestinal diseases, for example, main ingredients of food, minor ingredients, food additives, and health functional foods. Or it can be utilized easily as a functional drink.

The food means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through a certain processing step. It includes all additives, health foods and functional drinks.

Foods to which the food composition according to the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, functional foods, and the like. In addition, in the present invention, food includes special nutritional products (e.g., delicatessen, young, infant food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), breads, health supplements, seasonings. Foods (e.g. soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), candy, chocolates, gums, ice creams, dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, Pickled foods (various kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g. ramen soup, etc.) are not limited thereto. The food, beverage or food additives may be prepared by a conventional manufacturing method.

The health functional food refers to the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, or biotechnological techniques to act and express the function of the food for a specific purpose. It means a food that is designed and processed to express the body's regulation function to the living body sufficiently. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In the present invention, the functional drink is a generic term for drinking to quench thirst or to enjoy a taste, and other components other than the composition containing the composition for improving or preventing the symptoms of intestinal diseases as essential ingredients in the indicated ratios have special limitations. It may contain various flavors or natural carbohydrates and the like as an additional ingredient, as in normal beverages.

Furthermore, in addition to the above-mentioned foods containing food compositions for improving or preventing the symptoms of the bowel disease of the present invention, foods containing various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, such as colorants And fillers (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The components may be used independently or in combination.

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

The food composition of the present invention may be added to the Lactobacillus permanentum KBL 375 strain independently or in an acceptable carrier, or may be prepared in the form of a composition suitable for human or animal ingestion. That is, it can be used in addition to foods that do not contain other probiotic bacteria and foods that already contain some probiotic bacteria. For example, in the preparation of the food of the present invention, other microorganisms usable with the strain of the present invention are suitable for ingestion by humans or animals and can inhibit pathogenic harmful bacteria or improve microbial balance in the mammalian intestine upon ingestion. Those having probiotic activity 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 ( Streptococcus ), Gropionibacterium ( Propionibacterium ), Enterococcus , and bacteria belonging to the genus Pediococcus ( Pediococcus ). Specific examples of suitable probiotic microorganisms include Saccharomyces cerevisiae , Bacillus coagulans , Bacillus licheniformis , Bacillus subtilis , Bifidobacterium Bifidobacterium bifidum), Bifidobacterium Infante Tees (Bifidobacterium infantis), rongeom Bifidobacterium (Bifidobacterium longum), Enterococcus passive help (Enterococcus faecium ), Enterococcus faecalis , Lactobacillus acidophilus , Lactobacillus alimentarius , Lactobacillus casei , Lactobacillus casei , Lactobacillus curvatusus Lva , Lactobacillus delbruckii , Lactobacillus johnsonii , Lactobacillus farciminus , Lactobacillus gasseri , Lactobacillus helveticus , Lactobacillus Lactobacillus rhamnosus , Lactobacillus reuteri , Lactobacillus sakei , Lactococcus Lactococcus lactis ), Pediococcus acidilactici , and the like. Preferably, by further comprising a probiotic microbial mixed bacteria having excellent probiotic activity and excellent immune activity enhancement and anticancer activity in the food composition of the present invention can further enhance the effect. Examples of carriers that may be used in the food compositions of the present invention may be extenders, high fiber additives, encapsulating agents, lipids, and the like and examples of such carriers are well known in the art. The Lactobacillus permanent KBL 375 of the present invention may be in lyophilized or encapsulated form or in the form of a culture suspension or dry powder.

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

The additive for animal feed of the present invention may be in the form of a dry or liquid formulation, and may further include other non-pathogenic microorganisms in addition to the Lactobacillus permanentum KBL 375. Microorganisms that can be added include lactose, which have physiological activity and organic degradability under anaerobic conditions, such as Bacillus subtilis , which can produce proteolytic enzymes, lipolytic enzymes and sugar converting enzymes, and bovine stomach. bacilli (Lactobacillus) strains of fungi increase the weight of cattle, such as the Aspergillus duck, showing the effect of increasing the milk production of the milk to increase the absorption of digested food party (Aspergillus oryzae) (Slyter, LLJ Animal Sci.1976, 43. 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) and the like can be used.

