NOVEL LACTIC ACID BACTERIA AND USE THEREOF

Abstract

The present disclosure relates to Lactobacillus plantarum NK3 and Bifidobacterium longum NK49, which are novel lactic acid bacteria, and, more particularly, to a composition comprising novel lactic acid bacteria that are useful for the prevention or treatment of female menopausal disorders.

Claims

1. (canceled)

2. (canceled)

3. (canceled)

4. (canceled)

5. (canceled)

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. A method for preventing or treating female menopausal disorders comprising administering to a subject in need thereof an effective amount of Bifidobacterium longum NK49 (accession number: KCCM12088P).

13. The method of claim 12, wherein the method further comprises administering an effective amount of Lactobacillus plantarum NK3 (accession number: KCCM12089P).

14. The method of claim 12, wherein the Bifidobacterium longum NK49 (accession number: KCCM12088P) comprises a 16S rDNA sequence of SEQ ID NO: 2.

15. The method of claim 13, wherein Lactobacillus plantarum NK3 (accession number: KCCM12089P) comprises a 16S rDNA sequence of SEQ ID NO: 1.

16. The method of claim 12, wherein the female menopausal disorder is at least one selected from the group consist of hot flush, sweating, facial flushing, depression, anxiety, vaginal dryness, vaginal atrophy, urinary incontinence, obesity, hypertension, diabetes, skin aging and osteoporosis.

17. The method of claim 16, wherein the female menopausal disorder is osteoporosis, obesity, or depression.

18. The method of claim 12, the Bifidobacterium longum NK49 (accession number: KCCM12088P) is a live bacterial body thereof, a dead bacterial body thereof, a culture thereof, a lysate thereof, or an extract thereof.

19. The method of claim 13, the Lactobacillus plantarum NK3 (accession number: KCCM12089P) is a live bacterial body thereof, a dead bacterial body thereof, a culture thereof, a lysate thereof, or an extract thereof.

20. The method of claim 13, wherein a ratio of mixture of Lactobacillus plantarum NK3 (accession number: KCCM12089P) and Bifidobacterium longum NK49 (accession number: KCCM12088P) is 1:1 to 4:1 CFU.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The LP in drawings indicates the results of Lactobacillus plantarum NK3, while the BP indicates the results of Bifidobacterium longum NK49.

[0059] FIG. 1 is a graph showing an effect of increasing the weight of the womb with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0060] FIG. 2 is a graph showing an effect of increasing the weight of the thighbone with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0061] FIG. 3 is a graph showing an effect of recovering the length of the thighbone with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0062] FIG. 4 is a graph showing an inhibitory capacity of obesity with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0063] FIG. 5 is an image showing an effect of increasing the AMPK activity with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0064] FIG. 6 is a graph showing an effect on corticosterone concentrations with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0065] FIG. 7 is a graph showing an effect of alleviating depression through an elevated plus maze (EPM) experiment with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0066] FIG. 8 is a graph showing an effect of alleviating depression through a forced swimming test (FST) with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

[0067] FIG. 9 is a graph showing an effect of alleviating depression through a tail suspension test (TST) with regard to Lactobacillus plantarum NK3, Bifidobacterium longum NK49 and a mixture thereof, which are novel lactic acid bacteria.

MODE FOR INVENTION

[0068] Hereinafter, the present disclosure will be described in detail through preferred Examples for better understanding of the present disclosure. However, the following Examples are provided only for the purpose of illustrating the present disclosure, and thus the present disclosure is not limited thereto.

Example 1: Isolation and Identification of Lactic Acid Bacteria

[0069] (1) Isolation of Lactic Acid Bacteria from Human Feces

[0070] Human feces were placed and suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). After that, supernatant was taken and transplanted into BL agar medium (Nissui Pharmaceutical, Japan), and then anaerobically incubated at 37° C. for about 48 hours, after which colony-forming strains were isolated therefrom.

