Novel lactic acid bacteria and their uses

Abstract

The present invention relates to novel lactic acid bacteria, Lactococcus lactis P32 (KCCM13222P), Bifidobacterium bifidum P45 (KCCM13223P), or a mixture thereof, and uses thereof. The bacterial strains and mixtures thereof according to the present invention can be useful for the prevention or treatment of inflammatory diseases, osteoporosis, depression, or anxiety disorders.

Description

[Technical Field]

[0001] The present invention relates to novel lactic acid bacteria, Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof, and uses thereof. [Background technology]

[0002] Under normal conditions, the female vagina has a slightly acidic environment due to the abundance of lactobacilli present inside, which inhibits the invasion of pathogenic microorganisms and the proliferation of fungi. This slightly acidic environment can be disrupted by external factors, such as excessive vaginal douching, long-term use of antibiotics or contraceptives, or disruption of hormonal balance due to pregnancy, childbirth, or menopause, which can cause changes in the amount of lactobacilli, which are beneficial bacteria normally present in the vagina. This can lead to the development of vaginitis, which induces discharge, itching, and pain.

[0003] Many methods for preventing and treating vaginitis have been studied. Recently, oral or cream-based administration of vaginitis treatments, such as metronidazole, has been commonly used to treat vaginal bacterial infections. However, the use of broad-spectrum antibiotics such as metronidazole is considered undesirable because it not only poses the risk of antibiotic resistance but also may destroy a wide range of normal vaginal flora, including beneficial lactic acid bacteria. Furthermore, long-term use of antibiotics has been reported to be problematic, as it can cause systemic toxicity due to antibiotic absorption through the vagina. Therefore, there is an urgent need to develop a safe composition for preventing or treating vaginitis that can prevent and / or treat vaginal infections without causing side effects.

[0004] Osteoporosis is a complex disease that is influenced by genetic factors and environmental factors such as diet and lifestyle. In particular, in women, bone loss accelerates rapidly due to hormonal changes during menopause. Currently available osteoporosis treatments include vitamin D, female hormones, bisphosphonates, selective estrogen receptor modulators, and calcitonin. Most of these drugs prevent bone loss by reducing osteoclast activity and regulating bone resorption. However, osteoporosis treatments that suppress osteoclast activity are not fundamental treatments for osteoporosis, and therefore have limited potential for complete cure. Therefore, increased osteoblast activity, which can substantially increase bone density and strengthen bones, is essential.

[0005] Most of the osteoporosis treatments currently in use are estrogen-based substances, and long-term administration of these drugs poses the problem of side effects such as cancer, gallstones, and thrombosis. Since osteoporosis cannot be treated by short-term drug administration alone and requires long-term drug administration, there is a need for the development of new substances that have excellent pharmacological effects that can replace estrogen and do not cause the side effects described above even when administered long-term.

[0006] On the other hand, infectious diseases, hormone imbalance (menopausal) diseases, etc. may be accompanied by diseases such as depression and anxiety disorders. Drugs such as antidepressants used to treat such mental disorders are limited in use because they can cause serious side effects such as cardiovascular diseases and suicide. These mental disorders cannot be treated by short-term administration of drugs alone, but require long-term administration. Therefore, there is a need for the development of new substances that have excellent medicinal effects without side effects when administered long-term. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a Lactococcus lactis P32 KCCM13222P strain.

[0008] Another object of the present invention is to provide a Bifidobacterium bifidum P45 KCCM13223P strain.

[0009] It is still another object of the present invention to provide an antibacterial composition comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0010] It is yet another object of the present invention to provide a pharmaceutical composition comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0011] It is yet another object of the present invention to provide a food composition comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0012] It is yet another object of the present invention to provide a method for preventing or treating inflammatory diseases, osteoporosis, depression, or anxiety disorders, comprising the step of administering to an individual Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof. [Means for solving the problem]

[0013] To achieve the above object, one aspect of the present invention relates to Lactococcus lactis P32 KCCM13222P.

[0014] The Lactococcus lactis P32 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy individual.

[0015] The 16S rDNA base sequence for identification and classification of the Lactococcus lactis P32 of the present invention is shown in SEQ ID NO: 1 attached hereto. Therefore, the Lactococcus lactis P32 of the present invention may contain the 16S rDNA of SEQ ID NO: 1.

[0016] Analysis of the 16S rDNA base sequence of SEQ ID NO: 1 showed 99% homology with known Lactococcus lactis strains, indicating the highest molecular phylogenetic relationship with Lactococcus lactis (Figure 1). Therefore, the lactic acid bacterium was identified as Lactococcus lactis, named Lactococcus lactis P32, and deposited with the Korea Microorganism Collection on August 3, 2022 (KCCM13222P).

[0017] The Lactococcus lactis P32 of the present invention is a Gram-positive cocci. The physiological properties of Lactococcus lactis P32 can be analyzed by conventional methods in the relevant technical field. Specifically, Lactococcus lactis P32 can utilize D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, maltose, lactose, sucrose, trehalose, starch, and gentiobiose as carbon sources. Another aspect of the present invention relates to Bifidobacterium bifidum P45 KCCM13223P.

[0018] The Bifidobacterium bifidum P45 of the present invention is characterized as a novel lactic acid bacterium isolated and identified from the feces of a healthy person.

[0019] The 16S rDNA base sequence for identifying and classifying the Bifidobacterium bifidum P45 of the present invention is shown in SEQ ID NO: 2 attached hereto. Therefore, the Bifidobacterium bifidum P45 of the present invention may contain the 16S rDNA of SEQ ID NO: 2.

[0020] Analysis of the 16S rDNA base sequence of SEQ ID NO: 2 showed 99% homology with known Bifidobacterium bifidum strains, indicating the highest molecular phylogenetic relationship with Bifidobacterium bifidum (Figure 3). Therefore, the lactic acid bacterium was identified as Bifidobacterium bifidum, named Bifidobacterium bifidum P45, and deposited with the Korea Microorganism Collection on August 3, 2022 (KCCM13223P).

