NOVEL PROBIOTIC COMPOSITION FOR REGULATION OF INTESTINAL IMMUNITY
20220409677 · 2022-12-29
Inventors
Cpc classification
A23V2002/00
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A23V2200/32
HUMAN NECESSITIES
A23V2002/00
HUMAN NECESSITIES
A61P1/06
HUMAN NECESSITIES
A61P1/00
HUMAN NECESSITIES
A23V2200/32
HUMAN NECESSITIES
A23V2200/3204
HUMAN NECESSITIES
A23V2200/3204
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to a novel probiotic composition for regulation of intestinal immunity and, more specifically, to a composition including Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis as active ingredients for suppressing inflammation and/or preventing, ameliorating, or treating inflammatory bowel diseases.
Claims
1. A pharmaceutical composition for preventing or treating inflammatory bowel disease, comprising Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis as an active ingredient.
2. The composition according to claim 1, wherein the composition comprises a Lactobacillus johnsonii IDCC9203 strain, a Lactobacillus plantarum IDCC3501 strain, and a Bifidobacterium animalis subspecies lactis IDCC4301 strain.
3. The composition according to claim 1, wherein the Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis are live cells or dead cells, respectively.
4. A food composition for preventing or improving inflammatory bowel disease, comprising Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis as an active ingredient.
5. The composition according to claim 4, wherein the Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis are live cells or dead cells, respectively.
6. A pharmaceutical composition for anti-inflammatory, comprising Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis as an active ingredient.
7. A food composition for anti-inflammatory, comprising Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis as an active ingredient.
8. Use of a composition comprising Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis as an active ingredient for preparing a formulation for the prevention or treatment of inflammatory bowel disease.
9. A method of treating inflammatory bowel disease, comprising administering to a subject in need thereof an effective amount of a composition comprising Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis as an active ingredient.
10. The method according to claim 9, wherein the inflammatory bowel disease is one or more selected from the group consisting of Crohn's disease, ulcerative colitis, intestinal Behcet's disease, and ischemic enteritis.
11. Use of a composition comprising Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis as an active ingredient for preparing a formulation for preparing an anti-inflammatory agent.
12. A method of inhibiting inflammation comprising administering to a subject in need thereof an effective amount of a composition comprising Lactobacillus johnsonii, Lactobacillus plantarum and Bifidobacterium animalis subspecies lactis as an active ingredient.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
MODE FOR CARRYING OUT INVENTION
[0104] Hereinafter, the present invention will be described in detail.
[0105] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.
Example 1: Discovering a New Combination of Strain Mixture
[0106] For various lactic acid bacteria strains isolated from infant feces, kimchi, and cheese, the individual efficacy of each strain was screened in various aspects. In addition, as a result of performing efficacy evaluation in various aspects using various strains in various combinations, the present inventors confirmed an unexpected synergistic effect with respect to the combination of three strains of Lactobacillus johnsonii IDCC9203 (Isolated from infant feces/Accession number KCTC 10923BP), Lactobacillus plantarum IDCC3501 (Isolated from kimchi/Accession number KCTC 13586BP) and Bifidobacterium animalis subspecies lactis IDCC4301 (Isolated from infant feces/Accession number KCTC 13587BP) in anti-inflammatory efficacy, as well as confirmed that the actual therapeutic efficacy is remarkable in terms of pharmacology for inflammatory bowel disease. The data presented below demonstrate the unexpected discovery of the strain combination of the three lactic acid bacteria preparations of the present invention.
