Composition for improving respiratory diseases using Lactobacillus plantarum strain

Abstract

The present invention relates to a food composition for enhancing respiratory function and improving respiratory diseases, using Lactobacillus plantarum KF511 strain; a health functional food comprising the food composition; and a pharmaceutical composition for preventing or treating the respiratory diseases, comprising Lactobacillus plantarum strain, its culture solution, its concentrate, or its dried product, as an active ingredient.

Claims

1. A food composition for enhancing respiratory function and improving respiratory diseases, comprising: Lactobacillus plantarum KF511 strain (L. plantarum KF511); a culture solution comprising L. plantarum KF511; a concentrate comprising L. plantarum KF511; or a dried product comprising L. plantarum KF511; wherein the food composition comprises 10.sup.7 to 10.sup.12 CFU of the L. plantarum KF511, the L. plantarum KF511 is an active ingredient in the food composition and is assigned an accession number KCCM 12573P.

2. The food composition according to claim 1, wherein the respiratory diseases are pulmonary diseases accompanied by symptoms of cough, sputum, dyspnea, airway hypersensitivity, airway obstruction, mucus hypersecretion, decreased expiratory flow rate and/or gas exchange disorder.

3. The food composition according to claim 1, wherein the respiratory diseases are asthma, chronic obstructive pulmonary disease (COPD), diffuse interstitial pulmonary disease, acute respiratory distress syndrome (ARDS), or acute lung injury.

4. The food composition according to claim 3, wherein the respiratory diseases are the asthma or the COPD.

5. A health functional food comprising the food composition according to claim 1.

6. A pharmaceutical composition for preventing or treating respiratory diseases, comprising: Lactobacillus plantarum KF511 strain (L. plantarum KF511); a culture solution comprising L. plantarum KF511; a concentrate comprising L. plantarum KF511; or a dried product comprising L. plantarum KF511; wherein the pharmaceutical composition comprises 10.sup.7 to 10.sup.12 CFU of the L. plantarum KF511, the L. plantarum KF511 is an active ingredient in the pharmaceutical composition and is assigned an accession number KCCM 12573P.

7. The pharmaceutical composition according to claim 6, wherein the respiratory diseases are asthma, chronic obstructive pulmonary disease (COPD), diffuse interstitial pulmonary disease, acute respiratory distress syndrome (ARDS), or acute lung injury.

8. The pharmaceutical composition according to claim 7, wherein the respiratory diseases are the asthma or the COPD.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the total number of cells counted from bronchoalveolar lavage fluid (BALF) after administrating the Lactobacillus plantarum KF511 strain of the present invention and ROF as a positive control to a respiratory disease (COPD) mouse model induced by PPE/CSE.

(2) FIGS. 2 and 3 show the results of analyzing immune cells (macrophage, lymphocyte, neutrophil and eosinophil) in BALF through Diff-Quick staining.

(3) FIG. 4 shows the results of staining lung tissues extracted from a respiratory disease (COPD) mouse model.

(4) FIG. 5 shows the results of analyzing cytokines and chemokines (KC, MCP-1, MDC, MIP-2, TARC, GM-CSF, INF-gamma, IL-6, IL-10, IL-17) in BALF of a respiratory disease (COPD) mouse model.

(5) FIG. 6 shows the results of analyzing cytokines and chemokines (KC, MCP-1, MDC, MIP-2, TARC, GM-CSF, INF-gamma, IL-1beta, IL-6, IL-17) from lung tissues of a respiratory disease (COPD) mouse model.

(6) FIG. 7 shows the results of measuring ratios of B cells in lung tissues after treating the Lactobacillus plantarum KF511 strain to the lung tissues derived from a respiratory disease (COPD) mouse model for each concentration.

(7) FIG. 8 shows the results of measuring IgA contents in BALF after treating the Lactobacillus plantarum KF511 strain to a respiratory disease (COPD) mouse model for each concentration.

(8) FIG. 9 shows the results of measuring expression levels of MUC5AC after treating the Lactobacillus plantarum KF511 strain to NCI-H292 cells, a mucinous epithelial cancer cell line of human respiratory epithelial cells, for each concentration.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(9) Hereinafter, the present invention will be described in more detail through the following Examples. However, these Examples are intended to illustrate the present invention only, and not to limit the scope of the present invention to these Examples.

