Composition comprising <i>Lactobacillus reuteri </i>ATG-F4 for prevention or treatment of muscular disorder

Abstract

The present invention relates to a composition for the prevention or treatment of sarcopenia, the composition including a Lactobacillus reuteri ATG-F4 strain. More specifically, the strain exhibits an effect of improving muscle rehabilitation and motor ability by increasing the muscle mass in skeletal muscles, thereby being suitably used as an ingredient for compositions for the prevention or treatment of sarcopenia or muscular atrophy.

Claims

1. A method of preventing or treating a muscular disorder by administering to a subject in need thereof a composition comprising a Lactobacillus reuteri ATG-F4 strain deposited under Accession No. KCTC13717BP, or cells thereof, a lysate thereof, a culture thereof, a culture fluid resulting from removing the cells from the culture thereof, an extract of the cells thereof, an extract of the culture thereof, or an extract of the culture fluid resulting from removing the cells from the culture thereof.

2. The method of claim 1, wherein the muscular disorder is selected from the group consisting of sarcopenia, cardiac atrophy, atony, muscular dystrophy, muscular degeneration, and muscle weakness.

3. A method of increasing muscle mass by administering to a subject in need thereof a composition comprising a Lactobacillus reuteri ATG-F4 strain deposited under Accession No. KCTC13717BP, or cells thereof, a lysate thereof, a culture thereof, a culture fluid resulting from removing the cells from the culture thereof, an extract of the cells thereof, an extract of the culture thereof, or an extract of the culture fluid resulting from removing the cells from the culture thereof.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schedule for animal testing to see muscle recovery in mice according to Experimental Example 1;

(2) FIGS. 2A and 2B are graphs showing the increase in body weight and ratio of tibialis anterior (TA) to body weight as the result of recovery from muscle loss according to Experimental Example 1;

(3) FIGS. 3A through 3D are graphs for the comparison between the weight of skeletal muscles in the experimental groups and the sarcopenia-induced group according to Experimental Example 1;

(4) FIG. 4 is a graph representing the weight of skeletal muscles of healthy models of Experimental Example 2 when the healthy models were fed the L. reuteri ATG-F4 strain;

(5) FIG. 5 is a graph representing the speed, degree of incline, and duration of the treadmill test of Experimental Example 3-1;

(6) FIGS. 6A and 6B are graphs showing the results of the treadmill test for the experimental groups and the sarcopenia-induced group in Experimental Example 3-1;

(7) FIGS. 7A and 7B are graphs showing the results of Kondziela's inverted screen test for the experimental groups and the sarcopenia-induced group in Experimental Example 3-2;

(8) FIG. 8 represents the 16S rRNA sequence of L. reuteri ATG-F4 (SEQ ID NO: 1);

(9) FIG. 9 represents the 16S RNA sequence of L. casei ATG-F1 (SEQ ID NO: 2);

(10) FIG. 10 represents the 16S rRNA sequence of L. reuteri ATG-F3 (SEQ ID NO: 3);

(11) FIG. 11A shows the differences between the 16S rRNA sequences of the ATG-F3 (SEQ ID NO: 3) and F4 (SEQ ID NO: 1) strains; and

(12) FIG. 11B shows the differences between the 16S rRNA sequences of the ATG-F3 (SEQ ID NO: 3) and F4 (SEQ ID NO: 1) strains.

BEST MODE

(13) Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

Embodiment 1. Isolation and Identification of ATG-F1, ATG-F3, and ATG-F4 Lactic Acid Bacteria

(14) Samples were prepared through three times of donation of a newborn's feces, on Jan. 8, 2016, May 31, 2018, and Jun. 4, 2018, respectively, and were diluted with 0.9% saline through 10-fold serial dilutions, and the diluted samples were smeared on de Man, Rogosa, Sharpe (MRS) media and incubated at 37 C. for about 48 hours.

(15) Colonies of the lactic acid bacteria formed on the incubated MRS medium were observed under a microscope to select ones that showed the morphology of genus Bacillus and were catalase-negative. Whole genome sequencing was performed on them, and the strains were named ATG-F1, ATG-F3, and ATG-F4 (also referred to as F1, F3, and F4, respectively).

(16) The 16S rRNA sequencing of the ATG-F1, ATG-F3, and ATG-F4 strains was commissioned to Solgent Co., Ltd. (Daejeon). The primers 27F (5-AGA GTT TGA TCC TGG CTC AG-3), 518F (5-CCA GCA GCC GCG GTA ATA C-3), 907R (5-CCG TCA ATT CMT TTR AGT TT-3), and 1492R (5-GGT TAC CTT GTT ACG ACT T-3) were used for sequencing for a total of 4 sequence reads, and the contig sequences derived through sequence alignment of each reading were analyzed using the BLAST online tool (blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Center for Biotechnology Information (NCBI). The 16S rRNA sequences of the above strains are represented in FIGS. 8 through 10.