[0071] (2) Isolation of Lactic Acid Bacteria from Kimchi

[0072] Cabbage kimchi, radish kimchi or green onion kimchi was crushed respectively, after which crushed supernatant was taken and transplanted into MRS agar medium (Difco, USA), and then anaerobically incubated at 37° C. for about 48 hours, after which colony-forming strains were isolated therefrom.

[0073] (3) Identification of Isolated Lactic Acid Bacteria

[0074] Physiological properties and 16S rDNA sequences of the strains isolated from human feces or kimchi were analyzed to identify species of the strains, and then names were given to the strains. Strain names given to lactic acid bacteria are the same as shown in table 1 below. Specifically, the lactic acid bacteria isolated from kimchi were five species of Lactobacillus plantarum (Nos. 1 to 5 of table 1), five species of Lactobacillus brevis (Nos. 6 to 10 of table 1), five species of Lactobacillus sakei (Nos. 11 to 15 of table 1), and five species of Lactobacillus curvatus (Nos. 16 to 20 of table 1). The lactic acid bacteria isolated from human feces were five species of Lactobacillus rhamnosus (Nos. 21 to 25 of table 1), five species of Lactobacillus plantarum (Nos. 26 to 30 of table 1), five species of Lactobacillus reuteri (Nos. 31 to 35 of table 1), four species of Lactobacillus johnsonii (Nos. 36 to 39 of table 1), three species of Lactobacillus mucosae (Nos. 40 to 42 of table 1), three species of Bifidobacterium adolescentis (Nos. 43 to 45 of table 1), and five species of Bifidobacterium longum (Nos. 46 to 50 of table 1).

TABLE-US-00001 TABLE 1 No. Strain Name 1 Lactobacillus plantarum NK1 2 Lactobacillus plantarum NK2 3 Lactobacillus plantarum NK3 4 Lactobacillus plantarum NK4 5 Lactobacillus plantarum NK5 6 Lactobacillus brevis NK6 7 Lactobacillus brevis NK7 8 Lactobacillus brevis NK8 9 Lactobacillus brevis NK9 10 Lactobacillus brevis NK10 11 Lactobacillus sakei NK11 12 Lactobacillus sakei NK12 13 Lactobacillus sakei NK13 14 Lactobacillus sakei NK14 15 Lactobacillus sakei NK15 16 Lactobacillus curvatus NK16 17 Lactobacillus curvatus NK17 18 Lactobacillus curvatus NK18 19 Lactobacillus curvatus NK19 20 Lactobacillus curvatus NK20 21 Lactobacillus rhamnosus NK21 22 Lactobacillus rhamnosus NK22 23 Lactobacillus rhamnosus NK23 24 Lactobacillus rhamnosus NK24 25 Lactobacillus rhamnosus NK25 26 Lactobacillus plantarum NK26 27 Lactobacillus plantarum NK27 28 Lactobacillus plantarum NK28 29 Lactobacillus plantarum NK29 30 Lactobacillus plantarum NK30 31 Lactobacillus reuteri NK31 32 Lactobacillus reuteri NK32 33 Lactobacillus reuteri NK33 34 Lactobacillus reuteri NK34 35 Lactobacillus reuteri NK35 36 Lactobacillus johnsonii NK36 37 Lactobacillus johnsonii NK37 38 Lactobacillus johnsonii NK38 39 Lactobacillus johnsonii NK39 40 Lactobacillus mucosae NK40 41 Lactobacillus mucosae NK41 42 Lactobacillus mucosae NK42 43 Bifidobacterium adolescentis NK43 44 Bifidobacterium adolescentis NK44 45 Bifidobacterium adolescentis NK45 46 Bifidobacterium longum NK46 47 Bifidobacterium longum NK47 48 Bifidobacterium longum NK48 49 Bifidobacterium longum NK49 50 Bifidobacterium longum NK50 [0075] (4) Physiological Properties of Novel Lactic Acid Bacterium Lactobacillus plantarum NK3