[0021] The Bifidobacterium bifidum P45 of the present invention is a Gram-positive bacillus. More specifically, the physiological properties of Bifidobacterium bifidum P45 can be analyzed by conventional methods in the relevant technical field. Specifically, Bifidobacterium bifidum P45 can utilize D-glucose, D-lactose, D-sucrose, D-maltose, L-arabinose, gelatin, esculin-iron citrate, D-cellobiose, D-mannose, and D-raffinose as carbon sources.

[0022] Yet another aspect of the present invention relates to an antibacterial composition comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0023] Yet another aspect of the present invention relates to a pharmaceutical composition for preventing or treating infections caused by Gardnerella vaginalis or Staphylococcus aureus, comprising the antibacterial composition.

[0024] In one embodiment of the present invention, it has been confirmed that Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 inhibit Gardnerella vaginalis and / or Staphylococcus aureus (Table 6), and thus they can be used for antibacterial purposes, and therefore can also be used for the prevention or treatment of Gardnerella vaginalis or Staphylococcus aureus infections.

[0025] Yet another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases or osteoporosis, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0026] As used herein, the term "inflammatory disease" refers collectively to diseases whose primary pathology is inflammation. The inflammatory disease of the present invention may be any one or more selected from the group consisting of arthritis, gout, hepatitis, obesity, keratitis, gastritis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleuritis, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis. More specifically, the inflammatory disease may be, but is not limited to, vaginitis.

[0027] Vaginitis is an inflammatory disease that occurs in the female vagina and can be broadly divided into three main causes: bacterial vaginosis (BV), Candida vaginitis (CV), and Trichomonas vaginalis. Among these, bacterial vaginosis has recently been recognized as the most common type of vaginitis, and bacteria such as Atopobium vaginae, Gardnerella vaginalis, and Prevotella bivia have been reported to be found in vaginal secretions isolated from patients with bacterial vaginosis. Gardnerella vaginalis, in particular, is known to be the main causative bacterium of vaginitis. The novel lactic acid bacteria of the present invention exhibit effective inhibitory effects against Gardnerella vaginalis and are therefore effective in treating vaginitis.

[0028] In one embodiment of the present invention, it has been confirmed that Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 reduce the expression of inflammatory cytokines (Table 6), and thus can be used to treat inflammatory diseases.

[0029] In this invention, "osteoporosis" refers to a disease caused by an imbalance between osteoblasts and osteoclasts, resulting in increased bone resorption compared to bone formation. The bone mineral content decreases, thinning the bone mass, widening the marrow cavity, and weakening the bones as the condition progresses, making them susceptible to fractures even with minor impacts. Known causes of osteoporosis include old age, lack of exercise, low body weight, smoking, a low-calcium diet, menopause, and ovarian removal. Women, in particular, experience persistent bone loss from the age of 30 onward, and hormonal changes during menopause rapidly accelerate bone loss.

[0030] In one example of the present invention, it was confirmed that Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 showed an improving effect on osteoporosis indexes (Tables 9 and 10).

[0031] Yet another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of depression or anxiety disorders, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0032] The "depression" is a disease whose main symptoms are loss of motivation and melancholy, and which causes various cognitive and psychosomatic symptoms, resulting in a decline in daily functioning, and may be any one or more selected from the group including, but not limited to, major depressive disorder, persistent depressive disorder, dysthymia, disruptive mood dysregulation disorder, premenstrual dysphoric disorder, substance / medication-induced depressive disorder, depressive disorder due to another medical condition, other specified depressive disorder, and unspecified depressive disorder.

[0033] The "anxiety disorder" refers to a mental illness that causes impairment in daily life due to various forms of abnormal, pathological anxiety and fear, and may be any one or more selected from the group including, but not limited to, generalized anxiety disorder, specific phobia, agoraphobia, social anxiety disorder, panic disorder, separation anxiety disorder, and selective mutism.

[0034] Said "depression" or "anxiety disorder" may be caused by, but is not limited to, inflammatory diseases or female hormone imbalances.

[0035] In the examples of the present invention, it was confirmed through behavioral experiments and measurement of BDNF and Claudin-5 levels that Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 significantly improved depressive and anxious behaviors in mouse models in which inflammation and / or ovariectomy induced female hormone imbalance (Tables 8 to 10).

[0036] Specifically, the mixture in the pharmaceutical composition may be, but is not limited to, a mixture of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P in a colony forming unit (CFU) ratio of 1:1 to 4:1.

[0037] In the examples of the present invention, mice modeled on vaginitis and / or osteoporosis were treated with a mixture of Lactococcus lactis P32 and Bifidobacterium bifidum P45, and it was confirmed that the mixture had excellent preventive or therapeutic effects on osteoporosis (Tables 8 to 10).

[0038] More specifically, the Lactococcus lactis P32 KCCM13222P or Bifidobacterium bifidum P45 KCCM13223P may be in the form of live cells, killed cells, a culture thereof, a disrupted product thereof, or an extract thereof, but may be applied in any form that can achieve the desired effect.

[0039] In the present invention, "viable cells" refers to the novel lactic acid bacteria of the present invention themselves, "killed cells" refers to lactic acid bacteria that have been sterilized by heating, pressurization, chemical treatment, or the like, and "disrupted material" refers to lactic acid bacteria that have been disrupted by enzyme treatment, homogenization, ultrasonic treatment, or the like.

[0040] In the present invention, the term "extract" refers to a product obtained by extracting lactic acid bacteria with a known extraction solvent.

[0041] In the present invention, the term "waste" or "culture medium" refers to a product obtained by culturing lactic acid bacteria in a known medium, and the product may contain novel lactic acid bacteria. The medium may be selected from known liquid or solid media, such as, but not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium.