The additive for animal feed of the present invention may further include one or more enzyme preparations in addition to the Lactobacillus permanentum KBL 375. Enzyme preparation can be either dry or liquid. Enzyme preparations include lipases such as lipases, phytases that break down phytic acid to form phosphates and inositol phosphates, starches and Amylase, an enzyme that hydrolyzes α-1,4-glycoside bonds in glycogen, and phosphatase, cellulose, which hydrolyzes organic phosphate esters Carboxymethylcellulase that breaks down xylase, xylase that breaks down xylose, maltase and maltase that hydrolyzes maltose into two molecules of glucose Sugar-producing enzymes such as invertase, which hydrolyzes saccharose to form a glucose-fructose mixture, may be used.

In the Lactobacillus fermentum KBL 375 strain of the present invention is used as an additive for animal feed, raw materials for feed include peanuts, peas, beets, pulp, grain by-products, animal intestine powder, fish meal powder, etc. They may be used, and they may be used unrestricted or processed without limitation. The process is not necessarily limited to this, for example, a process in which a feed material is compressed to a predetermined outlet under pressure in a state of filling, and in the case of protein, an extrusion is used to denature and increase usability. It is preferable. Extrusion has the advantages of denaturing proteins and destroying antienzymatic factors through heat treatment. In addition, in the case of soy protein, it is possible to improve the digestibility of the protein through extrusion and inactivate antinutrients such as trypsin inhibitor, one of the inhibitors of protease present in soybean, Increasing digestion can increase the nutritional value of soy protein.

In yet another aspect, the present invention contains an effective amount of at least one selected from the group consisting of Lactobacillus latent KBL 375 (Accession No. KCTC 13381BP) strain, culture of the strain, lysate of the strain and extract of the strain. It relates to a pharmaceutical composition for the treatment or prevention of bowel disease.

The pharmaceutical composition of the present invention may be provided in a composition which can be combined with the cells of live cells, in the form of dry strains, cultures of strains, lysates of strains or pharmaceutically acceptable carriers or media thereof. Carriers or media used include solvents, dispersants, coatings, absorption accelerators, controlled release agents (ie, sustained release), and one or more inert excipients (starch, polyols, granules, microfine cellulose, microcrystalline cellulose). (Eg, including celpiers, celphere beads, diluents, lubricants, binders, disintegrants, etc.) If desired, tablet formulations of the disclosed compositions may be standard aqueous or It may also be coated by a non-aqueous technique.Excipients for use as pharmaceutically acceptable carriers and pharmaceutically acceptable inert carriers and examples of such additional ingredients include binders, fillers, disintegrants, lubricants, antimicrobial agents And coatings, but are not limited to these.

In the present invention, the bowel disease is in the group consisting of abdominal bloating, abdominal discomfort, infectious diarrhea caused by pathogenic microorganisms, gastroenteritis, inflammatory bowel disease, neuro enteritis syndrome, irritable bowel syndrome, small intestinal microflora hypergrowth and intestinal diarrhea It may be characterized by being selected, and include diseases caused by impaired normal barrier function of the intestine.

Inflammatory bowel disease (IBD) includes Crohn's disease, intestinal lesions associated with Behcet's disease, ulcerative colitis, hemorrhagic rectal ulcer and ileal cystitis, and refers to a group of diseases including Crohn's disease and ulcerative colitis. Ulcerative colitis affects only the large intestine. Inflammation and ulcers in ulcerative colitis are limited to the two innermost layers of the four layers of the large intestine, the mucosa and submucosa. In Crohn's disease, inflammation and ulcers can spread through all layers of the barrier in both the small and large intestine.

Irritable bowel syndrome, on the other hand, is a chronic disease that causes abdominal discomfort and abdominal pain, such as diarrhea and constipation, and changes in bowel habits such as diarrhea and constipation. It is often worsened by the social environment.

The term 'treating', unless stated otherwise, 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 condition. As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above. Thus, the treatment or therapy of intestinal disease in a mammal may comprise one or more of the following:

(1) inhibits the growth of intestinal diseases, that is, inhibits their development,

(2) prevent the spread of intestinal disease,

(3) relieve intestinal diseases.