[0076] Out of the strains described in above table 1, it was confirmed that Lactobacillus plantarum NK3 (accession number KCCM12089P) is a gram-positive bacillus. In addition, it was shown that 16S rDNA of Lactobacillus plantarum NK3 has a sequence of SEQ ID NO: 1. As a result of comparing the 16S rDNA sequence of Lactobacillus plantarum NK3 through BLAST search, a Lactobacillus plantarum strain having the same 16S rDNA sequence is not searched at all, and it was confirmed that the sequence is 99% homologous to the 16S rDNA sequence of a generally known Lactobacillus plantarum strain. Out of the physiological properties of Lactobacillus plantarum NK3, availability of carbon source was analyzed with a sugar fermentation test using API 50 CHL kit. The results thereof are as shown in table 2 below. In table 2 below, “+” indicates that the availability of carbon source is positive and “−” indicates that the availability of carbon source is negative.

TABLE-US-00002 TABLE 2 Carbon Source NK3 Carbon Source NK3 CONTROL − Esculin + Glycerol − Salicin + Erythritol − Cellobiose + D-arabinose − Maltose + L-arabinose + Lactose + D-ribose + Melibiose + D-xylose − Sucrose + L-xylose − Trehalose + D-adonitol − Inulin − Methyl-BD-xylopyranoside − Melezitose + D-galactose + Raffinose − D-glucose + Starch − D-fructose + Glycogen − D-mannose + Xylitol − L-sorbose − Gentiobiose + Rhamnosus − D-turanose + Dulcitol − D-lyxose − Inositol − D-tagatose − Mannitol + D-fucose − Sorbitol + L-fucose − α-methyl-mannoside ± D-arabitol − α-methyl-glucoside − L-arabitol − N-acetyl-glucosamine + Gluconate ± Amygdaline + 2-keto-gluconate − Albutin + 5-keto-gluconate −
(5) Physiological Properties of Novel Lactic Acid Bacterium Bifidobacterium longum NK49

[0077] Out of the strains described in above table 1, it was confirmed that Bifidobacterium longum NK49 (accession number KCCM12088P) is a gram-positive bacillus. In addition, it was shown that 16S rDNA of Bifidobacterium longum NK49 has a sequence of SEQ ID NO: 2. As a result of comparing the 16S rDNA sequence of Bifidobacterium longum NK49 through BLAST search, a Bifidobacterium longum strain having the same 16S rDNA sequence is not searched at all, and it was confirmed that the sequence is 99% homologous to the 165 rDNA sequence of a generally known Bifidobacterium longum strain. Out of the physiological proper-ties of Bifidobacterium longum NK49, availability of carbon source was analyzed with a sugar fermentation test using API 50 CHL kit. The results thereof are the same as shown in table 3 below. In table 3 below, “+” indicates that the availability of carbon source is positive and “−” indicates that the availability of carbon source is negative.

TABLE-US-00003 TABLE 3 Carbon Source NK49 Carbon Source NK49 CONTROL − Esculin + Glycerol − Salicin + Erythritol − Cellobiose − D-arabinose − Maltose + L-arabinose + Lactose + D-ribose + Melibiose + D-xylose ± Sucrose + L-xylose − Trehalose − D-adonitol − Inulin − Methyl-BD-xylopyranoside − Melezitose − D-galactose + Raffinose + D-glucose + Starch − D-fructose + Glycogen − D-mannose − Xylitol − L-sorbose − Gentiobiose − Rhamnosus − D-turanose ± Dulcitol − D-lyxose − Inositol − D-tagatose − Mannitol + D-fucose − Sorbitol + L-fucose − α-methyl-mannoside − D-arabitol − α-methyl-glucoside ± L-arabitol − N-acetyl-glucosamine − Gluconate − Amygdaline − 2-keto-gluconate − Albutin − 5-keto-gluconate −