[0042] The pharmaceutical composition according to the present invention can be prepared into a pharmaceutical dosage form using methods well known in the art so as to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. In preparing the dosage form, the pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable carrier within a range that does not inhibit the activity of the novel lactic acid bacterium.

[0043] Yet another aspect of the present invention relates to a method for preventing or treating inflammatory diseases, osteoporosis, depression, or anxiety disorders, comprising the step of administering to a subject a pharmaceutical composition comprising said strain or a mixture of said strains.

[0044] The "inflammatory disease", "osteoporosis", "depression", and "anxiety disorder" are as described above, and specifically, the inflammatory disease may be vaginitis.

[0045] The subject is an animal, typically a mammal that may benefit from treatment using the lactic acid bacteria of the present invention. Preferred examples of such subjects include primates such as humans.

[0046] One aspect of the present invention relates to a food composition for preventing or ameliorating inflammatory diseases or osteoporosis, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0047] The "inflammatory disease" and "osteoporosis" are as described above. Specifically, the inflammatory disease may be vaginitis.

[0048] Another aspect of the present invention relates to a food composition for preventing or ameliorating depression or anxiety disorders, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0049] Said "depression" or "anxiety disorder" may be caused by, but is not limited to, inflammatory diseases or female hormone imbalances.

[0050] Specifically, the mixture of bacterial strains may be, but is not limited to, a mixture of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P in a colony forming unit (CFU) ratio of 1:1 to 4:1.

[0051] The type of food is not particularly limited. Foods to which lactic acid bacteria can be added include sausages, meats, bread, chocolates, snacks, candies, confectioneries, ramen, pizza, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, supplemental drinks, alcoholic beverages, and vitamin complexes. When formulated into drinking water, liquid components added in addition to the novel lactic acid bacteria are not limited to the above, and may include various flavorings or natural carbohydrates, as in conventional beverages. The natural carbohydrates mentioned above may be monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, etc.), polysaccharides (e.g., common sugars such as dextrin, cyclodextrin, etc.), and sugar alcohols such as xylitol, sorbitol, and erythritol.

[0052] More specifically, the lactic acid bacteria contained in the pharmaceutical composition of the present invention may be viable cells, killed cells, a culture thereof, a disrupted product thereof, or an extract thereof, but any form of lactic acid bacteria that can achieve the preventive or therapeutic effect of inflammatory diseases or osteoporosis may be used without limitation. The "viable cells," "killed cells," "culture," "disrupted product," and "extract" are as described above.

[0053] In particular, the food may be a functional health food. The functional health food is a food that emphasizes the bioregulatory function of the food, and is a food that has been added with added value to act and express a specific purpose using physical, biochemical, and biotechnological methods. The ingredients of such functional health foods are involved in biodefense, biorhythm regulation, disease prevention, and recovery.

[0054] They are designed and processed to fully exert their body regulating functions on the living body, and may contain food-acceptable food supplement additives, sweeteners, or functional ingredients.

[0055] When the Lactococcus lactis P32 or Bifidobacterium bifidum P45 of the present invention is used as a functional health food (or functional health drink additive), the novel lactic acid bacteria can be added directly or in combination with other foods or food ingredients in a conventional manner. The amount of Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 added can be determined appropriately depending on the intended use (prevention, health or improvement, therapeutic treatment).

[0056] Yet another aspect of the present invention relates to a method for preventing or treating inflammatory diseases, osteoporosis, depression, or anxiety disorders, comprising the step of administering to an individual Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0057] The "inflammatory disease," "osteoporosis," "depression," and "anxiety disorder" are as described above. Specifically, the inflammatory disease may be vaginitis.

[0058] The "individual" includes an animal or human suffering from an inflammatory disease, osteoporosis, depression, or anxiety disorder whose symptoms can be improved by administration of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof according to the present invention. By administering the strain, the mixture of strains, or a composition containing the same according to the present invention to an individual, inflammatory disease, osteoporosis, depression, or anxiety disorder can be effectively prevented and treated.

[0059] The term "administration" refers to the introduction of a substance into a human or animal by a suitable method. The bacterial strain, bacterial strain mixture, or composition containing the same according to the present invention may be administered orally or parenterally via any common route, as long as it can reach the target tissue. The therapeutic composition according to the present invention may also be administered by any device capable of transferring the active ingredient to target cells.

[0060] The preferred dosage of the strain, strain mixture, or composition containing the same according to the present invention may vary depending on the condition and weight of the patient, the severity of the disease, the drug form, the route and duration of administration, but can be appropriately selected by those skilled in the art.

[0061] Yet another aspect of the present invention relates to the use of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof for the prevention or treatment of inflammatory diseases, osteoporosis, depression, or anxiety disorders.

[0062] The "inflammatory disease," "osteoporosis," "depression," and "anxiety disorder" are as described above. Specifically, the inflammatory disease may be vaginitis. [Effects of the Invention]

[0063] The Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof according to the present invention is highly effective in improving vaginitis, osteoporosis, depression, or anxiety disorders.

[0064] It should be understood that the effects of the present invention are not limited to the effects described above, but include all effects that can be inferred from the configuration of the invention described in the detailed description of the present invention or the claims. [Brief explanation of the drawings]

[0065] [Figure 1] FIG. 1 shows the 16S rDNA homology of Lactococcus lactis P32 KCCM13222P with known Lactococcus lactis strains. [Figure 2] Figure 1 shows the phylogenetic characteristics of Lactococcus lactis P32 KCCM13222P. [Figure 3] FIG. 1 shows the 16S rDNA homology of Bifidobacterium bifidum P45 KCCM13223P with known Bifidobacterium bifidum strains. [Figure 4] FIG. 1 shows the phylogenetic characteristics of Bifidobacterium bifidum P45 KCCM13223P. [Figure 5] FIG. 1 is a graph confirming the vaginitis-improving effect of the lactic acid bacteria of the present invention in Experimental Example 6 of the present invention. [Figure 6] FIG. 1 is a graph confirming the vaginitis-improving effect of the lactic acid bacteria of the present invention in Experimental Example 7 of the present invention. [Figure 7] FIG. 10 is a graph confirming the vaginitis-improving effect of the lactic acid bacteria of the present invention in Experimental Example 8 of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

[0066] The present invention will be described in detail below with reference to examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0067] Example 1. Isolation and identification of lactic acid bacteria

[0068] 1-1. Isolation of lactic acid bacteria from human feces

[0069] Feces from healthy individuals were suspended in GAM broth (Nissui Pharmaceutical, Japan), and the supernatant was transferred to GAM agar medium (Nissui Pharmaceutical, Japan), MRS agar medium (BD, USA), or BL agar medium (Nissui Pharmaceutical, Japan). After anaerobically culturing at 37°C for approximately 48 hours, colony-forming strains were isolated.