Example 1: Isolation and Identification of Lactobacillus plantarum KF511 Strain

(10) Lactobacillus plantarum strain according to the present invention was isolated from Kimhi that is traditionally prepared using Lactobacillus MRS agar medium (MRS medium, BD288130, Difco Company, USA), and the isolated strain was identified through 16S rDNA sequencing. As a result of the identification, the 16s rDNA base sequence (SEQ ID NO: 1) of the strain showed a homology of 99.93% with the existing Lactobacillus plantarum ATCC14917(T) strain. The medium composition for culturing the strain is a MRS medium, the culture condition includes a pH of 6.50.2, a temperature of 37 C. and a stirring culture for 24 hours, the oxygen demand is facultative anaerobic, and the strain can be preserved through a freeze-drying or a freezing of the cell suspension.

(11) The present inventors have named the above strain as Lactobacillus plantarum KF511. This strain has been deposited with the Korea Microorganism Conservation Center (KCCM), an international microbial depository authority, on Aug. 21, 2019, and has been given an accession number KCCM 12573P.

Experimental Example 1: PPE+CSE-Induced Respiratory Disease (COPD) Mouse Model

(12) BALB/C mouse (male, 5 weeks old) were purchased from Orient Bio Company, acclimatized for 1 week, and grouped into n=10. The sample treatment group was orally administered with a sample of 10.sup.7 to 10.sup.9 CFU/head for 24 days from one week before the start of COPD induction until dissection, and the same amount of PBS was administered to the naive and COPD groups. At this time, the Roflumilast (ROF) group, the positive control group, was orally administered in a concentration of 10 mg/kg every day after the start of induction. For the COPD induction, 1.2 unit of Porcine Pancreatic Elastase (PPE) and 100% Cigarette Smoke Extraction (CSE) were used. 1.2 unit of the PPE was injected intranasally for a total of 3 times once every 7 days, and 20 l of CSE was treated intranasally for 3 days from the next day after the PPE injection. After the last PPE injection, the dissection was performed the next day without treatment of the CSE. The dissection was performed after anesthesia with isoflurane using an inhalation anesthetic machine. During the period of experiment, a feed and a drinking water were provided in the form of self-feeding, and the groups were bred under the condition of 24 C. and 60% humidity.

Experimental Example 2: Total Number of Cells Infiltrated in BALF and Analysis of Immune Cells in BALF Through Diff-Quick Staining

(13) 2-1: Total Number of Cells Infiltrated in BALF

(14) After injecting 1 mL of a PBS through the airway of a respiratory disease (COPD) model mouse, 700 L of bronchoalveolar lavage fluid (BALF) was recovered by gently massaging. 10 uL of the recovered BALF solution was mixed with Accustain T solution in the same amount, and 12 uL of the mixed solution was injected into an Accuchip channel. Thereafter, total cells were automatically counted with a cell counter (ADAM-MC, NANOENTEK. INC, Korea) (FIG. 1).

(15) 2-2: Analysis of Immune Cells in BALF Through Diff-Quick Staining

(16) The recovered BALF was separated into a supernatant and a cell pellet through centrifugation under the condition of 300g and 5 minutes. The cell pellet was resuspended by adding 700 L of the PBS, and 150 L of this BALF suspension was centrifuged with a Cytospin device (Centrifuge 5403, Eppendorf, Hamburg, Germany) under the condition of 1000 rpm, 10 minutes and 4 C. to attach the BALF cells on a slide. Thereafter, the cells were stained using a Diff-Quick staining reagent according to the protocol of the manufacturer (1-5-1 Wakinohamakaigandori, chuo-ku, Kobe, Japan), and then observed with a microscope (FIGS. 2 and 3).

Experimental Example 3: Histological Examination

(17) A lung tissue was removed from the dissected respiratory disease (COPD) model mouse, and a mesenchyme of the lung was removed followed by fixed in 10% neutral buffered formalin for 3 days. The fixed tissue was dehydrated with ethyl alcohol and xylene, embedded in a paraffin, trimmed, and sliced to a thickness of 5 m. After attaching the prepared section to a slide, it was stained with H&E to confirm its general shape, and then observed with a microscope (FIG. 4).