Example 2: Therapeutic Effect of Lactic Acid Bacteria on Osteoporosis in Animal Model

[0078] (1) Preparation of Animal Model with Osteoporosis and Administration of Lactic Acid Bacteria

[0079] An experiment was performed by using six C57BL/6 mice (female, 21-23 g and 6 weeks old) per group after being acclimated in a laboratory for one week. After the mice were anesthetized with isoflurane, the ovaries were removed therefrom to create an animal model with osteoporosis. From one week after ovarian removal, Lactobacillus plantarum NK3, Bifidobacterium longum NK49, a 1:1 mixture thereof, or a 4:1 mixture thereof (a ratio of LP:BL is 4:1 and the same as below), which is a novel lactic acid bacterium, was orally administered in a daily amount of 1×10.sup.9 CFU for six days a week for five weeks. In addition, 17β-estradiol (PC) was intraperitoneally administered at 10 μg/kg/day to the experimental animals of the positive control group, and the sham group and the experimental group with removed ovaries were orally dosed with physiological saline solution instead of drugs.

[0080] On the day after the administration of lactic acid bacteria was completed, the experimental animals were sacrificed, and then blood, thighbone, colon, womb, and liver were separated therefrom so as to measure osteoporosis indices.

[0081] (2) Measurement of Osteoporosis Indices

[0082] Osteocalcin in blood was measured with R&D system ELISA Kit (Minneapolis, Minn., USA); Ca and P were measured with ASAN Ca-Lq Reagents and ASAN Pi-Lq Reagents of Asan Pharmaceutical Co., Ltd, respectively; and alkaline phosphatase (AP) activity was measured with Alkaline phosphatase activity colorimetric assay kit (Sigma, USA).

[0083] (3) Experimental Results

[0084] As a result of breeding the animal model with removed ovaries as described above for six weeks, the corresponding animal model showed a decrease in the weight of the womb (FIG. 1), a decrease in the weight of the thighbone (FIG. 2), and an increase in the length of the thighbone (FIG. 3). However, with regard to the groups dosed with Lactobacillus plantarum NK3 (LP), Bifidobacterium longum NK49 (BL), a 1:1 mixture thereof (M1:1), and a 4:1 mixture thereof (LP:BL=4:1, M4:1), it was confirmed that the weight of the womb and the thighbone is increased at a level similar to the group dosed with estradiol similar to estrogen (FIGS. 1 and 2) and the length of the thighbone is recovered (FIG. 3).

[0085] In addition, as a result of breeding the animal model with removed ovaries for six weeks as described in table 4 below, the corresponding animal model tended to show an increase in alkaline phosphatase (AP) activity, an increase in the concentration of phosphorus (P) and osteocalcin, and a decrease in calcium (Ca) in blood. Meanwhile, with regard to the groups dosed with Lactobacillus plantarum NK3 (LP), Bifidobacterium longum NK49 (BL), a 1:1 mixture thereof (M1:1), and a 4:1 mixture thereof (M4:1), according to the present disclosure, it was confirmed that the activity of alkaline phosphatase (AP) and the concentration of osteocalcin, phosphorus and calcium are recovered at a level similar to the group dosed with estradiol.

TABLE-US-00004 TABLE 4 Mixture Mixture of lactic of lactic Positive Normal Induced Control acid acid control group group group LP BL bacteria bacteria group (Nor) (Sham) (Con) NK3 NK49 (M1:1) (M4:1) (PC) AP (IU/L) 170.0 ± 3.4  174.4 ± 5.1  282.2 ± 10.3  217.2 ± 8.6  218.9 ± 5.1  215.5 ± 6.8  213.8 ± 8.6  220.6 ± 10.3  Ca  6.6 ± 0.2 6.4 ± 0.3 6.4 ± 0.4 6.3 ± 0.3 6.3 ± 0.3 6.3 ± 0.2 6.2 ± 0.2 6.2 ± 0.2 (mg/dL) P 11.2 ± 0.2 11.8 ± 0.9  12.5 ± 1.5  12.1 ± 1.4  11.5 ± 1.2  11.9 ± 1.1  11.8 ± 0.9  12.1 ± 1.1  (mg/dL) Osteocalcin 0.89 ± 0.2 0.98 ± 0.21 1.85 ± 0.32 1.46 ± 0.25 1.38 ± 0.15 1.41 ± 0.22 1.42 ± 0.19 1.53 ± 0.15 (μg/L)