Example 2: Confirmation of Anti-Inflammatory Synergistic Effect of Three Types of Lactic Acid Bacteria Preparations (Mixture) of the Present Invention, and Comparison of Strain Combination Specificity Thereof
[0107] 2-1. Confirmation of Specificity of the Composition of the Three Lactic Acid Bacteria Preparations (Mixture) of the Present Invention, and Comparison of Anti-Inflammatory Effects
[0108] Lactobacillus johnsonii IDCC9203 (No. 6 in
[0109] Various mixtures (i.e., mixtures of the same species but with different specific strains) were prepared in the same manner as in the present invention using other lactic acid bacteria of the same species as the constituent strains of the three lactic acid bacteria preparations (mixture) of the present invention, and efficacy was comparatively evaluated. As a representative example,
[0110] RAW 264.7 cells, which are mouse macrophages, were cultured at 37° C. and 5% CO.sub.2 condition using phenol red-free Dulbecco's modified Eagle medium (DMEM, Gibco-BRL, Gaithersburg, Md., USA) containing 10% fetal bovine serum (FBS, Atlas, Fort Collins, Colo., USA) and 1% penicillin (Sigma-Aldrich, St. Lousi, Mo., USA).
[0111] In order to confirm the effect of the three lactic acid bacteria preparations (mixture) of the present invention on inflammatory cytokine production, the cultured Raw 264.7 cells were recovered using a scraper or trypsinize, put into a 15 ml falcon tube, and centrifuged at 500 rpm for 5 minutes. After removing the supernatant, 1 ml of complete media was added and resuspended, and the number of cells per ml was counted. 2 ml of the diluted cell solution was added to each well of a 6-well plate and cultured overnight for 24 hours. After sucktion of the medium in each well in a 6-well plate, it was washed with PBS. After dispensing 2 ml of a new medium, 4 ul of LPS (final LPS concentration is 2 ul/ml) was treated in all wells and in the first well, 1 mM dexamethasone was treated with 20 ul (final concentration of 10 uM/ml) as a positive control. PBS was treated as a negative control, the remaining wells were treated with LPS, and the three lactic acid bacteria preparations of the present invention were treated at a concentration of 1×10.sup.5 to 1×10.sup.7 cell/mL, followed by incubation overnight for 24 hours. The culture solution of each well was transferred to a 15 ml falcon tube and centrifuged at 9,500 rpm for 5 minutes. The supernatant of each tube was separately recovered and used for cytokine experiments. As an inflammatory cytokine, it was confirmed by using an ELISA kit (R&D system) whether or not to inhibit the production of TNF-α, IL-6, and IL-1β.
[0112] As a result of the experiment, it was confirmed that when the three types of lactic acid bacteria preparations (mixture) of the present invention were treated, the inflammatory cytokine production inhibitory effect (i.e. anti-inflammatory effect) was significantly increased compared to when the individual strains constituting the same were used alone. As a representative example showing this, as shown in
[0113] In addition, as representatively shown in
[0114] 2-2. Comparison of NO Production Inhibitory Ability of Constituent Strains
[0115] As a result of comparing the individual efficacy of each strain in various aspects for various lactic acid bacteria strains isolated from infant feces, kimchi and cheese, representatively,
[0116] The experimental method is simply as follows. Each of the lactic acid bacteria was cultured by the above-mentioned general culture method, and after completion of the culture, the cells were obtained by centrifugation, and the cells were killed by heat-killed at 100° C. for 15 minutes. RAW 264.7 cells (1×10.sup.5/mL) were first cultured in DMEM, treated with LPS (2 μl/ml) and each heat-killed cells (1×10 cell.sup.5/mL), and then incubated for 24 hours. The concentration of NO was investigated by measuring the amount of nitrite in the cell culture supernatant using Griess reagent (Sigma, USA) according to the manufacturer's protocol. After mixing 150 μL of cell culture supernatant obtained from RAW264.7 cell medium treated as described above and incubated for 24 hours and 150 μL of Griess reagent, it was centrifuged at 1,000×g for 10 minutes, and then incubated for 10 minutes at room temperature. Absorbance was measured at 595 nm using a microplate reader, and was compared based on a calibration curve formed through sodium nitrite.