[0070] 1-2. Identification of isolated lactic acid bacteria

[0071] Strains isolated from human feces were identified and named by analyzing Gram staining, physiological characteristics, and 16S rDNA gene sequences. The names of the lactic acid bacteria strains are shown in Table 1 below. Specifically, six species of Lactobacillus plantarum (numbers 1 to 6 in Table 1), five species of Lactobacillus sakei (numbers 6 to 10 in Table 1), two species of Lactobacillus reuteri (numbers 11 and 12 in Table 1), three species of Lactobacillus rhamnosus (numbers 13 to 15 in Table 1), five species of Lactococcus lactis (numbers 16 to 20 in Table 1), three species of Bifidobacterium adolescentis (numbers 21 to 23 in Table 1), and Bifidobacterium bifidum. The three species of Bifidobacterium bifidum (numbers 24 to 26 in Table 1) and four species of Bifidobacterium longum (numbers 27 to 30 in Table 1) were:

[0072] [Table 1] JPEG2025528234000003.jpg98159

[0073] 1-3. Physiological properties of the novel lactic acid bacterium Lactococcus lactis P32

[0074] Among the strains listed in Table 1, Lactococcus lactis P32 is a Gram-positive cocci and did not exhibit hemolysis in blood culture medium. The 16S rDNA of Lactococcus lactis P32 has been shown to have the nucleotide sequence of SEQ ID NO: 1. When the 16S rDNA sequence was compared by BLAST search, no Lactococcus lactis strains were found to have the same 16S rDNA sequence, and it was confirmed that the 16S rDNA sequence of this strain is 99% identical to the 16S rDNA sequence of known Lactococcus lactis strains.

[0075] Among the physiological characteristics of Lactococcus lactis P32, carbon source utilization was analyzed in a sugar fermentation test using an API50 CHL kit (BioMérieux, USA). The results are shown in Table 2 below. In Table 2, "+" indicates a positive result for carbon source utilization, and "-" indicates a negative result for carbon source utilization.

[0076] [Table 2] JPEG2025528234000005.jpg126159

[0077] The novel strain Lactococcus lactis P32 was deposited at the Korea Microorganism Collection (Yu-lim Bldg. 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an accredited depository, and was assigned the accession number KCCM13222P.

[0078] 1-4. Physiological properties of the novel lactic acid bacterium Bifidobacterium bifidum P45

[0079] Bifidobacterium bifidum P45 is a Gram-positive bacillus that does not exhibit hemolysis in blood culture media. The 16S rDNA of Bifidobacterium bifidum P45 has been shown to have the base sequence of SEQ ID NO: 2. A BLAST search of the 16S rDNA sequence revealed that no other Bifidobacterium bifidum strains had the same 16S rDNA sequence, and confirmed that the 16S rDNA sequence exhibited 99% identity with the 16S rDNA sequence of known Bifidobacterium bifidum strains.

[0080] Among the physiological characteristics of Bifidobacterium bifidum P45, carbon source utilization was analyzed in a sugar fermentation test using an API20 CHL kit (BioMérieux, USA). The results and other physiological characteristics are shown in Table 3 below. In Table 3 below, "+" indicates a positive reaction and "-" indicates a negative reaction.

[0081] [Table 3] JPEG2025528234000007.jpg212159

[0082] The novel strain B. bifidum P45 has been deposited with the Korea Microorganism Collection (Yu-lim Bldg. 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an official depository, and has been assigned the accession number KCCM13223P.

[0083] Experimental Example 1. Confirmation of antibiotic susceptibility of Lactococcus lactis P32 and Bifidobacterium bifidum P45 strains

[0084] The MIC (minimum inhibitory concentration) of antibiotics was determined by adding 2 × 10 Lactococcus lactis P32 and Bifidobacterium bifidum P45 to 2 ml of antibiotic-containing BHI (Brain Heart Infusion) medium (BD Biosciences Korea, Seoul, Korea). 5 The cells were inoculated at CFU / ml and cultured anaerobically at 37°C for 24 hours, and the results are shown in Table 4 below.

[0085] [Table 4]

[0086] *AM, ampicillin; VM, vancomycin; GM, gentamicin; KM, kanamycin; SM, streptomycin; EM, erythromycin; ClM, clindamycin; TC, tetracycline; CM, chloramphenicol

[0087] Experimental Example 2: Confirmation of the antibacterial effect of the lactic acid bacteria of the present invention against bacteria causing vaginitis

[0088] Brain Heart Infusion (BHI) medium (BD Biosciences Korea, Seoul, Korea) was supplemented with 1% yeast extract (BD), 0.1% maltose, 0.1% glucose, and 10% horse serum to prepare BHIS medium. The isolated lactic acid bacteria (1 × 10 6 CFU / ml) and Gardnerella vaginalis or Staphylococcus aureus (1 × 10 6 CFU / ml) were inoculated and cultured for 24 hours under anaerobic conditions at 37°C. Bacteria were then harvested, and bacterial DNA was extracted using a Qiagen DNA purification kit (Qiagen, Germany). Gardnerella vaginalis and Staphylococcus aureus were quantified by qPCR.