[0086] The above results suggest that novel lactic acid bacteria and a mixture thereof according to the present disclosure have an effect of treating osteoporosis, which is one of symptoms of female menopausal disorders, at a level similar to estrogen.

Example 3: Effect of Lactic Acid Bacteria on Ameliorating Obesity in Animal Model

[0087] (1) Preparation of Animal Model with Induced Obesity and Administration of Lactic Acid Bacteria

[0088] An experiment was performed by using six C57BL/6 mice (female, 21-23 g and 6 weeks old) per group after being acclimated in a laboratory for one week as described above in (1) of Example 2. After the mice were anesthetized with isoflurane and the ovaries were removed therefrom, the resulting mice were raised for six weeks so as to create an animal model with obesity, showing a significant weight gain. From one week after ovarian removal, Lactobacillus plantarum NK3, Bifidobacterium longum NK49, a 1:1 mixture thereof, or a 4:1 mixture thereof, which is a novel lactic acid bacterium, was orally administered in a daily amount of 1×10.sup.9 CFU for six days a week for five weeks. In addition, 17β-estradiol (PC) was intraperitoneally administered at 10 μg/kg/day to the experimental animals of the positive control group, and the sham group and the experimental group with removed ovaries were orally dosed with physiological saline solution instead of drugs.

[0089] On the day after the administration of lactic acid bacteria was completed, the experimental animals were sacrificed, and then blood, thighbone, colon, womb, and liver were separated therefrom so as to measure obesity indices. The body weight was measured once a week.

[0090] (2) Measurement of AMPK Activity

[0091] The AMPK activity in the liver was measured by using an immunoblotting method. Specifically, the isolated liver was placed in a radio immunoprecipitation assay lysis buffer (RIPA, Pierce, Rockford, Ill., USA), pulverized, and centrifuged at 10,000 g for 10 minutes so as to obtain a supernatant. This supernatant was subjected to 10% SDS-PAGE gel electrophoresis and transferred to a membrane. After reacting with antibodies of AMPK, p-AMPK and β-actin overnight at 4° C., the resulting product was subjected to a reaction with a secondary antibody and washed. Then, the resulting product was confirmed by developing color with an enhanced chemiluminescence (ECL) reagent.

[0092] (3) Measurement of SREBP-1c and PGC-1α Expression

[0093] An expression of sterol regulatory element-binding transcription factor 1c (SREBP-1c) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was measured by using a quantitative reverse transcription polymerase chain reaction (qPCR).

[0094] Specifically, mRNA was extracted from the liver isolated from the animal model by using a Qiagen mRNA extract kit, and then qPCR was performed by using SYBER premix agents with a Takara thermal cycler. Thermal cycling was performed under the following conditions by using the primers in table 5 below: A DNA polymerase activation was performed at 95° C. for 30 seconds, and then amplified repeatedly 40 times while being subjected to a reaction at 95° C. at an interval of 15 seconds and at 60° C. at an interval of 30 seconds. The amount of final expressed genes was calculated in comparison with β-actin.

