COMPOSITION COMPRISING LACTOBACILLUS SAKEI CVL-001 OR CULTURE LIQUID THEREOF FOR ALLEVIATING, PREVENTING, OR TREATING BONE DISEASES OR METABOLIC DISEASES

Abstract

The present invention relates to a Lactobacillus sakei CVL-001 strain and a method for isolating same, wherein the strain is lactic acid bacteria which can be isolated from kimchi, and thus can be effectively used as a probiotic or food additive. Furthermore, the present invention relates to a composition for improving, preventing, or treating bone diseases or metabolic diseases, including Lactobacillus sakei CVL-001 strain (Lactobacillus sakei) or a culture medium thereof, wherein the Lactobacillus sakei strain isolated from kimchi and the culture medium thereof exhibit the effects of suppressing osteoclast differentiation, improving osteoporosis, suppressing adipocyte differentiation, and suppressing weight gain, and thus can be effectively used for the improvement, prevention, or treatment of osteoporosis or obesity related diseases.

Claims

1. A Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP.

2. The Lactobacillus sakei CVL-001 strain of claim 1, wherein the strain includes a 16S rRNA sequence represented by SEQ ID NO: 1.

3.-4. (canceled)

5. A method for isolating a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP, the method comprising a step of culturing a kimchi extract.

6. The method of claim 5, wherein the kimchi extract is a kimchi-ground juice obtained by grinding kimchi and filtering the ground kimchi.

7. The method of claim 5, wherein the culturing step comprises a first culturing step for incubating the kimchi extract; and a second culturing step for culturing a bacterial strain isolated in the first culturing step.

8. The method of claim 7, wherein the first culturing step is conducted in a medium containing calcium carbonate.

9. The method of claim 7, wherein the first culturing step is conducted at 20 to 32° C. for 36 to 60 hours.

10. The method of claim 7, wherein the second culturing step is a step in which selection is made of a Gram-positive lactic acid bacterium from the isolated strains.

11. The method of claim 7, wherein the second culturing step is a step in which selection is made of a catalase-negative lactic acid bacterium from the isolated strains.

12. The method of claim 7, wherein the second culturing step is conducted at 20 to 32° C. for 12 to 36 hours.

13. A composition comprising the Lactobacillus sakei CVL-001 strain of claim 1, or a culture liquid thereof.

14.-16. (canceled)

17. The composition of claim 13, wherein the composition contains the Lactobacillus sakei CVL-001 strain at a concentration of 5×10.sup.5 to 5×10.sup.12 CFU/ml.

18. (canceled)

19. The composition of claim 13, wherein the composition contains the culture liquid at a concentration of 50 to 400 μl/ml.

20. The composition of claim 13, wherein the culture liquid has a pH of 6.5 to 8.5.

21.-28. (canceled)

29. The composition of claim 13, wherein the strain includes a 16S rRNA sequence represented by SEQ ID NO: 1.

30. A Method for preventing or treating a bone disease, the method comprising: administering to a subject in need thereof the composition of claim 13.

31. The method of claim 30, wherein the bone disease is selected from the group consisting of osteoporosis, bone metastatic cancer, osteomalacia, rickets, fibrous osteitis, adynamic bone disease, metabolic disease, and periodontal disease.

32. The method of claim 30, wherein the composition contains the Lactobacillus sakei CVL-001 strain at a concentration of 5×10.sup.5 to 5×10.sup.12 CFU/ml.

33. The method of claim 30, wherein the composition contains the culture liquid at a concentration of 50 to 400 μl/ml.

34. The method of claim 30, wherein the culture liquid has a pH of 6.5 to 8.5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1a is a photographic image showing an inhibitory effect of a Lactobacillus sakei CVL-001 strain culture liquid on osteoclast differentiation.

[0083] FIG. 1b is a graph showing an inhibitory effect of a Lactobacillus sakei CVL-001 strain culture liquid on osteoclast differentiation.

[0084] FIG. 2 is a photographic image showing western blots that account for an inhibitory effect of a Lactobacillus sakei CVL-001 strain culture liquid on the phosphorylation of osteoclast differentiation-inducing proteins.

[0085] FIG. 3 shows graphs that account for inhibitory effects of a Lactobacillus sakei CVL-001 strain culture liquid on the expression of osteoclast differentiation-inducing genes.

[0086] FIG. 4 is a graph showing the increasing effect of a Lactobacillus sakei CVL-001 strain and a culture liquid thereof on bone mineral density in osteoporosis-induced mouse models.

[0087] FIG. 5 is a graph showing an inhibitory effect of a Lactobacillus sakei CVL-001 strain culture liquid on adipocyte differentiation.

[0088] FIG. 6 is a plot showing inhibitory effects of a Lactobacillus sakei CVL-001 strain on weight gain in high fat diet-fed experimental groups with time.

[0089] FIG. 7 is a graph showing reductive effects of a Lactobacillus sakei CVL-001 strain on epididymal fat weight in high fat diet-fed experimental groups.

[0090] FIG. 8 is a plot showing inhibitory effects of a Lactobacillus sakei CVL-001 strain culture liquid on weight gain in high fat diet-fed experimental groups with time.