Experimental Example 4: Analysis of Cytokines and Chemokines in BALF

(18) After injecting 1 mL of PBS through the airway of a respiratory disease (COPD) model mouse, 700 L of bronchoalveolar lavage fluid (BALF) was recovered by gently massaging. The recovered BALF was centrifuged under the condition of 300g and 5 minutes to separate a supernatant, and then used to analyze cytokines and chemokines (KC, MCP-1, MDC, MIP-2, TARC, GM-CSF, INF-gamma, IL-6, IL-10, IL-17). After taking 30 uL of the supernatant for the analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer (Quansis Biosciences Company) of a Q-plex ELISA array kit. The results were shown for each of the cytokines or the chemokines in FIG. 5.

Experimental Example 5: Analysis of Cytokines and Chemokines in Lung Tissue

(19) To a lung tissue of a respiratory disease (COPD) model mouse, a PBS was added in an amount corresponding to 10 times the weight of the lung tissue, crushed with a homogenizer, and centrifuged under the condition of 12,000 rpm and 20 min. The recovered supernatant was used to analyze cytokines and chemokines (KC, MCP-1, MDC, MIP-2, TARC, GM-CSF, INF-gamma, IL-1beta, IL-6, IL-17). After taking 30 uL of the supernatant for the analysis of cytokines and chemokines, the experiment was performed according to the protocol of the manufacturer (Quansis Biosciences Company) of a Q-plex ELISA array kit. The results were shown for each of the cytokines or the chemokines in FIG. 6.

Experimental Example 6: Measurement of a Ratio of Immune Cells in Lung Tissue

(20) A lung tissue was recovered from a respiratory disease (COPD) model mouse, and minced using dissecting scissors. The sliced lung tissues were put into a GentleMACS C tube, and were treated with enzymes D and A according to the protocol of the manufacturer (Miltenyi Biotec Company) to unicellularize the tissues with a MACS dissociator. After the unicellularized cells were subjected to red blood cell lysis, the number of cells in each of the groups was measured. The cells were made in a concentration of 110.sup.7 cells/mL, and 200 L per well was dispensed in a 96-well V-bottom plate.

(21) Before staining immune cell-related surface markers, Fc Block was treated with 0.8 L/20 L/well using a FACS buffer (PBS+8% NaHCO.sub.3+1% FBS+10% NaN.sub.3), and then pipetted and left in a refrigerate at 4 C. for 15 minutes. For washing, the supernatant was removed by centrifugation at 1800 rpm for 3 minutes. In order to stain the surface markers of various immune cells, related antibodies were calculated according to the recommended amount of a staining concentration from the manufacturer (Biolegend Company), respectively, and prepared by diluting in the FACS buffer.

(22) The experiment was performed to use FITC anti-mouse I-A/I-E, PE anti-mouse CD11c, PerCP/Cy5.5 anti-mouse Ly-6C antibody, APC anti-mouse CD64 (FcRI), Alexa Fluor 700 anti-mouse Ly-6G, APC/Fire 750 anti-mouse CD45 antibody, Brilliant Violet 421 anti-mouse CD24, and Brilliant Violet 510 anti-mouse/human CD11b antibody. The diluted antibodies were treated with 20 L/well and reacted at a room temperature for 30 minutes. Then, they were washed with a PBS, and the cells were suspended in the PBS. The suspended cells were analyzed using Cytoflex (Beckman Coulter Company), a flow cytometer. The results were shown in FIG. 7 according to the treatment concentration of each Lactobacillus plantarum KF511 (LPKF511) strain.