TABLE-US-00005 TABLE 5 SEQ ID NO Primer Sequence 3 SREBP-1c forward 5′-AGC TGT CGG GGT AGC GTC TG-3′ 4 SREBP-1c reverse 5′-GAG AGT TGG CAC CTG GGC TG-3′ 5 PGC-1α forward 5′-CCG CCA CCT TCA ATC CAG AG-3′ 6 PGC-1α reverse 5′-CAA GTT CTC GAT TTC TCG ACG G-3′ 7 β-actin forward 5′-TGT CCA CCT TCC AGC AGA TGT-3′ 8 β-actin reverse 5′-AGC TCA GTA ACA GTC CGC CTA GA-3′

[0095] (4) Experimental Results

[0096] As a result of breeding the animal model with removed ovaries as described above for six weeks, the corresponding animal model showed a significant weight gain. Meanwhile, with regard to the groups dosed with Lactobacillus plantarum NK3 (LP), Bifidobacterium longum NK49 (BL), a 1:1 mixture thereof (M1:1), and a 4:1 mixture thereof (M4:1) according to the present disclosure, it was confirmed that a weight loss occurs at a level similar to the group dosed with estradiol, which is in particular less than a weight gain of the normal animal without removed ovaries (FIG. 4).

[0097] In addition, as a result of breeding the animal model with removed ovaries for six weeks, the AMPK activity was decreased (FIG. 5), while the expression of SREBP-1c was induced and the expression of PGC-1a was inhibited as shown in table 6 below. However, with regard to the groups dosed with Lactobacillus plantarum NK3 (LP), Bifidobacterium longum NK49 (BL), a 1:1 mixture thereof (M1:1), and a 4:1 mixture thereof (M4:1) according to the present disclosure, the AMPK activity occurred at a level similar to the group dosed with estradiol (FIG. 5), which was higher than that of the control group with removed ovaries. In addition, it was confirmed as shown in table 6 below that the expression of SREBP-1c is inhibited and the expression of PGC-1a is induced the present disclosure compared to the mouse model with removed ovaries, thereby confirming that obesity is inhibited.

TABLE-US-00006 TABLE 6 Mixture Mixture of lactic of lactic Positive Normal Induced Control acid acid control group group group LP BL bacteria bacteria group (Nor) (Sham) (Con) NK3 NK49 (M1:1) (M4:1) (PC) SREBP-1c 1.0 ± 0.1 1.2 ± 0.2 4.5 ± 0.5 2.5 ± 0.6 2.2 ± 0.5 2.3 ± 0.3 2.4 ± 0.4 2.7 ± 0.3 PGC-1α 1.0 ± 0.1 1.1 ± 0.1 0.6 ± 0.2 0.8 ± 0.2 0.9 ± 0.2 0.8 ± 0.1 0.9 ± 0.2 0.8 ± 0.2

[0098] The above results suggest that novel lactic acid bacteria and a mixture thereof according to the present disclosure have an effect of ameliorating obesity or weight gain, which is one of symptoms of female menopausal disorders, at a level similar to estrogen.

Example 4: Therapeutic Effect of Lactic Acid Bacteria on Depression in Animal Model

[0099] (1) Preparing of Restraint Stress-Induced Mouse Model of Depression

[0100] A mouse model of depression was fixed to a 3×10 cm cylindrical-shaped restraint stress apparatus, such that the mouse may not move therein at all. Then, the mouse was given restraint stress 12 hours each for two days in a row. From the next day, Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof was administered (NK3, NK49, MIX: 1×10.sup.9 CFU/mouse) for five days to measure the indices of depressive behaviors on the day after the final administration.

[0101] (2) Measurement of Plasma Corticosterone Concentrations

[0102] With regard to the restraint stress-induced mouse model of depression prepared as above, the plasma concentration of the stress hormone corticosterone was significantly higher than that of the control group, thus confirming that the animal model is made suitable for this experiment. In case of administering Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof (MIX), it was confirmed that the plasma concentration of corticosterone becomes all low (FIG. 6).