[0091] FIG. 9 is a graph showing reductive effects of a Lactobacillus sakei CVL-001 strain culture liquid on epididymal fat weight in high fat diet-fed experimental groups.

[0092] FIG. 10 is a plot showing antidiabetic effects of a Lactobacillus sakei CVL-001 strain culture liquid in high fat diet-fed experimental groups as measured by a glucose tolerance test.

[0093] FIG. 11 is a plot showing antidiabetic effects of a Lactobacillus sakei CVL-001 strain in high fat diet-fed experimental groups as measured by a glucose tolerance test.

[0094] FIG. 12 is a plot showing antidiabetic effects of a Lactobacillus sakei CVL-001 strain culture liquid in high fat diet-fed experimental groups as measured by an insulin tolerance test.

[0095] FIG. 13 is a plot showing antidiabetic effects of a Lactobacillus sakei CVL-001 strain in high fat diet-fed experimental groups as measured by an insulin tolerance test.

BEST MODE FOR CARRYING OUT THE INVENTION

[0096] The present disclosure is concerned with a Lactobacillus sakei CVL-001, deposited under accession number KCTC13816BP, having activity of alleviating, preventing, or treating a bone disease.

DETAILED DESCRIPTION

[0097] A better understanding of the present disclosure may be obtained via the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.

[0098] Unless stated otherwise, “%”, used to indicate concentrations of particular substances, stands for (wt./wt.) % for solid/solid, (wt./vol.) % for solid/liquid, and (vol./vol.) % for liquid/liquid throughout the specification.

Example 1: Isolation of Strain

[0099] Napa cabbage kimchi just after being prepared was collected from all parts of Korea. While being stored at 6±0.5° C. from week 0 to week 5, the collected kimchi was used as kimchi samples for isolating lactic acid bacteria. A hand blender (Hanil Co, Korea) was used to grind 500 g of the kimchi, followed by filtration through sterile gauze. The filtrate was diluted with sterile water in a serial manner from 101 to 10.sup.7 fold. Finally, the 10.sup.7-fold diluted kimchi juice was spread over an agar.

[0100] The diluted kimchi juice was spread over an MRS agar (Difco Co., France) containing 2% of calcium carbonate (CaCO.sub.3) and incubated at 30° C. for 48 hours before picking up colonies with halo. The picked colonies were subjected to Gram staining (Gram stain kit, BD Co., USA) and catalase testing (Biomerieux Co., France) to tentatively select Gram-positive, catalase-negative colony as lactic acid bacteria.

[0101] The lactic acid bacterium strains thus isolated were inoculated into an MRS broth, incubated at 30° C. for 24 hours, and added with 25% (v/v) of glycerol to prepare glycerol stocks which were stored at −70° C. until use.

Example 2: Identification of Lactic Acid Bacteria

[0102] The finally selected lactic acid bacterium Lactobacillus sakei CVL-001 was identified by 16S rRNA sequencing. After purely isolated, the strains spread on MRS plates were delivered via refrigerated parcel service to SolGent (Daejeon, Korea) in which the strains were analyzed by 16S rRNA sequencing with a universal primer set of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).

[0103] The isolated strain Lactobacillus sakei CVL-001 was determined to have the 16S rRNA coding nucleotide sequence consisting of a total of 1,439 bp shown in Table 1, below.

TABLE-US-00001 TABLE 1 SEQ ID NO: Name Sequence 1 16S rRNA GCAGTCGAACGCACTCTCGTTTAGATTGAAGGAGCTTGCTCCTGATTGA coding TAAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCT nucleotide GCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAA sequence of AACCTAACACCGCATGGTGTAGGGTTGAAAGATGGTTTCGGCTATCACT Lactobacillus TTAGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCA sakei CVL-001 CCAAGACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTG GGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAAT CTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAG AAGGTTTTCGGATCGTAAAACTCTGTTGTTGGAGAAGAATGTATCTGATA GTAACTGATCAGGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAAC TACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGA TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAA AGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAG TGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGA TATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTG ACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGG TAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCC CTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGA CCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTT GACATCCTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAG TGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAG TTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGG GACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCT ACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCT CTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGA AGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGT TCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACA CCCAAAGCCGGTGAGGTAACCCTTCGGGGAGCCAGCCG 2 27F forward AGAGTTTGATCCTGGCTCAG primer 3 1492R backward GGTTACCTTGTTACGACTT primer

[0104] With respect to the nucleotide sequence obtained above, sequence similarity search was run with the Blast program (www.ncbi.nlm.nhi.gov) for the registered database (GenBank database). As a result, the strain was finally identified as Lactobacillus sakei LZ217 because of a similarity of 99% therewith.

[0105] In light of the results obtained above, that is, morphological traits, sugar metabolism ability, and 16S rRNA sequencing results, the Lactobacillus sakei CVL-001 strain with excellent mannitol productivity and viability (predominancy) in kimchi juice was identified as Lactobacillus sakei LZ217.

[0106] The isolated strain was termed Lactobacillus sakei CVL-001 and deposited with the Korean Collection for Type Culture located at 823, Shinjung-dong, Jeongeup City, Jeollabuk-do, Korea.