(4) prevent recurrence of intestinal disease, and

(5) to relieve symptoms of bowel disease

The composition for preventing or treating intestinal diseases according to the present invention comprises a pharmaceutically effective amount of Lactobacillus Permanum KBL 375 (Accession No. KCTC 13381BP) strain alone or includes one or more pharmaceutically acceptable carriers, excipients or diluents. It may include.

In the present invention, the term "effective amount (or effective amount)" means an amount sufficient to deliver the desired effect but small enough to sufficiently prevent serious side effects within the medical judgment. The amount of 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 of administration.

The composition of the present invention may be administered to the subject individual at least once daily. Unit dosage refers to units that are physically separated, suitable for unit administration for human subjects and other mammals, each unit comprising a suitable pharmaceutical carrier and containing a predetermined amount of the microorganism of the present invention having a therapeutic effect. . The dosage unit for oral administration of an adult patient preferably contains at least 0.001 g of the microorganism of the present invention, and the oral dosage of the composition of the present invention is 0.001 to 10 g, preferably 0.01 to 5 g at a time. The pharmaceutically effective amount of the microorganisms of the present invention is 0.01 to 10 g / day. However, the dosage will vary depending on the severity of the patient's intestinal disease and the microorganisms and auxiliary active ingredients used. In addition, the total daily dose may be divided into several times and administered continuously as needed. Thus, the dosage range does not limit the scope of the invention in any way.

In addition, "pharmaceutically acceptable" as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like when administered to a human.

Compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin 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 intramuscular, and the dosage of the active ingredient is determined by the route of administration, age, sex, The composition for the prevention or treatment of gastrointestinal diseases according to the present invention may be appropriately selected according to various factors such as the weight and the severity of the patient, in parallel with known compounds having the effect of preventing, ameliorating or treating the symptoms of intestinal diseases. May be administered.

The pharmaceutical compositions of the present invention may in particular be provided as enteric coated enteric preparations as oral unit dosage forms. "Enteric coating" as used herein includes all known types of pharmaceutically acceptable coatings that are not degraded by gastric acid so that the coating is maintained but is sufficiently degraded in the small intestine to allow the active ingredient to be released into the small intestine. do. The "entertaining coating" of the present invention is maintained for at least 2 hours when contacting artificial gastric juice, such as HCl solution of pH 1 at 36 ℃ to 38 ℃, preferably after the artificial such as KH₂PO₄ buffer solution of pH 6.8 Refers to a coating that degrades within 30 minutes in enteric juice.

The enteric coating of the present invention is coated in one core in an amount of about 16 to 30, preferably 16 to 20 or 25 mg or less. The case where the thickness of the enteric coating of this invention is 5-100 micrometers, Preferably it is 20-80 micrometers shows a satisfactory result as an enteric coating. The material of the enteric coating is suitably selected from known polymeric materials. Suitable polymeric materials are described in a number of known publications (L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373; H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359 Hagers Handbuchder pharmazeutischen Praxis, 4th edition, Vol. 7, pp. 739-742, and 766-778, (Springer Verlag, 1971); and Remington's Pharmaceutical Sciences, 13th edition, pp. 1689-1691 (Mack Publ., Co. , 1970), and cellulose ester derivatives, cellulose ethers, methylacrylate copolymers of acrylic resins and copolymers of maleic and phthalic acid derivatives can be included therein.

The enteric coating of the present invention can be prepared using conventional enteric coating methods that spray the enteric coating solution onto the core. Suitable solvents used in the enteric coating process are alcohols such as ethanol, ketones such as acetone, halogenated hydrocarbon solvents such as dichloromethane (CH ₂ Cl ₂ ) and mixed solvents of these solvents may be used. Softeners such as di-n-butylphthalate or triacetin are added to the coating solution at a ratio of 1 to about 0.05 to about 0.3 (coating material to softener). It is appropriate to carry out the spraying process continuously and it is possible to adjust the spraying amount taking into account the conditions of the coating. Spray pressure can be varied and generally satisfactory results are obtained with spray pressures of about 1 to about 1.5 bar.

In another aspect the present invention provides the use of said strain or composition for use in the prevention or treatment of intestinal disease and the use of said strain or composition for the preparation of an agent for treating bowel disease.