[0089] Specifically, qPCR was performed using extracted DNA (100 ng), Sybr premix (Takara Bio Inc., Japan), and primers in a thermal cycler (QIAGEN NV, Germany). The primers listed in Table 5 below were used, and the PCR was performed at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 72°C for 30 seconds. The results of the antibacterial effect are shown in Table 5 below.

[0090] [Table 5]

[0091] Experimental Example 3: Confirmation of the anti-inflammatory effect of the lactic acid bacteria of the present invention

[0092] Ten C57BL / 6 mice (male, 6 weeks old, 19-22 g) were used per group. 2 mL of sterilized 4% thioglycolate was administered intraperitoneally to the mice. Four days later, the mice were anesthetized and 8 mL of RPMI 1640 medium was administered intraperitoneally. Five to ten minutes later, the RPMI medium (containing macrophages) was removed from the mouse peritoneal cavity, centrifuged at 1,000 rpm for 10 minutes, and then washed twice with RPMI 1640 medium. 0.5 × 10 macrophages were added to each well. 6 The lactic acid bacteria and heat-inactivated G. vaginalis (1 × 10), an inflammatory response inducer, were plated on a 24-well plate. 5 The cells were cultured in a CO₂ / air incubator for 24 hours. The supernatant was collected, and the expression levels of TNF-α, IL-1β, and IL-10 cytokines were measured using an Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (eBioscience, San Diego, CA, USA).

[0093] Specifically, 0.05 mL of the supernatant was placed in a 96-well plate coated with antibodies against TNF-α, IL-1β, and IL-10, and incubated at room temperature for 2 hours. After washing with phosphate buffered saline-Tween (PBS-Tween), a coloring agent was added and allowed to develop for 20 minutes. The absorbance was measured at 450 nm and calculated. The amount of cells attached to the well plate was then measured as viable cell mass using the MTT method. The results of the anti-inflammatory efficacy test of lactic acid bacteria are shown in Table 6 below.

[0094] Experimental Example 4. Effect of Claudin-1 protein expression in Caco2 cells

[0095] Caco2 cells were provided by the Korea Cell Line Bank and cultured in RPMI1640 medium for 48 hours. After that, 2 × 10 Caco2 cells were cultured per well. 6 Then, the isolated lactic acid bacteria (1 × 10 4 CFU / ml) and G. vaginalis (1 × 10 5 The cells were cultured in a CO₂ / air incubator with 20 ng / ml (CFU / ml) of lactic acid bacteria. The cells were then collected, lysed in RIPA buffer, and centrifuged (10,000 rpm, 10 min). The supernatant was separated, and the amount of claudin-1 protein was measured using an ELISA kit (eBioscience, San Diego, CA, USA). The method used in Experimental Example 3 was repeated. The expression levels of claudin-1 for each lactic acid bacteria are shown in Table 6 below.

[0096] [Table 6] JPEG2025528234000011.jpg220159JPEG2025528234000012.jpg221159JPEG2025528234000013.jpg105159

[0097] 1) Inhibition rate: -, <5% inhibition; +, 5-20% inhibition; ++, 21-40% inhibition; +++, 41-60% inhibition; ++++, 61-80% inhibition; +++++, 81-100% inhibition. Inhibition rate (%) = 100 × ([group treated with G. vaginalis or S. aureus only - group treated with lactic acid bacteria and G. vaginalis or S. aureus] / [group treated with G. vaginalis or S. aureus only - group treated with vehicle only]).

[0098] 2) Increase rate: -, <5% increase; +, 5-20% increase; ++, 21-40% increase; +++, 41-60% increase; ++++, 61-80% increase; +++++, 81-100% increase. Induction / increase (%) = 100 × ([group treated with Lactobacillus and G. vaginalis - group treated with G. vaginalis only] / [group treated with vehicle only - group treated with G. vaginalis only]).

[0099] As shown in Table 6, among the isolated lactic acid bacteria, Lactococcus lactis P32 and Bifidobacterium bifidum P45 exhibited excellent antibacterial effects against Gardnerella vaginalis and Staphylococcus aureus. Furthermore, they reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β) compared with anti-inflammatory cytokines (IL-10) in mice with inflammatory responses. This suggests that Lactococcus lactis P32 and Bifidobacterium bifidum P45 are lactic acid bacteria with excellent antibacterial and anti-inflammatory effects.

[0100] Experimental Example 5. Selection of lactic acid bacteria that improve vaginitis

[0101] 5-1. Creation of a vaginitis model mouse and administration of lactic acid bacteria

[0102] C57BL / 6 mice (female, 18-21 g, 6 weeks old) were allowed to adapt to the laboratory for one week, then divided into seven groups of five mice each. The mice were injected subcutaneously with 0.125 mg of β-estradiol 17-benzoate (Sigma, MO, USA) dissolved in olive oil. After 72 hours, they were inoculated with Gardnerella vaginalis (1 × 10 8The mice were intravaginally infected with 1 × 10 CFU / mouse / 20 μL of the lactic acid bacteria Lactobacillus plantarum P4, Lactobacillus sakei P11, Lactococcus lactis P32, Bifidobacterium bifidum P45, or Bifidobacterium longum P51 once daily from day 8 post-infection for 14 days. 9 CFU / mouse were orally administered. Lactobacillus was administered for 14 days, and behavioral tests were performed 24 hours later for two days. The mice were sacrificed the next day, and blood was collected. The vaginal lavage fluid was collected. Vaginal, colonic, and brain tissues were then isolated.

[0103] 5-2. Confirmation of vaginitis occurrence and quantification of Gardnerella vaginalis strains

[0104] The degree of inflammation accompanied by edema in the vagina and uterus infected with Gardnerella vaginalis was visually confirmed. DNA was isolated from the lavage fluid collected from the mice using a Qiagen DNA purification kit (Qiagen, Germany), and Gardnerella vaginalis was quantified by PCR. PCR was performed as in Experimental Example 2. The measurement results are shown in Table 7 below.