Example 3: Preparation of Lactobacillus sakei CVL-001 Strain Culture Liquid

[0107] A culture for use in application to cells was obtained by culturing Lactobacillus sakei CVL-001 for 24 hours in alpha-MEM medium, centrifuging the cell culture, and adjusting the separated supernatant into a pH of 7.4.

Example 4: Assay for Inhibitory Activity of Lactobacillus sakei CVL-001 Strain Culture Liquid Against Osteoclast Differentiation

[0108] Mouse macrophages were seeded at a density of 2×10.sup.5 cells/well into 12-well plates and incubated for 24 hours. After the medium was changed with a medium supplemented with fetal bovine serum (hereinafter referred to as “FBS”), 1% penicillin-streptomycin (hereinafter referred to as “PS”), and macrophage colony stimulating factor (hereinafter referred to as “M-CSF”), the cells were incubated with the culture liquid (50, 100, and 200 μl/ml) for 2 hours. Then, the cells were treated with 100 ng/ml RANKL (receptor activator of nuclear factor kappa-B ligand) for 24 hours and allowed to differentiate for 6 days in the manner described above.

[0109] Subsequently, a chromogenic substrate for the cytochemical enzyme marker tartrate-resistant acid phosphatase (hereinafter referred to as “TRAP”) was added to stain the nuclei. Multinucleated cells with three or more nuclei were observed and imaged.

[0110] When applied to macrophages, RANKL binds to RANK to cause differentiation into TRAP positive cells. Stimulation of TRAP-positive cells with an inflammatory factor such as RANKL, TNF-α, etc. renders cell fusion into multinuclear TRAP positive cells.

[0111] As shown in FIGS. 1a and 1b and Table 2, treatment of macrophage with RANKL increased differentiation into osteoclasts and the culture liquid was observed to significantly decrease the count of osteoclasts in a dose-dependent manner. From the result, it was understood that the Lactobacillus sakei CVL-001 culture liquid can inhibit osteoclast differentiation.

TABLE-US-00002 TABLE 2 RANKL − − + + + + Culture liquid − 200 − 50 100 200 Counts of osteoclast 0 0 1,165 151 10 0 (Avg.)

Example 5: Assay for Inhibitory Activity of Lactobacillus sakei CVL-001 Strain Culture Liquid Against Expression of Osteoclast Differentiation-Inducing Protein

[0112] Examination was made of the MAPK (mitogen-activated protein kinase) and NF-κB signaling mechanism when mouse macrophages were treated with RANKL and the culture liquid. To this end, Mouse macrophages were seeded at a density of 4×10.sup.5 cells/well into 12-well plates and incubated for 24 hours. After the medium was changed with a medium supplemented with FBS, 1% PS, and M-CSF, the Lactobacillus sakei CVL-001 strain culture liquid (100 μl/ml) was incubated for 2 hours. Then, the cells were treated with 100 ng/ml RANKL for 0, 5, 15, and 30 min.

[0113] After the medium was removed, proteins were extracted with a protein lysis buffer and quantitated and 30 μg of the proteins was separated in SDS-PAGE gel. The proteins were transferred onto a membrane, incubated with primary and secondary antibodies (p-jnk (Cell Signaling Technology, #9251S), p-p38 (Cell Signaling Technology, 9211S), p-ERK (Santa Cruz, sc-7383), Ikbalpha (Cell Signaling Technology, 9242S), p-p65 (Cell Signaling Technology, 3031 S)), sequentially. Color development was made using a kit (BIO-RAD, ECL solution detection kit) and measured by a device (Chemidoc).

[0114] RANKL binds to RANK to upregulate the expression of MAPK and NF-κB via TRAF6 and induce the activation of various transcription factors, leading to the promotion of osteoclast differentiation. Examination was thus made to see whether the Lactobacillus sakei culture liquid intervenes with the mechanisms to inhibit osteoclast differentiation. In the regard, phosphorylation of the MAPK proteins ERK, JNK, and p38 was analyzed by western blotting.

[0115] As a result, increased phosphorylation levels of JNK, P38, and ERK were observed 5 and 15 min after incubation with RANKL whereas the MAPK proteins were dephosphorylated by treatment with the culture liquid. In addition, inhibition was observed on both the degradation of IkB alpha and the phosphorylation of p65.

[0116] As is understood from the data of FIG. 2, the Lactobacillus sakei culture liquid inhibits the phosphorylation of JNK, P38, and ERK, thereby suppressing differentiation into osteoclasts.

Example 6: Assay for Effect of Lactobacillus sakei Culture Liquid on Expression of Osteoclast Differentiation-Inducing Gene

[0117] After being treated with the Lactobacillus sakei culture liquid, the mouse macrophages were assayed for gene expression of TRAP, DC-STAMP, Cathepsin K, and NFATc1, which are genes involved in osteoclastogenesis, through real-time PCR. Bone marrow-derived macrophages (hereinafter referred to as “BMDMs”) were seeded at a density of 1×10.sup.5 cells/well into 12-well plates and incubated for 24 hours. Next, the cells were treated with a FBS-free medium supplemented with 1% PS only for 1 hour and then with 100 μl/ml Lactobacillus sakei culture liquid for 2 hours, followed by incubation with RANKL for 24 hours. In this manner, the cells were subjected to differentiation for 3 days before RNA isolation from the cells with the aid of TRIzol solution. On the basis of RNA quantitation, RT premix was used to synthesize cDNA which was then amplified by real-time PCR using primers.