In the present invention, the term 'prevention' relates to preventing, delaying, impeding or hindering the disease.

In the present invention, the term 'treatment' relates to caring for a diseased subject 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 intestinal disease, comprising administering a pharmaceutically effective amount of the strain or composition to an individual in need of preventing or treating intestinal disease or improving the health of the intestine.

Since the pharmaceutical composition and the method of administration used in the method of preventing or treating intestinal diseases have been described above, the common contents between the two are omitted in order to avoid excessive complexity of the present specification.

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

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Screening of Probiotic Strains with Immunomodulatory Function

Probiotic strains with immunomodulatory functions were screened using leukemia monocytes, THP-1 cell line and peripheral blood monocytes (PBMC). In each of the two cell lines, strains derived from human gut or vagina are dispensed so that the cell number is 1: 100, and inflammation of the cytokine IL-6, which is a major indicator of the inflammatory response, is distributed. The ratio of IL-10, a cytokine that is a major indicator of regulation, was measured (IL-10 / IL-6). Screening is Lactobacillus fern ( Lactobacillus gasseri ) 2 types , Lactobacillus ruteri ( Lactobacillus reuteri ) 1 type, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) 5 species , Lactobacillus fermentum , 2 species, Lactobacillus paracazei ( Lactobacillus paracasei ) Four, Lactobacillus salivarius ( Lactobacillus salivarius ) Four, Lactobacillus plantarum ( Lactobacilus plantarum ) Type 1, Lactobacillus ashdophyllus ( Lactobacillus acidophilus ) 2 kinds and Lactococcus lactis ( Lactococcus lactis ) Two species , a total of 23 strains were used

Screening revealed that the Lactobacillus latent KBL 375 (KCTC 13381BP) strain had very high immunomodulation with IL-10 / IL-6 value of 4.94 in THP-1 cell line and 7.79 IL-10 / IL-6 value in peripheral blood monocytes. The effect was shown. Table 1 below is a result of comparing IL-10 / IL-6 values between two Lactobacillus fermentum strains of the 23 probiotic strains used in the present screening.

Cell line Strain IL-10 / IL-6 THP-1 Lactobaciilus fermentum KBL 374 1.766582 Lactobaciilus fermentum KBL 375 4.941038 PBMC Lactobaciilus fermentum KBL 374 2.981526 Lactobaciilus fermentum KBL 375 7.7890503

As can be seen in Table 1, the Lactobacillus latent KBL 375 strain exhibited a very high IL-10 / IL-6 value, which predicted that the strain was very high in immunomodulatory function. KBL 375 strain was used for further experiments.

Example  2. Lactobacillus Permanent Validation of immunomodulatory effect of KBL 375 strain in vitro  test)

In order to verify the immunomodulatory effect by the Lactobacillus latent KBL 375 (Accession No. KCTC 13381BP) strain, THP-1 and RAW 264.7 cells were treated with Lactobacillus latent KBL 375 strains involved in immune regulation. The cytokine production ability and the expression of marker genes for T cell differentiation when treated with Lactobacillus latent KBL 375 strain in PBMC were confirmed.

First, THP-1 cell lines were dispensed into each well of a 24-well plate by 1 × 10 ⁵ cells and differentiated into mature macrophages, followed by a fresh replacement of the culture medium, and 3 hours later Lactobacillus fermentation. KBL 375 strain was dispensed into each well by 1 × 10 ⁷ cells based on viable cell count. As a control, Lactobacillus permanentment under the same conditions Instead of KBL 375 strain, LPS was treated at a concentration of 1 μg / ml to induce an inflammatory response. After 24 hours, the cultures were collected and measured for each cytokine according to the manufacturer's method using a BD cytometric bead array (CBA) human inflammation kit (Cat No. 551811).

As a result, as shown in FIG. 1, in the THP-1 cell line, Lactobacillus percentage The group treated with the KBL 375 strain showed significantly lower levels of inflammatory cytokines IL-6, TNF, IL1-b and IL-8 than those treated with LPS. Therefore, Lactobacillus Percentum It was confirmed that the KBL 375 strain has an effect of significantly inhibiting the production of inflammatory cytokines in THP-1 cells.