(17) As a result of comparing the nucleotide sequences of SEQ ID NOs: 1 to 3 obtained through 16S rRNA sequencing to the BLAST database of NCBI, the 16S rRNA sequences of the ATG-F3 and ATG-F4 strains were shown to match 99.9% with that of the L. reuteri strain IRT and therefore taxonomically belong to L. reuteri. In addition, the ATG-F1 strain was found to belong to L. casei.

(18) Accordingly, the strain of the present invention was named L. reuteri ATG-F4. The strain is specifically disclosed in PCT/KR2019/006937 (Korean Patent No. 10-1951919, Title of invention: NOVEL LACTOBACILLUS REUTERI ATG-F4 STRAIN HAVING FUNCTION OF ENHANCING DOPAMINE SECRETION AND COMPOSITION COMPRISING SAME FOR PREVENTION OR TREATMENT OF PSYCHOPATHY)

(19) Next, the differences between the 16S rRNA sequences of the strains ATG-F3 and F4, which are of the same species, were analyzed.

(20) The differences between sequences were analyzed using Clustal Omega of EMBL-EBI (www.ebi.ac.uk/Tools/msa/clustalo), and this is shown in FIGS. 11A and 11B below.

(21) Referring to FIGS. 11A and 11B, although the ATG-F3 and F4 strains are of the same species, differences in the 16S rRNA sequences have been found, and based on this, it can be expected that there would be differences in the physiological characteristics.

(22) Next, a API50 CH test (BioMerieux, France) was performed for identification and characterization of the bacteria based on the carbohydrate fermentation patterns. To put it briefly, pure cultured lactic acid bacteria was suspended in 10 ml of API50 CHL medium to an OD.sub.600 of about 0.5, and the suspension culture was inoculated into each cupule of the API50 CH test strip and incubated at 37 C. The carbohydrate fermentation results were checked at 24, 48, and 72 hours after inoculation. In the fermentation pattern of lactic acid bacteria ATG-F1, F3, and F4 for the 49 carbohydrates using API50 CH, + represents a positive reaction, w represents a weak positive reaction, and represents a negative reaction.

(23) TABLE-US-00001 TABLE 1 L. casei L. reuteri L. reuteri Carbohydrates ATG-Fl ATG-F3 ATG-F4 Glycerol Erythritol D-Arabinose w L-Arabinose + + Ribose w + + D-Xylose w L-Xylose Adonitol w w Methyl-D- Xylopyranoside Galactose + + + Glucose + + + Fructose + Mannose + Sorbose + Rhamnose w Dulcitol Inositol w Mannitol + Sorbitol + w Methyl-D- Mannopyranoside Methyl-D- w Glucopyranoside N-Acetylglucosamine + Amygdalin w Arbutin + w Esculin + + Salicin + w Cellobiose + w Maltose w + + Lactose + + Melibiose + + Sucrose + + + Trehalose + Inulin + Melezitose + Raffinose + + Starch Glycogen Xylitol Gentiobiose + w Turanose + Lyxose Tagatose + D-Fucose L-Fucose + D-Arabitol w L-Arabitol w Gluconate w w + 2-keto-glugonate 5-keto-gluconate

(24) As a result of examining the glycolytic capacity of ATG-F1 lactic acid bacteria, weak positive reactions (changes from blue to green within 48 hours) were observed for ribose, adonitol, amygdalin, maltose, and gluconate, and positive reactions (changes from blue to yellow within 48 hours) were observed for galactose, glucose, fructose, mannose, mannitol, sorbitol, N-acetylglucosamine, arbutin, esculin, salicin, cellobiose, sucrose, trehalose, inulin, melechtose, gentiobiose, turanose, and tagatose. The ATG-F1 bacteria was found to have the ability to break down a total of 24 types of carbohydrates. As a result of examining the glycolytic capacity of ATG-F3 lactic acid bacteria, a weak positive reaction (a change from blue to green within 48 hours) was observed for gluconate, and positive reactions (changes from blue to yellow within 48 hours) were observed for L-arabinose, ribose, galactose, glucose, maltose, lactose, melibiose, sucrose and raffinose. The ATG-F3 bacteria was found to have the ability to break down a total of 10 types of carbohydrates.