[0118] The primers used were designed for Mouse TRAP, DC-STAMP, Cathepsin K, and NFATc1, which were assayed in relative amounts to the control gene GAPDH.

[0119] Macrophages undergo differentiation into TRAP-positive, multinucleated osteoclasts when treated When treated with RANKL, which binds to RANK. In addition, treatment with RANKL activates NFATc1, which is a transcription factor playing an important role in differentiation into osteoclasts, and upregulates the expression of TRAP, Cathepsine K, and DC-STAMP, which are involved in osteoclastogenesis.

[0120] As can be seen in FIG. 3 and Table 3, the expression levels of TRAP, DC-STAMP, Cathepsin K, and NFATc1, which were elevated by treatment with RANKL, were decreased by treatment with Lactobacillus sakei culture liquid. These data imply that the Lactobacillus sakei culture liquid inhibits the activity of the transcription factor NFATc1 to downregulate the expression of TRAP, DC-STAMP, and Cathepsin K, which are genes involved in osteoclastogenesis, thereby suppressing osteoclast differentiation.

TABLE-US-00003 TABLE 3 RANKL − + − + Culture liquid − − + + TRAP 1 104,133 201.9 7,122 DC-STAMP 1 38,698 72 8,539 Cathepsin K 1 3,983 210 709 NFATc1 1 913,838 922 85,284

Example 7: Construction of Osteoporosis Animal Model by Mouse Ovariectomy

[0121] C57BL/6 female mice (7 weeks old) were housed in plastic cages maintained at a temperature of 22±2° C. and a relative humidity of 50±10% with a photoperiod of 12-h light and 12-h dark. The mice were acclimated to the same environment for about one week before ovariectomy.

[0122] The mice were anesthetized using an intramuscular injection of Zoletil and Lumpun, after which the ovariectomy region was shaved and sterilized. A 1-cm incision was made on the skin. Then, the ovary was detected along the uterus with care to exert no injuries on other organs and ligated with a suture so as to resect the opposite ovaries. After ovariectomy, the organs were each repositioned within the abdomen and the incision was sutured with a thread. From day 10, a therapeutic substance was administered for 8 weeks.

[0123] Administration of lactic acid bacteria: After pre-culture and main culture, Lactobacillus sakei was divided into aliquots of 1×10.sup.7, 1×10.sup.8, and 1×10.sup.9 cells/ml and orally administered.

[0124] Administration of culture liquid: After pre-culture and main culture, Lactobacillus sakei was divided into aliquots of 1×10.sup.7, 1×10.sup.8, and 1×10.sup.9 cells/ml. The cell aliquots were 10-fold diluted and cultured in an MRS medium at 30° C. for 24 hours. The cells were settled by centrifugation, and the culture liquid supernatant was collected and adjusted into a pH of 7.4 before oral administration at a dose of 200 μl.

[0125] Test groups were divided as follows:

[0126] C57BL/6 mice; 80 heads

[0127] (1) G1: non-ovariectomized sham, 10 heads, MRS medium (control) administered

[0128] (2) G2: non-ovariectomized sham, 10 heads, culture liquid administered

[0129] (3) G3: non-ovariectomized sham, 10 heads, Lactobacillus sakei administered at a dose of 1×10.sup.9 cells/ml

[0130] (4) G4: ovariectomized test group (OVX), 10 heads, MRS medium (control) administered

[0131] (5) G5: ovariectomized test group (OVX), 10 heads, culture liquid (1/4 diluted) administered

[0132] (6) G6: ovariectomized test group (OVX), 10 heads, culture liquid (1/2 diluted) administered

[0133] (7) G7: ovariectomized test group (OVX), 10 heads, culture liquid (stock) administered

[0134] (8) G8: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×10.sup.7 cells/ml

[0135] (9) G9: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×10.sup.8 cells/ml

[0136] (10) G10: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×10.sup.9 cells/ml

Example 8: Evaluation of Bone Mineral Density in Osteoporosis-Induced Mice According to Administration of Lactobacillus sakei and Culture Liquid Thereof

[0137] Bone mineral densities were measured using microcomputed tomography (micro-CT), which can provide high-definition images than simple radiography or computed tomography.

[0138] After completion of the experiment, the mouse legs were fixed with formalin, and analyzed for bone mineral density in the Laboratory Animal Center of Daegu-Gyeongbuk Medical Innovation Foundation.

[0139] Ovariectomized mice are known to decrease in bone mineral density due to estrogen inefficiency and as such, are widely used for studying osteoporosis diseases.

[0140] As can be seen in FIG. 4 and Table 4, the ovariectomized mouse group (G4) was observed to significantly decrease in bone mineral density, compared to the sham groups. Meanwhile, the Lactobacillus sakei strain and the culture liquid thereof increased the bone mineral density in a dose-dependent manner. Particularly, the groups to which the culture liquid stock (G7) and a high concentration of the bacteria (G10) were administered recovered the bone mineral density to levels as high as those in the non-ovariectomized sham groups.