Next, PBMC cells were dispensed into each well of a 24-well plate by 5 × 10 ⁵ cells, and then Lactobacillus permanent KBL 375 strain was added to 5 × 10 ⁶ cells by viable cell count. As a control, Escherichia coli isolated from the intestine coli O157: H7 EC4115) was added to each well by 5 × 10 ⁶ cells based on viable cell count, or LPS was added at a concentration of 500ng / ml. PBMC cells experimental group prepared under the above conditions were obtained after culturing for 5 days to collect the cells. To determine the amount of gene expression, RNA was extracted using easy-spin ™ (DNA free) Total RNA Extraction Kit (Intron) and cDNA was synthesized using High Capacity RNA-to-cDNA Kit (Thermofisher). Using the Rotor-Gene® Q (Qiagen) equipment, the synthesized cDNA was subjected to real-time PCR using the Rotor-Gene SYBR Green PCR kit (Qiagen) according to the manufacturer's method, and T-, the effector cell marker gene of Th1. Expression of bet, Th2 marker gene GATA3, Th17 marker gene RORrt and Immune regulatory T cells (Treg cells) marker gene FOXP3 mRNA expression was measured. At this time, 5'-CCA GCA GAG AAT GGA AAG TC-3 '(reverse) 5'-GAT to measure the expression level of the B2M gene as an internal control to correct the relative gene expression between each experimental group. The expression level was measured using a primer having a sequence of GCT TCT TAC ATG TCT CG-3 ′. The sequence of the primer for confirming each gene expression is 5'-CCC CAA GGA ATT GAC AGT TG-3 '(reverse) 5'-GGG AAA CTA AAG CTC ACA AAC-3 ′ for amplification of T-bet. 5′-CTG CAA TGC CTG TGG GCT C-3 ′ (reverse) 5′-GAC TGC AGG GAC TCT CGC T-3 ′ for amplification of GATA3, 5′- for amplification of RORrt AAG ACT CAT CGC CAA AGC AT-3 ′ (reverse) 5′-TCC ACA TGC TGG CTA CAC A-3 ′, for amplification of FOXP3 5′-TCA AGC ACT GCC AGG CG-3 ′ (reverse) 5′-CAG GAG CCC TTG TCG GAT-3 ′ was used.

As a result, as shown in Figure 2, Lactobacillus Permanum KBL375 strain significantly lowered the expression of T-bet, GATA3, RORrt genes, T-effector cell markers compared to Escherichia coli, while Foxp3, a marker of Treg cells, mRNA was found to increase significantly.

In addition, each RAW 264.7 cell line was dispensed 1 × 10 ⁵ cells into each well of a 24-well plate, and treated with LPS at a concentration of 2 ng / ml to induce an inflammatory response, followed by KCTC 5467, a representative subspecies of Lactobacillus fermentum. Alternatively, the Lactobacillus permanent KBL 375 strain was further dispensed into each well by 1 × 10 ⁶ cells based on the number of viable cells and incubated together for 24 hours to determine the concentration of TNF and IL-10 in an ELISA kit (Mouse TNF ELISA Set II (Cat No. 558534, BD OptEIATM), Mouse IL-10 ELISA Set (Cat No. 555252, OptEIATM)) and measured according to the manufacturer's method. The experimental group was not treated with Lactobacillus permanent strain under the same experimental conditions, and the measured values were compared. As a result, as shown in FIG. 3, the anti-inflammatory cytokine IL-10 concentration was significantly increased in the cells treated with the Lactobacillus latent KBL 375 strain compared to the Lactobacillus latent KCTC 5467 strain, which was a control group and a comparative experiment group. I could confirm it. The immunomodulatory ability was determined by IL-10 / TNF-α, which is a ratio with the pro-inflammatory cytokine TNF-α concentration.

As a result, it was confirmed that IL-10 / TNF-α values significantly higher than those of the control and comparative experimental groups Lactobacillus permanent KCTC 5467 in cells treated with the Lactobacillus permanent KBL 375 strain, which was the Lactobacillus permanent KBL. 375 strains show higher immunomodulatory and anti-inflammatory effects compared to known Lactobacillus permentum strains.