[0117] Lactobacillus johnsonii IDCC9203, (No. 6 in
Example 3: Confirmation of In Vivo Inflammatory Bowel Disease (IBD) Treatment Efficacy of Three Kinds of Probiotic Preparations of Lactic Acid Bacteria of the Present Invention & Comparison of Efficacy with Sulfasalazine, a Compound for Treating IBD
[0118] Experimental Method
[0119] 1) Preparation of Three Kinds of Probiotic Preparations of Lactic Acid Bacteria of the Present Invention
[0120] The three lactic acid bacteria probiotic preparations of the present invention used in the experiment were prepared by mixing three live strains of Lactobacillus johnsonii IDCC9203 (KCTC 10923BP), Lactobacillus plantarum IDCC3501 (KCTC 13586BP) and Bifidobacterium animalis subspecies lactis IDCC4301 (KCTC 13587BP) in a CFU (colony-forming unit) standard 1:1:1 ratio. Each live strain contained 2×10.sup.12 CFU/g. Each bacteria was cultured in a growth medium at 37° C. for 16 hours until the exponential phase, and then the bacterial pellet was stored in a freeze dryer and freeze-dried (Condition at −80° C. under vacuum, overnight). The freeze-dried three kinds of lactic acid bacteria preparations of the present invention were stored at 4° C. until use.
[0121] 2) Experimental Animals
[0122] Female BALB/c mice (8 weeks old) were purchased from Orient Bio (Seongnam, Republic of Korea). Five animals per cage were housed in an environmentally controlled facility (temperature 22° C.±2° C., humidity 55%±5%) with a 12/12 h light/dark cycle.
[0123] Animal care and treatment was carried out in accordance with the guidelines established by the National Institutes of Health Animal Research and Care, and it was approved by the Animal Experiment Ethics Committee (Institutional Animal Care and Use Committee, Approval No.: A1611-2) of Ildong Pharmaceutical Co., Ltd.
[0124] 3) Mouse Colitis Model
[0125] A mouse model of colitis induced by DSS (molecular weight 36-50 kDa/MP biochemicals, Santa Ana, Calif., USA) was used in this study. After 7 days of adaptation period, as shown in Table 1, mice were divided into 7 groups (10 mice per group), and the average of the initial body weight (BW) was similarly adjusted. DSS was dissolved in drinking water at a concentration of 3.5% (w/v), and all mice except the normal group were supplied from day-0 to day-8. Animals received water and food ad libitum.
[0126] 200 μl of distilled water (DW) was orally administered to vehicle group mice. The three lactic acid bacteria preparations of the present invention have a dose range of 10.sup.6 CFU/mice/day-10.sup.9 CFU/mice/day, and sulfasalazine (Tokyo chemical industry, Tokyo, Japan) as a positive control is at a concentration of 500 mg/kg (BW)/day, after suspending in DW, it was administered in the same volume and in the same manner as the vehicle group. Table 1 below summarizes the material treatment conditions for each experimental group and control group.
TABLE-US-00001 TABLE 1 Drinking Group water Administration Normal DW DW Vehicle 3.5% DSS DW ID-JPL 10.sup.6 3.5% DSS ID-JPL 10.sup.6 CFU per mouse per day ID-JPL 10.sup.7 3.5% DSS ID-JPL 10.sup.7 CFU per mouse per day ID-JPL 10.sup.8 3.5% DSS ID-JPL 10.sup.8 CFU per mouse per day ID-JPL 10.sup.9 3.5% DSS ID-JPL 10.sup.9 CFU per mouse per day Sulfasalazine 3.5% DSS Sulfasalazine 500 mg per kg BW per day BW, body weight; CFU, colony-forming units; DSS, dextran sulfate sodium; DW, distilled water.
[0127] BW (weight) and stool status were observed once a day for each mouse, and a DAI (disease activity index) score was calculated according to the criteria described in Table 2 below as in a previously known method. The maximum value that could be calculated according to Table 2 below was 12. After euthanasia with carbon dioxide gas, the large intestine (colon) from the ileocecal junction to the anus was removed and the length was measured, and was washed with phosphate buffered saline (PBS) in order to remove the intestinal residue. One third of the distal colon was fixed with 10% formaldehyde solution and used for histopathological analysis. The remaining two-thirds of the distal colon tissue was used for protein extraction to measure proinflammatory cytokine levels and stored at −80° C. until use.