TABLE-US-00004 TABLE 4 G1 G2 G3 G4 G5 G6 G7 G8 G9 G10 Bone Mineral 245.9 260.9 241.7 215.3 221.7 230.3 238.6 217.6 229.1 234.8 Density (mean) (mg/cc)

Example 9: Assay for Inhibitory Effect of Culture Liquid of Lactobacillus sakei CVL-001 Isolated from Kimchi on Adipocyte Differentiation (In Vitro)

[0141] 9-1. Preparation of Lactobacillus sakei CVL-001 Culture Liquid

[0142] Lactobacillus sakei CVL-001 was cultured at a density of 1×10.sup.8 CFU/ml in DMEM medium for 24 hours, followed by centrifugation. The supernatant thus obtained was adjusted into a pH of 7.4 to afford a Lactobacillus sakei CVL-001 culture liquid.

[0143] 9-2. Adipose Differentiation Using Undifferentiated Preadipocyte Strain 3T3-L1

[0144] Preadipocyte 3T3-L1 purchased from ATCC (American type culture collection, USA) was maintained by passage in DMEM supplemented with 10% bovine calf serum (BCS) and 1% PS. The day at which the cells reached 100% confluence after being seeded into plates was designated day −2. While the cells were cultured for an additional two days, the cell cycle was arrested at G1 phase in all the cells cultured in the plates.

[0145] For day 0, the cells were allowed to start differentiation in the presence of IBMX (3-isobutyl-1-methylxanthine), dexamethasone, and insulin (MDI). On day 2, the medium was changed with a medium containing insulin only. From day 4, the medium was changed with 10% fetal bovine serum (FBS)-supplemented medium every two days until differentiation.

[0146] 9-3. Assay for Inhibitory Activity of Lactobacillus sakei CVL-001 Culture Liquid Against Adipocyte Differentiation (Oil Red O Staining)

[0147] Adipocyte differentiation was induced when bovine calf serum (BCS) in the medium composition was substituted with FBS. 3T3-L1 cells were seeded at a density of 5×10.sup.5 cells/well into 12-well plates containing a medium supplemented with 10% FBS and 1% PS. On two days after the cells reached 100% confluence, that is, on day 0, the cells were treated with the culture liquid (12.5, 25.0, and 50.0%) for 2 hours and then with the adipogenesis inducer MDI.

[0148] The culture liquid was prepared as a result of culturing L. sakei at a concentration of 1×10.sup.8 cfu/ml in DMEM (High glucose)+FBS (10%)+PS (1%) medium for 24 hours, and dilution of the culture liquid was conducted with DMEM medium.

[0149] On day 2, the cells were incubated with the culture liquid (12.5, 25.0, and 50.0%) for 2 hours while the medium was changed, followed by treatment with insulin. On day 4, the cells were incubated with the culture liquid (12.5, 25.0, and 50.0%) while the medium was changed. The same procedure was repeated every two days until differentiation.

[0150] When differentiated adipocytes were observed, the medium was changed with 4% formalin to fix the cells for 10 min. For adipocyte staining, the cells were washed twice with D.W. and incubated with a mixture of 6:4 Oil red O staining: D.W. for 30 min. Washing with D.W. was followed by microscopic observation. Then, Oil red O was extracted with 100% isopropanol and quantitative analysis was performed by reading absorbance at 510 nm.

[0151] As is understood from the data of FIG. 5 and Table 5, 3T3-L1 cells underwent adipogenic differentiation when MDI was added to the cell culture medium. In this study result, exposure of 3T3-L1 cells to MDI increased adipogenic differentiation whereas the culture liquid was observed to significantly reduce counts of adipocytes in a dose-dependent manner.

TABLE-US-00005 TABLE 5 Culture liquid (%) — 12.5 25.0 50.0 Degree of adipogenic 5.7052 4.1410 2.0922 2.0940 differentiation

[0152] Taken together, the data imply that the Lactobacillus sakei CVL-001 culture liquid can inhibit adipogenic differentiation.

Example 10: Construction of Obesity Mouse Animal Model for Assaying Reductive Effect on Obesity (In Vivo)

[0153] 10-1. Preparation of Live Lactobacillus sakei CVL-001

[0154] Live bacteria to be applied to mice were prepared. In brief, the bacteria were cultured for 24 hours at 30° C. in MRS agar plates, and a single colony thus formed was picked and pre-cultured in MRS broth. The pre-culturing was conducted in 5 ml of the medium at 30° C. for 24 hours while shaking at 150 rpm. Subsequently, a 10-fold dilution of the culture was amplified for 3 hours in the same conditions.

[0155] In order to keep a balance between cell counts, the cultures finally obtained were diluted in PBS until the optical density at 600 nm (OD600) reached 0.6 as measured by a spectrophotometer. The O.D. value accounts for the existence of the lactic acid bacteria at a concentration of 0.89×10.sup.9 CFU/ml. With the administration of 200 μl to each mouse (1×10.sup.8 CFU/mouse, 1×10.sup.9 CFU/mouse) in mind, live bacterial media were prepared by centrifugation at 3,000 RPM for 15 min.