Example  3. Lactobacillus Permanent Of KBL 375 strain Spectacular wall  Tight junction strengthening effect

To confirm the intestinal wall tight junction strengthening effect of the Lactobacillus fermentum KBL 375 (KCTC 13381BP) strain, a TEER (Transepithelial / transendothelial electrical resistance) assay was performed. For comparison, the Lactobacillus permanentment SNUV 417 strain derived from human vagina was used as a comparative experimental group.

Caco-2 (ATCC® HTB-37 ™) cell line for TEER assay, 20% FBS, 1% non-essential amino acids solution, 1% HEPES, 1.5% sodium bicarbonate solution, 50 μg / ml gentamicin, 10 U / ml MEM medium containing penicillin-streptomycin was incubated at 37 ° C. and 5% CO ₂ .

Caco-2 cells were dispensed in 3 wells 3 × 10 ⁴ cells per well in a 12 well plate and the medium was changed every other day. After 6 days, the cells were replaced with medium without FBS and antibiotics and cultured overnight. After the plate was placed at room temperature for at least 20 minutes on day 7, the temperature of the plate was adjusted to room temperature, and then the TEER was measured using a VOM resistance meter (World Precision Instruments, Sarasota, FL, USA) (based on 0h). After washing the medium of the Lactobacillus Permentum KBL 375 strain of the present invention and the Lactobacillus Permentum SNUV 417 strain of the comparative experimental group with PBS, it was released to MEM. Live / dead cell counting was performed using FACS to treat the strain to 3 × 10 ⁷ cells per well based on viable cell counts in Caco-2 cells. After the strain was treated, the TEER values were measured at 12 h and 24 h. 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, the following equation was applied after conversion to 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 Figure 4, it was confirmed that the cells treated with the Lactobacillus Permanum KBL 375 strain significantly increased compared to the Lactobacillus Permantum SNUV 417 strain of the control and comparative experiment groups. This suggests that the Lactobacillus latent KBL 375 strain has an effect of strengthening the tight junction of the intestinal wall and can be used for the treatment of irritable bowel syndrome.

Example  4. Lactobacillus Permanent Validation of Enteritis Mitigation Effect of KBL 375 Strain in vivo test)

Lactobacillus permanent KBL 375 (Accession No. KCTC 13381BP) strain is in vivo We also wanted to check whether the bowel function was improved in the stomach. To this end, C57BL / 6 mice were divided into groups of 10 animals, and 2% DSS was dissolved in tap water, followed by drinking with drinking water for 9 days to induce enteritis. At the same time, the control mice were orally administered with PBS 200 μl daily, and the experimental mice were diluted with Lactobacillus permanent KBL 375 strain to 2 × 10 ¹⁰ CFU / ml in PBS and then orally administered 200 μl daily. Thereafter, during the 9 days in which enteritis was induced by feeding DSS, the weight change of the control and experimental mice was measured every day, and the length of the colon was measured by necropsy of the mice on the 9th day after the feeding of DSS.

As a result, as shown in FIG. 5, the weight of the mouse on the 9th day induced by enteritis by DSS was reduced to 87% of the initial weight in the control group, but Lactobacillus percentage In the experimental group administered orally with the KBL 375 strain, the body weight of the mice was 97% of the initial weight, indicating a slight change in body weight. In addition, as a result of measuring the length of the colon, the length of the colon was less than 80% of the control group on day 9 in mice induced with DSS, but in the experimental group administered orally to the Lactobacillus fermentum KBL 375 strain The length represents 90% of the control group, confirming that Lactobacillus Permantum KBL 375 strain can exert a beneficial effect on intestinal health.