[0103] (3) Elevated Plus Maze (EPM) Experiment

[0104] An elevated plus maze experimental apparatus was a black plexiglass apparatus, in which two open arms (30×7 cm) and two enclosed arms (30×7 cm) with 20 cm-high walls stand 50 cm high above a floor, each extending 7 cm away from a center platform. A mouse movement in the elevated plus maze was recorded after being placed in a room with a video camera of 20 Lux brightness installed high thereon.

[0105] Specifically, a C57BL/6 mouse (male, 19-22 g) was placed in the middle of the elevated plus maze with the head of the mouse facing toward an open arm. A time spent in open and closed arms and the number of entries thereinto were measured for five minutes. A time spent in open arms (OT) out of a total test time was calculated via [a time spent in open arms/(a time spent in open arms (OT)+a time spent in closed arms)]×100. And open arm entries (OE) were calculated via [open arm entries/(open arm entries+closed arm entries)]×100, and the results thereof are shown in FIG. 7.

[0106] As a result of measuring a ratio of the time spent in open arms (OT) and the open arm entries (OE) as shown in FIG. 7, the groups dosed with Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof (MIX) showed a value significantly higher than that of the non-dosed group of mice with induced restraint stress, thus confirming that the anxiety symptoms are alleviated among the mice dosed with the strains.

[0107] (4) Forced Swimming Test (FST)

[0108] A water tank with a height of 40 cm and a diameter of 20 cm was filled up to 30 cm with water having a temperature of 25±1° C. according to the conventionally known method of Porsolt et al. (1997), after which each of the experimental mice was placed into the water tank and forced to stay there for six minutes, out of which first two minutes were considered as an adaptive time without measurement, and then for the last four minutes, an immobility time for the experimental animal was measured (in this case, the immobility means a state that the mouse is standing upright and floating in the water motionlessly except the least movement to keep its head only above the water). The results of measurement are shown in FIG. 8.

[0109] As a result of measuring a ratio of immobile mice as shown in FIG. 8, the groups dosed with Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof (MIX) showed a value significantly lower than that of the non-dosed group of mice with induced restraint stress, thus confirming that the sudden depressive state of the mice that give up survival is alleviated when being dosed with the strains.

[0110] (5) Tail Suspension Test (TST)

[0111] According to the conventionally known method of Steru et al. (1985), a fixture was attached about 1 cm to the end of the tail of the mouse, and then hung 50 cm away from the ground. Then, an immobility time for the experimental animal was measured for a total of six minutes and the results thereof are shown in FIG. 9.

[0112] As a result of measuring a ratio of immobile mice as shown in FIG. 9, the groups dosed with Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof (MIX) showed a value significantly lower than that of the non-dosed group of mice with induced restraint stress, thus confirming that the depressive state of the mice is alleviated when being dosed with the strains.

[0113] The above results suggest that novel lactic acid bacteria and a mixture thereof according to the present disclosure have an effect of ameliorating depression, which is one of symptoms of female menopausal disorders.

[0114] Accession Information of Lactic Acid Bacteria

[0115] The present inventors deposited Lactobacillus plantarum NK3 for the purpose of patent to the Korean Culture Center of Microorganisms, a certified depository institution (address: Yurim B/D, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea) on Aug. 4, 2017, and received an accession number of KCCM12089P.

[0116] Also, the present inventors deposited Bifidobacterium longum NK49 for the purpose of patent to the Korean Culture Center of Microorganisms, a certified depository institution (address: Yurim B/D, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea) on Aug. 4, 2017, and received an accession number of KCCM12088P.

INDUSTRIAL APPLICABILITY

[0117] Lactobacillus plantarum NK3, Bifidobacterium longum NK49, or a mixture thereof, which are novel lactic acid bacteria according to the present disclosure, have an effect of treating female menopausal disorders, such as treatment of osteoporosis, amelioration of obesity, or alleviation of depression, and thus may be used as a composition for preventing or treating female menopausal disorders and may be expected to be useful in a related industry field of drug medicines and foods.