[0156] 10-2. Preparation of Lactobacillus sakei CVL-001 Culture Liquid

[0157] An MRS medium in which the amplification of Lactobacillus sakei had been completed was 10-fold diluted was incubated at 30° C. for 24 hours while shaking at 150 rpm. After centrifugation at 3,000 RPM for 15 min, the supernatant was separated and adjusted into a pH of 7.4. Filtration through 0.45 μm membrane filter paper was conducted before storage at 4° C.

[0158] 10-3. Assay for Anti-Obesity Activity of Lactobacillus sakei CVL-001 Strain and Culture Liquid (In Vivo)

[0159] To C57BL/6 male mice at 7 weeks of age were administered the live bacteria (single daily dose 200 μl/subject), the culture liquid (single daily dose 200 μl/subject), and a high fat diet. Subsequently, the mice were weighed every two weeks. At week 11, the mice were measured for blood sugar levels. At week 14, an autopsy was performed on the mice, after which blood was sampled and the organs were weighed.

[0160] Information on the groups used for the strain test was as follows:

[0161] G1: Control group (normal diet fed), PBS (phosphate buffered saline) orally administered (n=6)

[0162] G2: Control group, 10.sup.9 L. sakei orally administered (n=6)

[0163] G3: High fat group (high fat diet fed), PBS orally administered (n=8)

[0164] G4: High fat group, 10.sup.8 L. sakei orally administered (n=8)

[0165] G5: High fat group, 10.sup.9 L. sakei orally administered (n=8)

[0166] Information on groups used for culture liquid testing was as follows:

[0167] G1: Control group (normal diet fed), MRS orally administered (n=6)

[0168] G2: Control group, culture liquid (stock) orally administered (n=6)

[0169] G3: High fat group (high fat diet fed), MRS orally administered (n=8)

[0170] G4: High fat group, culture liquid (1/2 diluted) orally administered (n=8)

[0171] G5: High fat group, culture liquid (stock) orally administered (n=8)

Example 11: Assay for Effect of Lactobacillus sakei CVL-001 Strain Isolated from Kimchi and Culture Liquid Thereof (In Vivo)

[0172] 11-1. Change in Body Weight by Lactobacillus sakei CVL-001 Strain or Culture Liquid Thereof (In Vivo)

[0173] Oral administration of a high fat diet including 60% of fats is known to further increase the weight of mice than that of a normal diet. Thus, high fat diet-induced obesity animal models are widely used for studies into obesity diseases.

[0174] As can be seen in FIG. 6, significant weight gain was observed in the high fat diet-fed group (G3), compared to the normal diet-fed group (G1). Particularly, the groups (G4 and G5) to which the Lactobacillus sakei CVL-001 strain was fed at dose of 10.sup.8 and 10.sup.9 cells, respectively, together with the high fat diet were observed to significantly decrease in weight, compared to G3.

TABLE-US-00006 TABLE 6 G1 G2 G3 G4 G5 Diet Normal Normal High fat High fat High fat Strain PBS L. sakei 10.sup.9 PBS L. sakei 10.sup.8 L. sakei 10.sup.9 Weight (g) 0.80 0.85 2.29 2.23 1.93

[0175] As shown in FIG. 7 and Table 6, G3 to which the high-fat was fed significantly increased in epididymal fat weight, compared to G1 while a significant reduction of epididymal fat weight was observed in G5 to which the strain had been fed at a dose of 10.sup.9 cells, together with a high fat diet. As shown in FIG. 8, the high fat diet-fed group (G3) was observed to significantly increase in body weight, compared to the normal diet-fed group (G1). Particularly, a significant reduction of body weight was detected in the group to which the Lactobacillus sakei CVL-001 culture liquid stock (G5) was fed, together with the high fat diet, compared to G3. The high fat diet-induced weight gain was not suppressed by administering the 1/2 diluted culture liquid (G4).

TABLE-US-00007 TABLE 7 G1 G2 G3 G4 G5 Diet Normal Normal High fat High fat High fat Culture MRS Culture liquid MRS Culture liquid Culture liquid liquid stock 1/2 diluted stock Weight (g) 0.744 0.691 2.579 2.065 1.768

[0176] As is understood from data of FIG. 9 and Table 7, the epididymal fat weight was also significantly increased in the high fat diet-fed group G3 and significantly decreased in G5 to which the culture liquid stock had been fed, along with the high fat diet, compared to the normal diet fed group G1.

[0177] 11-2. Fasting Blood Sugar Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid

[0178] After being fasted for 12 hours from p.m. 9 before the day of experiment to a.m. 9 on the day of experiment, mice were subjected to a blood sugar test. The high fat-fed mouse animal models were measured to maintain high fasting blood sugar levels. Tests for experimental groups were conducted in the same conditions as in Example 10-3, and the groups to which the strain and the culture liquid were administered are listed in Tables 8 and 9, respectively.