(25) As a result of examining the glycolytic capacity of ATG-F4 lactic acid bacteria, weak positive reactions (changes from blue to green within 48 hours) were observed for D-arabinose, D-xylose, adonitol, rhamnose, inositol, sorbitol, methyl-D-glucopyranoside, arbutin, salicin, cellobiose, gentiobiose, L-arabitol, and D-arabitol, and positive reactions (changes from blue to yellow within 48 hours) were observed for L-arabinose, ribose, galactose, glucose, esculin, maltose, lactose, melibiose, sucrose, raffinose, L-fucose, and gluconate. In addition, the ATG-F4 bacteria was found to have the ability to break down a total of 25 types of carbohydrates.

(26) It can be seen on the basis of the different results of examining the glycolytic capacities of ATG-F1, F3, and F4 lactic acid bacteria that their respective physiological characteristics are different.

Embodiment 2. Culture of Lactic Acid Bacteria of the Genus Lactobacillus

(27) In the present embodiment, Lactobacillus casei ATG-F1, Lactobacillus reuteri ATG-F3, and Lactobacillus reuteri ATG-F4 (Accession No. KCTC13717BP) were used. Each lactic acid bacteria was cultured in MRS broth (Difco Lactobacilli MRS Broth) at 37 C. for 24 hours, and the cultured lactic acid bacteria were centrifuged at 12,000 g for 10 minutes to obtain only the cells, which were then suspended in 0.85% (v/v) saline for use in each experiment.

Experimental Example 1. Confirmation of Activation of Muscle Recovery in Sarcopenia-Induced Model

(28) Prior to all animal experiments conducted according to the present experimental example, the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of AtoGEN Co., Ltd. (Approval No.: ATG-IACUC-REV-180810) in accordance with the appropriate procedures and guidance on ethical animal experiments was provided. The animals used in the present experiment were 4-week-old C57BL/6J, purchased from Central Lab Animal Inc. (Seoul, Korea) for experiments.

(29) In the present experimental example, an animal experiment was performed to investigate the improvement of muscle rehabilitation and motor ability depending on the intake of L. casei ATG-F1, L. reuteri ATG-F3 or L. reuteri ATG-F4 lactic acid bacteria in a sarcopenia-induced model (a model of skeletal muscle atrophy induced by immobilization using a skin stapler in mice).

(30) A total of 25 4-week-old C57BL/6J mice (male) were tested using 5 mice per experimental group, and the experiment was performed according to the schedule shown in FIG. 1. First, the mice were given an acclimation period of 1 week and then fed lactic acid bacteria for 2 weeks from the age of 5 weeks. The control group (Ctrl) and the sarcopenia-induced group (Ctrl-Disuse) were given ad libitum access to sterile drinking water and conventional feed. The experimental groups (the groups that were fed lactic acid bacteria after inducing sarcopenia (F1, F3, and F4)) were given ad libitum access to lactic acid bacteria which had been diluted in sterile drinking water at 10.sup.7 CFU/ml. At 7 weeks of age, both hind legs of the mice were immobilized for 10 days using surgical skin staplers (Covidien Royal 35W skin stapler) (Caron, A. Z. et al., 2009; Du et al., 2011). After 10 days of immobilization of the legs, the staples were removed, and a recovery period of 3 days was given.

(31) After the recovery period, to examine the muscle rehabilitation capacity of the control group, sarcopenia-induced group, and experimental groups, the body weight (BW), TA weight per body weight (TA/Body weight), and the weights of 4 types of skeletal muscles (tibialis anterior; gastrocnemius; extensor digitorum longus; and soleus which will be referred to as TA, GA, EDL, and SOL, respectively hereinafter) were compared by removing the muscles, the results of which are provided in FIGS. 2 and 3.

(32) Referring to FIGS. 2A and 2B, the weights of the experimental groups with lactic acid bacteria intake increased compared to the sarcopenia-induced group (Ctrl-Disuse). TA/Body weight also increased in the experimental groups with lactic acid bacteria intake. In particular, BW and TA/Body weight of the experimental group with L. reuteri ATG-F4 lactic acid bacteria intake increased the most, indicating an outstanding muscle rehabilitation effect.

(33) Referring to FIGS. 3A through 3D, the weights of all 4 types of skeletal muscles (TA, GA, EDL, and SOL) increased in the experimental groups with lactic acid bacteria intake compared to the sarcopenia-induced group. In particular, the weights of TA, GA, and SOL of the experimental group with L. reuteri ATG-F4 lactic acid bacteria intake increased 1.21 times compared to the sarcopenia-induced group and the weight of EDL increased 1.62 times compared to the sarcopenia-induced group.