TABLE-US-00002 TABLE 2 Weight Visible loss Stool blood Score (%) consistency in feces 0 None Normal None 1 1-5 2 6-10 3 11-20 Pasty stoools Slight bleeding 4 >20 Diarrhea Gross bleeding
[0128] 4) Histological Analysis
[0129] Colonic tissue was fixed with a 10% neutral buffered formaldehyde solution, praffin embedding was carried out in a conventional manner. Paraffin-embedded tissue sections were cut to a thickness of 4 μm and stained with hematoxylin & eosin (H & E). H&E-stained colon tissues were observed at 100× and 200× magnifications using a Nikon ECLIPSE 50i optical microscope (Nikon, Tokyo, Japan), and as described in Table 3 (Histological Scoring System), histological evaluation was performed by two pathologists according to commonly known parameters. According to Table 3, the maximum score calculated by summing each of the three parameters was 10.
TABLE-US-00003 TABLE 3 Feature Score Description Severity of inflammation 0 None 1 Mild 2 Moderate 3 Severe Extent of inflammation 0 None 1 Mucosa 2 Mucosa and submucosa 3 Transmural Crypt damage 0 None 1 ⅓ damaged 2 ½ damaged 3 Crypts lost, surface epithelium present 4 Crypts and surface epithelium lost
[0130] 5) Measurement of Colonic Pro-Inflammatory Cytokine Levels
[0131] Colonic proteins were extracted using RIPA buffer (Upstate, Temesula, Calif., USA) added with a protease inhibitor (Sigma-Aldrich, St. Louis, Mo., USA). After adding 300 μl of RIPA buffer to colon tissue, the tissue was homogenized on ice for 1 minute, and centrifuge at 20,000 g for 15 min at 4° C. After collecting the supernatant, quantification was performed using a bicinchoninic acid protein assay reagent kit (Pierce, Rockford, Ill., USA). An ELISA kit (R&D Systems, Minneapolis, Minn., USA) was used according to the manufacturer's protocol to detect the levels of proinflammatory cytokines such as TNF-a, IL-1b and IL-6 in colon tissues. Each cytokine value was corrected for the total amount of each colon protein sample.
[0132] 6) Statistical Analysis
[0133] Results are presented as mean±standard error, and each result was processed using a statistical analysis system, Prism 7 (GraphPad Software, San Diego, Calif., USA). All results represent the average of at least three independent experiments. Statistical comparisons between the vehicle group and each treatment group were performed by one-way analysis of variance and subsequent Dunnett's test. P value <0.05 was considered statistically significant.
[0134] Experimental Results
[0135] 3-1. Effects of 3 Types of Lactic Acid Bacteria Preparations of the Present Invention on Colitis Symptoms In Vivo
[0136] To determine the severity of colitis, the BW and defecation status of each mouse were observed once a day and the DAI score was recorded. As the amount of DSS provided to the mice increased, the incidence of bloody stool and diarrhea increased, whereas body weight (BW) gradually decreased. In the vehicle group, from the 4th day of the experiment, the DAI score was shown to rise steadily. Administration of the three lactic acid bacteria preparations of the present invention dose-dependently decreased the DAI score (see
[0137] In mice induced with colitis, the length of the colon was shorter than in the normal group. This symptom of shortening of the colon length was prevented in a dose-dependent manner by the three types of lactic acid bacteria preparations of the present invention, and it showed an equivalent effect pattern between 10.sup.8 CFU/mice/day or 10.sup.9 CFU/mice/day dose administration group of the three lactic acid bacteria preparations of the present invention and the sulfasalazine administration group (see
[0138] 3-2. Effect of 3 Types of Lactic Acid Bacteria Preparations of the Present Invention on Colon Histological Parameters
[0139] To evaluate the severity of histological damage of DSS-induced colitis, H&E-stained colon tissues were observed using a microscope, and histologic scores were measured. As shown in
[0140] 3-3. Effects of the Three Lactic Acid Bacteria Preparations of the Present Invention on the Expression of Proinflammatory Cytokines in the Colon