TABLE-US-00008 TABLE 8 Group Fasting blood glucose (mg/dL) G1: ND + control 106.13 ± 12.81.sup.a G2: ND + L. sakei 10{circumflex over ( )}9  96.00 ± 16.49.sup.a G3: HFD + control 147.75 ± 19.71.sup.b G4: HFD + L. sakei 10{circumflex over ( )}9 117.63 ± 25.04.sup.a G5: HFD + L. sakei 10{circumflex over ( )}9 108.50 ± 30.52.sup.a

[0179] As can be seen in Table 8, the high fat-fed group G3 maintained a significantly high fasting blood glucose level, compared to the normal diet-fed group G1 in this experiment. Particularly, blood glucose levels in G4 and G5 to which the Lactobacillus sakei CVL-001 strain had been administered at different doses, together with the high fat diet, were lower than that of G3 and similar to that of the normal diet-fed group G1.

TABLE-US-00009 TABLE 9 Group Fasting blood glucose (mg/dL) G1: ND + MRS 104.00 ± 15.6.sup.a G2: ND + sup 110.25 ± 20.8.sup.a G3: HFD + MRS 140.00 ± 28.7.sup.b G4: HFD + sup(1/2)  129.38 ± 23.52.sup.b G5: HFD + sup  106.75 ± 15.23.sup.ab

[0180] As can be seen in Table 9, a significantly high fasting blood glucose level was maintained in the high fat diet-fed group G3, compared to the normal diet-fed group G1 in this experiment. Particularly, the blood glucose level in G5 to which the Lactobacillus sakei CVL-001 culture liquid stock had been fed, together with the high fat diet was observed to be low, compared to that of the culture liquid stock-administered group G3, and similar to that of the normal diet-fed group G1.

[0181] 11-3. Glucose Tolerance Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid

[0182] Tests for experimental groups were conducted in the same conditions as in Example 10-3, and mice were subjected to a glucose tolerance test after being fasted for 12 hours from p.m. 9 before the day of experiment to a.m. 9 on the day of experiment. A PBS solution containing 10% glucose, sterilized by 0.2 μm filtration) was intraperitoneally administered (27-gauge sterile needle) at a dose of 2 mg (glucose/g. volume (μl)=body weight (g)×20) to each mouse. The mice were housed again in cages and measured for blood glucose level at time intervals of 0, 30, 60, 90, and 120 min. For the glucose test, blood was taken from tails. Measurements are given in Tables 10 and 11 and FIGS. 10 and 11.

TABLE-US-00010 TABLE 10 Group 0 30 60 90 120 (min) Normal Diet 110.60 ± 239.60 ± 170.40 ± 114.60 ± 97.00 ± MRS 17.52.sup.a 35.70.sup.a 32.99.sup.a 5.75.sup.a 12.38.sup.a Normal Diet 113.20 ± 236.40 ± 166.00 ± 119.40 ± 108.40 ± sup 17.38.sup.a 13.99.sup.a 13.81.sup.a 12.63.sup.a 8.43.sup.ab High Fat Diet 159.00 ± 392.60 ± 280.80 ± 221.40 ± 179.80 ± MRS 25.01.sup.b 45.12.sup.c 75.26.sup.b 37.10.sup.b 30.93.sup.c High Fat Diet 121.20 ± 407.20 ± 252.40 ± 213.20 ± 171.60 ± sup(1/2) 25.05.sup.a 36.35.sup.c 66.90.sup.b 47.41.sup.b 52.83.sup.c High Fat Diet 119.80 ± 327.00 ± 246.80 ± 185.20 ± 145.40 ± sup 19.23.sup.a 45.83.sup.b 36.99.sup.b 16.09.sup.b 9.69.sup.bc

[0183] As shown in Table 10 and FIG. 10, the high fat diet-fed group (G3) was observed to maintain a significantly high blood sugar level, compared to the normal diet-fed group (G1). Particularly, a significant reduction of blood glucose level was detected in the group to which the Lactobacillus sakei CVL-001 culture liquid stock (G5) was administered, together with the high fat diet, compared to G3, from 30 min after administration. The high fat diet-induced blood glucose increase was not suppressed by administering the 1/2 diluted culture liquid (G4

TABLE-US-00011 TABLE 11 Group 0 30 60 90 120 (min) Normal Diet 89.00 ± 261.80 ± 175.40 ± 136.60 ± 114.40 ± PBS 11.58.sup.a 41.70.sup.a 28.08.sup.a 21.00.sup.a 10.59.sup.a Normal Diet 90.60 ± 285.60 ± 188.80 ± 137.00 ± 114.20 ± L. sakei 10{circumflex over ( )}9 10.15.sup.a 55.98.sup.ab 35.72.sup.ab 17.94.sup.a 14.01.sup.a High Fat Diet 163.20 ± 352.60 ± 236.00 ± 194.00 ± 155.40 ± PBS 21.23.sup.c 68.44.sup.b 54.17.sup.b 39.28.sup.b 3.72.sup.b High Fat Diet 124.00 ± 335.00 ± 195.60 ± 167.20 ± 136.20 ± L.sakei 10{circumflex over ( )}8 26.51.sup.b 38.30.sup.ab 20.55.sup.ab 34.32.sup.ab 13.42.sup.b High Fat Diet 106.80 ± 296.00 ± 213.60 ± 163.60 ± 145.80 ± L. sakei 10{circumflex over ( )}9 18.15.sup.ab 33.75.sup.ab 25.42.sup.ab 21.27.sup.ab 19.20.sup.b

[0184] As can be seen in Table 11 and FIG. 11, a significant high level of blood glucose was observed in the high fat diet-fed group (G3), compared to the normal diet-fed group (G1). Particularly, the groups (G4 and G5) to which the Lactobacillus sakei CVL-001 strain was fed at dose of 10.sup.8 and 10.sup.9 cells, respectively, together with the high fat diet were observed to significantly decrease in blood glucose, compared to G3.