Experimental Example 7: Measurement of IgA Content in BALF

(23) IgA was measured according to the experimental method of the ELISA kit (eBioscience, USA). A capture antibody was diluted with a coating buffer, and 100 l per well was dispensed into a 96-well Immunoplate, and then left at 4 C. for about a day. The next day, after washing twice using a washing buffer, a blocking buffer was dispensed in an amount of 250 per well and left at a room temperature for 2 hours. After ending the blocking process followed by washing 4 times, the standard sample and the BALF solution of each group were diluted with an assay buffer, dispensed in an amount of 100 per well, and left at the room temperature for 2 hours. After washing the solution 4 times, a detection-antibody was diluted with the assay buffer, dispensed in an amount of 100 L per well, and left at the room temperature for 1 hour. After washing the solution 4 times, a substrate solution was dispensed in an amount of 100 L per well and left at the room temperature for 15 minutes, and a stop solution was added in an amount of 100 L per well to terminate the reaction. Thereafter, an absorbance was measured at a wavelength of 450 nm and quantified by a standard curve using Standard. The results were shown in FIG. 8 according to the treatment concentration of each Lactobacillus plantarum KF511 (LPKF511) strain.

Experimental Example 8: Confirmation of MUC5AC Inhibitory Ability Using NCI-H292 Cells

(24) Mucin, a glycoprotein in mucus, is produced by a mucin gene, and in particular, expression of MUC5AC, secretory mucin, plays an important role in the respiratory region. Accordingly, this experiment was performed to confirm inhibitory ability of MUC5AC of Lactobacillus plantarum KF511 strain using NCI-H292 cells, a mucinous epithelial cancer cell line of human respiratory epithelial cells.

(25) 8-1: Culture of H292 Cells

(26) H292 cells were cultured to use RPMI 1640 medium containing 10% Fetal bovine serum (FBS), 100 unit/ml of penicillin and 100 mg/ml of streptomycin. The H292 cells were inoculated in a 48-well cell culture plate in a concentration of 410.sup.4 cells/well, and cultured for 24 hours under the condition of providing 5% carbon dioxide (CO.sub.2) at 37 C. After culturing for 24 hours, the culture medium was replaced with a medium in which a concentration of the FBS was lowered to 0.2%, and starvation was performed for 24 hours under the condition of providing 5% carbon dioxide (CO.sub.2) at 37 C. Thereafter, all of the sample and a stimulant were treated to use a RPMI1640 medium without addition of a serum. A PMA (Phorbol 12-myristate 13-acetate) was used as the stimulant, and the final concentration was made to become 2 ng/mL, co-treated with KF511 10.sup.6-9 cells/well, and cultured for 24 hours. A culture supernatant was recovered and used to measure MUC5AC by an ELISA analysis method.

(27) 8-2: Measurement of MUC5AC

(28) The culture supernatant was dispensed into a 96-well immunoplate by 100 L, dried in a dry oven at 50 C., and washed 3 times with a PBS to which 0.05% tween 20 was added. Thereafter, the same washing method as above was used between all the steps. 1% bovine serum albumin (BSA) was dispensed in an amount of 200 L per well, and blocking was performed for 1 hour followed by being washed. MUC5AC antibody (abcam) was diluted 500-fold, dispensed in an amount of 100 L per well, and reacted for 2 hours. After washing, a goat anti-mouse IgG HRP (abcam) was diluted 2000-fold, dispensed in an amount of 100 L per well, and then reacted for 1 hour after blocking light. A substrate solution (BD) was dispensed in an amount of 100 L per well, and then reacted for 30 minutes after blocking the light, followed that the reaction was stopped by adding a stop solution in an amount of 50 L per well. An absorbance was measured at 450 nm using an ELISA reader. An inhibitory ability of MUC5AC showed a degree of inhibition during sample treatment compared to a production ability of MUC5AC by a PMA, based on untreated control cells.

(29) It was confirmed from the production ability of MUC5AC that the PMA increased production of MUC5AC by 2.5 times compared to the control cells that were not stimulated. In contrast, when the Lactobacillus plantarum KF511 (LPKF511) strain was treated, it was confirmed that the production of MUC5AC was significantly reduced depending on the treatment concentration thereof (FIG. 9).

(30) From the above description, a person who has an ordinary knowledge in the technical field to which the present invention pertains will understand that the present invention can be embodied in other certain embodiments without changing the technical spirit or essential features thereof. In this regard, it should be understood that the Examples described above are intended to be illustrative and not to be restrictive in all respects. It should be construed that the scope of the present invention includes all changes or modifications derived from the meaning and scope of the claims described below, and equivalents thereof, rather than the above detailed descriptions.