[0141] To detect the levels of colonic cytokines associated with the inflammatory response, ELISA was performed. Representatively, the expression patterns of three pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6, were investigated, and they are known to play an important role in the pathology of colitis. As shown in
Example 4: Comparative Evaluation of the Inflammatory Bowel Disease Treatment Efficacy of the Existing Commercial Probiotic Products Approved for IBD and the Three Types of Lactic Acid Bacteria Preparations of the Present Invention
[0142] Experimental Method
[0143] The effect on the improvement of inflammatory bowel disease (IBD) was compared with the three probiotic preparations (mixture) of the present invention using company V's lactic acid bacteria preparations, which is the first and only probiotic product in Korea that has been individually recognized for ‘helping intestinal health by regulating intestinal immunity’ by the Ministry of Food and Drug Safety, currently. The lactic acid bacteria preparation of V Company is known to consist of a total of about 450 billion live bacteria in one pouch (4.5×1011 bacteria/pack), and specifically, it is known that 8 types of live bacteria strains of Lactobacillus paracasei DSM 24734, Lactobacillus plantarum DSM 24730, Lactobacillus acidophilus DSM 24735, Lactobacillus delbrueckii subsp. bulgaricus DSM 24734, Bipidobacterium longum DSM 24736, Bipidobacterium breve DSM 24732, Bipidobacterium infantis DSM 24737, Streptococcus salivarius subsp. thermophilus DSM 24731 are mixed.
[0144] The test group composition was performed as shown in Table 4 below, at this time, the three strains described above as a live cell mixture of three types of lactic acid bacteria of the present invention in a ratio of 1:1:1 based on CFU was typically used for the experiment. Except for the conditions described in Table 4 below, the rest of the experimental method was performed in the same manner as in Example 3.
TABLE-US-00004 TABLE 4 Number of Drinking Route of Group animals water administration Dose amount Dosage Normal Control 10 Primary p.o 10 mL/kg/day 0 mg/kg/day Distilled water Vehicle 10 Primary p.o 10 mL/kg/day 0 mg/kg/day V Company lactic 10 distilled p.o 200 ul/mouse/ 5.4 × 10.sup.8 CFU/ acid bacteria water day day preparation with 3.5% Present invention 10 DSS p.o 200 ul/mouse/ 10.sup.8 CFU/day 10.sup.8 added day Present invention 10 p.o 200 ul/mouse/ 10.sup.9 CFU/day 10.sup.9 day
[0145] Experimental Results
[0146] 4-1. Comparison of Effects on Colitis Symptoms In Vivo
[0147] As shown in
[0148] In addition, as shown in
[0149] 4-2. Comparison of Colon Histological Parameters
[0150] As representatively shown in
[0151] 4-3. Comparison of Effects on the Expression of Proinflammatory Cytokines in the Colon
[0152] Representatively, as shown in
INDUSTRIAL APPLICABILITY
[0153] As described above, the present invention relates to a novel probiotic composition for regulating intestinal immunity, and more particularly, it relates to a composition for inhibiting inflammation and/or preventing, improving, or treating inflammatory bowel disease, comprising Lactobacillus johnsonii, Lactobacillus plantarum, and Bifidobacterium animalis subspecies lactis as an active ingredient.
[0154] The composition comprising the unique combination of three types of lactic acid bacteria of the present invention has remarkable preventive and therapeutic effects for inflammatory bowel disease (IBD), and in particular, these effects are pharmacologically superior compared to known IBD therapeutic drugs (e.g., sulfasalazine). In addition, since the composition of the present invention is a probiotic composition, it has a great advantage as a safe composition with very little risk of side effects, and thus has high industrial applicability.
[Accession Number]
[0155] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
[0156] accession number: KCTC10923BP
[0157] deposit date: 2006.03.15
[0158] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
[0159] accession number: KCTC13586BP
[0160] deposit date: 2018.07.17
[0161] Name of deposit institution: Korea Research Institute of Bioscience and Biotechnology
[0162] accession number: KCTC13587BP
[0163] deposit date: 2018.07.17