[0185] 11-4. Insulin Tolerance Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid

[0186] Tests for experimental groups were conducted in the same conditions as in Example 10-3, and mice were subjected to an insulin tolerance test after being fasted for 4 hours from a.m. 9 to p.m. 1 on the day of experiment. To each mouse, 2 mg/ml insulin (in D.W. adjusted into a pH of 3 by diluted HCl) was intraperitoneally administered (27-gauge sterile needle) at a dose of 1 U (0.03846 mg/kg. Volume (ul)=Body weight (g)×20). The mice were housed again in cages and measured for blood glucose level at time intervals of 0, 30, 60, 90, and 120 min. For the glucose test, blood was taken from tails.

TABLE-US-00012 TABLE 12 Group 0 30 60 90 120 (min) Normal Diet 130.00 ± 74.60 ± 67.20 ± 66.80 ± 70.80 ± MRS 10.06.sup.a 9.77.sup.a 15.45.sup.a 14.19.sup.a 8.30.sup.a Normal Diet 130.20 ± 75.60 ± 68.80 ± 65.00 ± 74.60 ± L. sakei sup 6.58.sup.a 11.48.sup.a 24.21.sup.a 19.83.sup.a 19.30.sup.a High Fat Diet 179.40 ± 119.80 ± 117.00 ± 145.40 ± 183.60 ± MRS 17.29.sup.b 14.40.sup.b 27.09.sup.b 22.22.sup.b 34.42.sup.c High Fat Diet 180.40 ± 114.80 ± 95.60 ± 129.40 ± 119.40 ± L. sakei sup(1/2) 25.83.sup.b 21.83.sup.b 18.68.sup.ab 62.94.sup.b 38.71.sup.b High Fat Diet 156.60 ± 110.20 ± 89.00 ± 71.00 ± 83.40 ± L. sakei sup 13.00.sup.b 18.45.sup.b 25.34.sup.ab 20.00.sup.a 18.52.sup.ab

[0187] As shown in Table 12 and FIG. 12, the high fat diet-fed group (G3) was observed to maintain a significantly high blood sugar level, compared to the normal diet-fed group (G1). Particularly, a significant reduction of blood glucose level was detected in the group to which the Lactobacillus sakei 9VL-001 culture liquid stock (G5) was administered, together with the high fat diet, compared to G3, at 90 and 120 mi after administration. The group to which the 1/2 diluted culture liquid was administered (G4)

TABLE-US-00013 TABLE 13 Group 0 30 60 90 120 (min) Normal Diet 140.40 ± 96.60 ± 75.60 ± 65.80 ± 76.80 ± PBS 16.14.sup.a 13.48.sup.ab 8.69.sup.a 7.76.sup.a 10.91.sup.a Normal Diet L. 126.40 ± 77.20 ± 69.00 ± 56.20 ± 66.20 ± sakei 10{circumflex over ( )}9 13.20.sup.a 9.41.sup.a 13.80.sup.a 14.30.sup.a 12.89.sup.a High Fat Diet 180.60 ± 142.80 ± 114.20 ± 153.60 ± 171.40 ± PBS 22.66.sup.b 31.04.sup.c 16.08.sup.b 37.32.sup.b 23.14.sup.c High Fat Diet 178.00 ± 98.60 ± 81.20 ± 83.20 ± 115.80 ± L. sakei 10{circumflex over ( )}8 12.54.sup.b 10.95.sup.ab 19.05.sup.a 17.68.sup.a 34.08.sup.b High Fat Diet L. 177.20 ± 121.00 ± 82.20 ± 85.20 ± 80.00 ± sakei 10{circumflex over ( )}9 28.22.sup.b 19.17.sup.bc 23.13.sup.a 13.12.sup.a 18.48.sup.a

[0188] As can be seen in Table 13 and FIG. 13, a significant high level of blood glucose was observed in the high fat diet-fed group (G3), compared to the normal diet-fed group (G1). Particularly, the groups (G4 and G5) to which the Lactobacillus sakei CVL-001 strain was fed at dose of 10.sup.8 and 10.sup.9 cells, respectively, together with the high fat diet were observed to significantly decrease in blood glucose, compared to G3, at all times except for 0 min after administration.

INDUSTRIAL APPLICABILITY

[0189] The present disclosure relates to a Lactobacillus sakei(Lactobacillus sakei) CVL-001 strain and an isolation method therefor and, more specifically, to a Lactobacillus sakei CVL-001 strain isolated and identified from kimchi.

[0190] In addition, the present disclosure relates to a composition comprising the Lactobacillus sakei CVL-001 strain or a culture liquid thereof for alleviation, prevention, or treatment of a bone disease and, more specifically, to a composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP or a culture liquid thereof for alleviation, prevention, or treatment of a bone disease.