[0105] 5-3. Cytokine analysis of vagina, colon, and hippocampus

[0106] The vagina, colon, and hippocampus isolated from the mice were homogenized in RIPA lysis buffer and centrifuged to obtain the supernatant, and the levels of TNF-α, IL-1β, IL-6, IL-10, and BDNF were measured using an ELISA kit. Protein levels were measured using a Bradford assay. Corticosterone and IL-6 levels in the blood were also measured using an ELISA kit. The measurement method was the same as in Experimental Example 3. The measurement results are shown in Table 7 below.

[0107] 5-4. Behavioral experiments

[0108] 5-4-1. Elevated plus maze (EPM) test

[0109] The elevated plus maze experimental apparatus consisted of two open arms (30 × 7 cm) and two enclosed arms (30 × 7 cm) with 20 cm-high walls, each extending 7 cm (7 × 7 cm) from a central platform. The walls were made of black Plexiglas, and were stacked 50 cm above the floor. The movements of mice placed in the elevated plus maze were recorded in a room illuminated at 20 lux and equipped with a video camera above.

[0110] Mice were placed in the center of the elevated plus maze (EPM) with their head facing the open arm, and the time and number of times spent in the open and enclosed arms were measured for 5 minutes. An arm entry was defined as an entry with all four paws. The time spent in the open arms during the entire experimental period was calculated for each mouse as (time spent in open arms) / (time spent in open arms + time spent in enclosed arms) × 100. After each behavioral experiment, any remaining odor was removed with 70% ethanol. The measurement results are shown in Table 7 below.

[0111] 5-4-2. Tail Suspension Test (TST)

[0112] The mouse was suspended 1 cm from the tip of its tail in the center of a 35 cm diameter, 50 cm high tube using a restraining device. The mouse's immobility time was measured for a total of 6 minutes. Immobility refers to a state in which the mouse remains motionless and exhibits minimal activity. The measurement results are shown in Table 7 below.

[0113] 5-4-3. Forced Swimming Test (FST)

[0114] A cylindrical tank measuring 40 cm in height and 20 cm in diameter was filled with water at 25±1°C to a height of 30 cm. Each mouse was placed in the tank individually. The first 2 minutes of the 6-minute period were not measured as an adaptation period, and the last 4 minutes were used to measure the immobility time of the experimental animals. "Immobility" refers to a state in which the animal floats upright and motionless, making only minimal movements to keep only its head above the water surface. The measurement results are shown in Table 7 below.

[0115] [Table 7] JPEG2025528234000015.jpg38159

[0116] 1) Inhibition rate: -, <5% inhibition; +, 5-20% inhibition; ++, 21-40% inhibition; +++, 41-60% inhibition; ++++, 61-80% inhibition; +++++, 81-100% inhibition. Inhibition rate (%) = 100 × ([group treated with G. vaginalis or S. aureus only - group treated with lactic acid bacteria and G. vaginalis or S. aureus] / [group treated with G. vaginalis or S. aureus only - group treated with vehicle only]).

[0117] As shown in Table 7, among the isolated lactic acid bacteria, Lactococcus lactis P32 and Bifidobacterium bifidum P45 had excellent growth inhibitory effects on Gardnerella vaginalis in the vagina and significantly reduced the levels of inflammatory cytokines in the vagina, blood, colon, and hippocampus.

[0118] Furthermore, the lactic acid bacteria-administered group showed an increase in the time and frequency spent in the open aisle in the elevated plus maze test, and a significant decrease in the immobility time in the forced swimming test and tail suspension test.

[0119] This suggests that Lactococcus lactis P32 and Bifidobacterium bifidum P45 are not only highly effective in preventing and treating inflammatory diseases, but are also effective strains in improving depression, anxiety disorders, and other conditions.

[0120] Experimental Example 6: Confirmation of the vaginitis-improving effects of Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains in mice with induced vaginitis

[0121] 6-1. Creation of mice with vaginitis and administration of lactic acid bacteria

[0122] C57BL / 6 mice (female, 18-21 g, 6 weeks old) were divided into five groups of five mice per group and allowed to adapt to the laboratory for one week. The mice were subcutaneously injected with 0.125 mg of β-estradiol 17-benzoate (Sigma, MO, USA) dissolved in olive oil. After 72 hours, they were inoculated with Gardnerella vaginalis (1 × 10 8 The mice were infected intravaginally with 20 μL of lactic acid bacteria (CFU / mouse / 20 μL) once daily for 7 days. The negative control group (NC) was treated with only the saline solution used to suspend Gardnerella vaginalis, and the positive control group (GV) was infected with Gardnerella vaginalis but was not administered any lactic acid bacteria.

[0123] The experimental group received 1 × 10 Lactococcus lactis P32, Bifidobacterium bifidum P45, and a mixed strain (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) isolated from the lactic acid bacteria once daily for 14 days from day 8 after infection. 9 CFU / mouse were orally administered.

[0124] Lactic acid bacteria were administered for 14 days, and behavioral tests were conducted 24 hours later for two days. The next day, the mice were sacrificed, blood was collected, and vaginal lavage fluid was collected. Blood, vagina, colon, and hippocampus were then isolated.

[0125] 6-2. Confirmation of the effect of improving vaginitis and quantification of Gardnerella vaginalis strains

[0126] The occurrence of inflammation and the improvement effect of lactic acid bacteria administration were confirmed from vaginal samples isolated from mice. Gardnerella vaginalis strains were also quantified from the vagina. This was done in the same manner as in Experiment 5, and the measurement results are shown in Figure 5 and Table 8 below.

[0127] As shown in Figure 5 and Table 8 below, inflammation occurred in the positive control group (GV) infected with Gardnerella vaginalis, and administration of the lactic acid bacteria of the present invention improved the inflammation. Furthermore, administration of the lactic acid bacteria of the present invention reduced Gardnerella vaginalis strains in the vagina. This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are effective strains for preventing or improving inflammation and Gardnerella vaginalis infection.