Example 5 Lactobacillus Percentum Productivity Verification of KBL 375 Strain

Two representative subspecies of Lactobacillus fermentum ( L. fermentum KCTC5049, L. fermentum KCTC5467) registered in the BRC were compared to confirm the productivity of Lactobacillus fermentum KBL 375 (KCTC 13381BP) strain. When three Lactobacillus fermentum strains were cultured in De Man, Rogosa and Sharpe (MRS) media, respectively, as shown in FIG. 6, the Lactobacillus fermentum KBL 375 strain had a faster growth rate than the other two Lactobacillus fermentum strains. I could confirm it. The specific growth rate of the Lactobacillus latent KBL375 strain was 0.756, confirming that the growth was at least 1.1 to 1.3 times faster than the other two Lactobacillus latents (Table 2). In addition, the doubling time required to double the growth of one cell was 0.91 hours for the Lactobacillus permanent KBL 375 strain, whereas 1.16 hours for the Lactobacillus fermentum KCC5049 and 1.02 hours for the Lactobacillus fermentum KCTC5467 strain. It was confirmed (Table 2). Accordingly, the Lactobacillus latent KBL 375 strain was confirmed that the productivity is more than 10% better than the other two reported Lactobacillus latent strains.

Specific growth rate and doubling time of Lactobacillus fermentum KBL375, Lactobacillus fermentum KCTC5049 and Lactobacillus fermentum KCTC5467 Strains Specific growth rate (h ^-1 ) Doubling time (h) Character L. fermentum KBL375 0.756 0.916 Isolated from feces adult L. fermentum KCTC5049 0.677 1.163 Isolated from saliva L. fermentum KCTC5467 0.596 1.024 Isolated from intestine

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC13381BP

Trust Date: 20171023

<110> Ko BioLabs, Inc. <120> Lactobacillus Fermentum KBL 375 and Use Thereof <130> P17-B271 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1120 <212> DNA <213> lactobacillus fermentum <400> 1 gtcctgttac ggtgctatac atgcaagtcg aacgcgttgg cctaattgat tgatggtgct 60 tgcacctgat tgattttggt cgccaacgag tggcggacgg gtgagtaaca cgtaggtaac 120 ctgcccagaa gcgggggaca acatttggaa acagatgcta ataccgcata acaacgttgt 180 tcgcatgaac aacgcttaaa agatggcttc tcgctatcac ttctggatgg acctgcggtg 240 cattagcttg ttggtggggt aacggcctac caaggcgatg atgcatagcc gagttgagag 300 actgatcggc cacaatggga ctgagacacg gcccatactc ctacgggagg cagcagtagg 360 gaatcttcca caatgggcgc aagcctgatg gagcaacacc gcgtgagtga agaagggttt 420 cggctcgtaa agctctgttg ttaaagaaga acacgtatga gagtaactgt tcatacgttg 480 acggtattta accagaaagt cacggctaac tacgtgccag cagccgcggt aatacgtagg 540 tggcaagcgt tatccggatt tattgggcgt aaagagagtg caggcggttt tctaagtctg 600 atgtgaaagc cttcggctta accggagaag tgcatcggaa actggataac ttgagtgcag 660 aagagggtag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg aagaacacca 720 gtggcgaagg cggctacctg gtctgcaact gacgctgaga ctcgaaagca tgggtagcga 780 acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctagtg ttggagggtt 840 tccgcccttc agtgccggag ctaacgcatt aagcactccg cctggggagt acgaccgcaa 900 ggttgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 960 cgaagctacg cgaagacctt accaggtctt gacatcttgc gccaacccta gagatagggc 1020 gtttccttcg ggacgcaatg acaggtggtg catggtcgtc gtcagctcgt gtcgtggaga 1080 tgttgggtta agtcccgcaa cgaagcgcca ccctggtact 1120

Claims (7)

Lactobacillus permanent KBL 375 (Accession No. KCTC 13381BP) strain.
The strain according to claim 1, wherein the strain has a 16s rDNA sequence represented by SEQ ID NO: 1.
A composition containing an effective amount of at least one selected from the group consisting of the cells of the strain of claim 1, the culture of the strain, the crushed product of the strain, and the extract of the strain.
The method of claim 3,
The composition is a composition, characterized in that the composition for food or food additives.
The method of claim 4, wherein
The food is a composition characterized in that the health functional food for improving enteritis, inflammatory bowel disease or irritable bowel syndrome.
The intestine selected from the group consisting of enteritis, inflammatory bowel disease and irritable bowel syndrome containing an effective amount of at least one selected from the group consisting of the cells of the strain of claim 1, the culture of the strain, the crushed product of the strain and the extract of the strain. Pharmaceutical compositions for the treatment or prevention of diseases.
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