(34) Through this, weight and muscle mass increase as the result of recovery of sarcopenia or muscular atrophy was confirmed, and the increase in muscle mass and the rehabilitation effect were found to be the best in the experimental group with L. reuteri ATG-F4 lactic acid bacteria intake.

Experimental Example 2. Confirmation of the Effect of Muscle Mass Increase Due to Intake of F4 Lactic Acid Bacteria in a Healthy Model

(35) The groups were divided into a control group (Ctrl) fed with conventional feed and sterile drinking water and an experimental group (F4) fed with conventional feed and sterile drinking water including L. reuteri ATG-F4 at about 10.sup.7 CFU/ml, and 5 mice were used for each experimental group. After stabilizing 4-week-old mice for 1 week, the experiment was conducted for 4 weeks.

(36) After 4 weeks, 4 types of skeletal muscles (TA, GA, EDL, and SOL) were removed and the weights were compared to determine the difference in muscle mass due to intake of L. reuteri ATG-F4 lactic acid bacteria.

(37) Referring to FIG. 4, the muscle mass of the skeletal muscle of the experimental group (F4) fed with the L. reuteri ATG-F4 strain increased. Compared to the control group, the muscle mass of TA increased 1.08 times, GA 1.03 times, EDL 1.01 times, and SOL 1.48 times, confirming that there is an effect of increasing muscle mass even in a healthy model.

Experimental Example 3. Confirmation of Activation of Muscle Recovery and Improvement of Motor Ability in Sarcopenia-Induced Mice

Experimental Example 3-1. Treadmill Test

(38) A treadmill test was performed to evaluate motor ability and endurance, and the 5 lane treadmill for mice (HARVARD) was used for testing. A control group, a sarcopenia-induced group, and experimental groups (the groups that were fed lactic acid bacteria after inducing sarcopenia (F1, F3, and F4)) were set as in Experimental Example 1 and the experiment was performed. In addition, the treadmill test was performed as shown in FIG. 5 by referring to the protocol used for the running test.

(39) Referring to FIG. 5, first, the mice were made to warm up for 5 minutes at a speed of 8 m/min. At the starting point, electric stimulation of 1.25 mA was applied to induce running, and the speed was increased by 2 m/min every 15 minutes, starting at a speed of 10 m/min. Running time was measured up to a maximum of 2 hours in 8 steps. After 1 hour, the angle was raised 5, and after an hour and a half, the angle was raised to 10. Mice that did not run for 5 seconds despite the application of electrical stimulation were considered to be physically exhausted and the experiment was terminated. The running time and distance of the mice measured were compared after the termination of the experiment, which is provided in FIG. 6.

(40) Referring to FIGS. 6A and 6B, compared to the sarcopenia-induced group, the experimental groups with lactic acid bacteria intake showed a longer running time and distance. In particular, the experimental group with intake of L. reuteri ATG-F4 lactic acid bacteria showed increased motor ability and endurance based on a running time which was 1.17 times longer and a running distance which was 1.25 times longer compared to the sarcopenia-induced group, confirming outstanding effects of activating muscle regeneration and rehabilitation.

Experimental Example 3-2. Kondziela's Inverted Screen Test

(41) To evaluate muscle strength and endurance, Kondziela's inverted screen test was performed, and as in Experimental Example 1, a control group, a sarcopenia-induced group, and experimental groups (the groups that were fed lactic acid bacteria after inducing sarcopenia (F1, F3, and F4)) were set.

(42) For Kondziela's inverted screen test, a wire mesh screen (specification: 440330 mm, width/length of each grid being 1 mm respectively) was used (Deacon, R. M., 2013; Reekes, T. H. et al, 2016). After placing a mouse at the center of the grid and turning over the grid, the time was measured. Bedding material was laid on the floor to absorb shock from the fall, and the wire mesh screen was fixed at least 40 cm above the floor. The experiment was independently repeated three times, the fall time of the mouse was recorded, and the average value was obtained. Referring to Table 2 below, one point was given for every 60 seconds, and after 241 seconds, the maximum score of 5 was given across the board. The fall time and the score results according to the fall time are shown in FIG. 7.

(43) TABLE-US-00002 TABLE 2 Fall time of mouse (Unit:seconds) Score 1-60 1 61-120 2 121-180 3 181-240 4 241 and over 5

(44) Referring to FIGS. 7A and 7B, the experimental groups with lactic acid bacteria intake held on for a longer period of time than the sarcopenia-induced group. In particular, outstanding effects of activating muscle regeneration and rehabilitation were shown through increased muscle strength and endurance, which is exhibited by the hanging time and the score based on Table 2 of the experimental group with L. reuteri ATG-F4 intake being 2.4 times and 1.67 times greater, respectively, than those of the sarcopenia-induced group.