[0128] 6-3. Cytokine analysis of vagina, colon, blood, and hippocampus

[0129] The vagina, colon, and hippocampus isolated from mice were homogenized in RIPA lysis buffer and centrifuged to obtain the supernatants, which were then used for cytokine analysis. Expression levels of TNF-α, IL-1β, IL-6, and IL-10 were measured in the vaginal supernatant, TNF-α, IL-1β, IL-6, and IL-10 in the colonic supernatant, and TNF-α, IL-1β, IL-6, IL-10, and BDNF in the hippocampal supernatant using ELISA kits. Blood corticosterone and IL-6 levels were also measured using ELISA kits. The measurement methods were similar to those in Experimental Example 3.

[0130] In addition, the expression levels of claudin-1 in the vaginal and colonic supernatants and claudin-5 in the hippocampal supernatants were measured by immunoblotting, and the protein levels were measured by Bradford assay.

[0131] Specifically, 50 μg of the supernatant was electrophoresed on a 10% (w / v) SDS polyacrylamide gel for 1 hour 30 minutes. The electrophoresed sample was transferred to nitrocellulose paper at 100 V and 400 mA for 1 hour 10 minutes. The nitrocellulose paper with the transferred sample was blocked with 5% skim milk for 30 minutes, washed three times for 5 minutes with phosphate-buffered saline-Tween, and incubated overnight with claudin-1 or claudin-5 antibody (Santa Cruz Biotechnology, USA) at a 1:100 ratio. The paper was then washed three times for 10 minutes, and incubated with secondary antibody (Santa Cruz Biotechnology, USA) at a 1:1000 ratio for 1 hour 20 minutes. The gel was then washed three times for 15 minutes, developed, and the intensity of the colored bands was measured. The measurement results are shown in Table 8 below.

[0132] 6-4. Behavioral experiments

[0133] The elevated plus maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were performed in the same manner as in Experimental Example 5. The measurement results are shown in Table 8 below.

[0134] [Table 8] JPEG2025528234000017.jpg167159

[0135] 1) The rate of increase was calculated relative to the negative control (NC) group treated with vehicle only, with the rate set at 1.

[0136] As shown in Table 8, Lactococcus lactis P32, Bifidobacterium bifidum P45, or a combination of these strains reduced the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines in the vagina, colon, blood, and hippocampus.

[0137] Furthermore, when the lactic acid bacteria and their mixed strains were administered, the time and frequency spent in the open aisle in the elevated plus maze test increased, the time spent in the immobility state in the forced swimming test and tail suspension test decreased significantly, and the levels of BDNF and Claudin-5 in the hippocampus increased.

[0138] This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are not only highly effective in preventing or treating vaginitis, but are also effective strains in improving depression or anxiety disorders caused by inflammatory diseases.

[0139] Experimental Example 7: Confirmation of the osteoporosis-improving effect of a mixed strain of Lactococcus lactis P32 and Bifidobacterium bifidum P45 in osteoporosis-induced mice

[0140] 7-1. Creation of osteoporotic mice and administration of lactic acid bacteria

[0141] C57BL / 6 mice (female, 18-21 g, 6 weeks old) were divided into three groups of seven mice and allowed to adapt to the laboratory for one week. After anesthesia with isoflurane, the ovaries were removed (ovariectomy) to induce osteoporosis.

[0142] The negative control group (NC) had not had the ovaries removed, and the positive control group (OV) had the ovaries removed but was not administered Lactobacillus.

[0143] The experimental group received 1 × 10 9 A mixed strain of Lactococcus lactis P32 and Bifidobacterium bifidum P45 (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) was orally administered to mice. 24 hours after the last dose, behavioral tests were performed for two days. The mice were sacrificed the next day and blood samples were collected. The vagina, brain, and femur were then isolated.

[0144] 7-2. Confirmation of occurrence of vaginitis and improvement effect

[0145] The occurrence of inflammation and the improvement effect of lactic acid bacteria administration were confirmed using vaginas isolated from mice. The same procedures were carried out as in Experiment 5, and the results are shown in Figure 6 below.

[0146] As shown in Figure 6 below, inflammation occurred in ovariectomized mice, and administration of the lactic acid bacteria of the present invention improved the inflammation. This suggests that the mixed strain of Lactococcus lactis P32 and Bifidobacterium bifidum P45 is an effective strain for preventing or improving inflammation.

[0147] 7-3. Cytokine analysis of vagina, colon, blood, and hippocampus and bone weight measurement

[0148] The vagina, colon, and hippocampus isolated from the mice were homogenized in RIPA lysis buffer and centrifuged to obtain the supernatant, which was then used for cytokine analysis. Expression levels of TNF-α and IL-6 were measured in the vaginal supernatant, and TNF-α, IL-1β, and BDNF in the hippocampal supernatant using an ELISA kit. The measurement methods were similar to those described in Experimental Example 3. Blood corticosterone and IL-6 were measured using an ELISA kit, and osteocalcin was measured using an R&D system ELISA kit (Minneapolis, MN, USA). Calcium (Ca) was measured using ASAN Ca-Lq Reagents (ASAN PHARM CO., LTD.). Femoral bone weights were also measured. The results are shown in Table 9 below.

[0149] 7-4. Behavioral experiments

[0150] The elevated plus maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were performed as in Experimental Example 5. The measurement results are shown in Table 9 below.

[0151] [Table 9] JPEG2025528234000019.jpg20159

[0152] As shown in Table 9, when the mixed strain of Lactococcus lactis P32 and Bifidobacterium bifidum P45 was administered, blood calcium and osteocalcin levels increased, and bone weight increased compared to the positive control group not administered lactic acid bacteria.

[0153] Furthermore, when the mixed strains of lactic acid bacteria were administered, the time and frequency spent in the open aisle in the elevated plus maze test increased, the time spent immobile in the forced swimming test and tail suspension test decreased significantly, and BDNF levels increased in the hippocampus.

[0154] This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains have excellent effects in preventing or treating osteoporosis, and are effective strains in improving depression and anxiety disorders caused by female hormone imbalance.

[0155] Experimental Example 8: Confirmation of the effects of Lactococcus lactis P32, Bifidobacterium bifidum P45, and a mixed strain on improving vaginitis and osteoporosis in mice with concomitant vaginitis and osteoporosis

[0156] 8-1. Creation of mice with vaginitis and osteoporosis and administration of lactic acid bacteria

[0157] Seven C57BL / 6 mice (female, 21-23 g, 6 weeks old) were acclimated to the laboratory for 1 week. The mice were anesthetized with isoflurane and then ovaries were removed to induce osteoporosis. The next day, the mice were subcutaneously injected with 0.125 mg of β-estradiol 17-benzoate (Sigma, MO, USA) dissolved in olive oil. 72 hours later, the mice were infected with Gardnerella vaginalis (1 × 10 8 The mice were infected intravaginally with 20 μL of 1000 ng / mouse (CFU / mouse / 20 μL) once daily for 7 days.

[0158] The negative control group (NC) was treated with only the saline solution used to suspend Gardnerella vaginalis, and the positive control group (GV / OV) was infected with Gardnerella vaginalis but was not administered any lactic acid bacteria.

[0159] The experimental group received 1 × 10 9CFU / mouse of isolated Lactococcus lactis P32, Bifidobacterium bifidum P45, and a mixed strain (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) were orally administered. 24 hours after the last Lactobacillus administration, behavioral tests were performed for two days. The mice were sacrificed the following day, and blood was collected. The vagina was washed and the vaginal wash was collected. Blood, vagina, colon, and brain were then isolated.

[0160] 8-2. Confirmation of the effect of improving vaginitis and quantification of Gardnerella vaginalis strains

[0161] The occurrence of inflammation and the improvement effect of lactic acid bacteria administration were confirmed using vaginal samples isolated from mice. Gardnerella vaginalis strains were also quantified in the vagina. This was done in the same manner as in Experiment 5, and the measurement results are shown in Figure 7 and Table 10 below.

[0162] As shown in Figure 7 and Table 10 below, inflammation occurred in the positive control group (GV / OV) in which mice were infected with Gardnerella vaginalis and then ovarian removed, and administration of the lactic acid bacteria of the present invention improved the inflammation. Furthermore, administration of the lactic acid bacteria of the present invention reduced Gardnerella vaginalis strains in the vagina. This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are effective strains for preventing or improving inflammation and Gardnerella vaginalis infection.

[0163] 8-3. Cytokine analysis of vagina, colon, blood, and hippocampus and bone weight measurement

[0164] The vagina, colon, and hippocampus isolated from mice were homogenized in RIPA lysis buffer and centrifuged to obtain the supernatants, which were then used for cytokine analysis. Expression levels of TNF-α, IL-1β, IL-6, and IL-10 were measured in the vaginal supernatant, TNF-α, IL-1β, IL-6, and IL-10 in the colonic supernatant, and TNF-α, IL-1β, IL-6, IL-10, and BDNF in the hippocampal supernatant using an ELISA kit. The measurement methods were similar to those in Experimental Example 3.

[0165] The expression levels of claudin-1 in the vaginal and colonic supernatants and claudin-5 in the hippocampal supernatants were measured by immunoblotting, and the protein levels were measured by Bradford assay. The measurement methods were the same as those in Experimental Example 6.

[0166] Blood corticosterone and IL-6 were measured using an ELISA kit, and osteocalcin was measured using an R&D system ELISA kit (Minneapolis, MN, USA). Calcium (Ca) and phosphorus (P) were measured using ASAN Ca-Lq Reagents (ASAN PHARM CO., LTD.) and ASAN Pi-Lq Reagents (ASAN PHARM CO., LTD., Korea). Protein content was measured using the Bradford assay. Femoral bone weights were also measured. The measurement results are shown in Table 10.

[0167] 8-4. Behavioral experiments

[0168] The elevated plus maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were performed as in Experimental Example 5. The measurement results are shown in Table 10 below.

[0169] [Table 10] JPEG2025528234000021.jpg196159JPEG2025528234000022.jpg33159

[0170] 1) The increase rate was calculated relative to the negative control group (NC) treated with vehicle only, with the value set at 1.

[0171] As shown in Table 10, Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains decreased the levels of inflammatory cytokines and increased the levels of anti-inflammatory cytokines in the vagina, colon, blood, and hippocampus. They also increased the levels of calcium, phosphorus, and osteocalcin in the blood, and increased body weight.

[0172] On the other hand, when the lactic acid bacteria and mixed strains were administered, the time and frequency spent in the open aisle in the elevated plus maze test increased, the time spent in the immobility state in the forced swimming test and tail suspension test decreased significantly, and the levels of BDNF and Claudin-5 in the hippocampus increased.

[0173] This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are highly effective in preventing or treating vaginitis and osteoporosis, and are effective strains in improving inflammatory diseases or depression and anxiety disorders caused by female hormone imbalance.

[0174] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. For example, each component described as a single component may be implemented in a distributed form, and components described as distributed may also be implemented in a combined form.

[0175] The scope of the present invention is defined by the claims that follow, and all modifications and variations that fall within the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

[0176] [Accession number] Depository institution: Korea Microorganism Collection (overseas) Accession number: KCCM13222P Date of acceptance: 20220803 Depository institution: Korea Microorganism Collection (overseas) Accession number: KCCM13223P Date of acceptance: 20220803

Claims

1. Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P.

2. The Lactococcus lactis P32 KCCM13222P according to claim 1, wherein the Lactococcus lactis P32 comprises the 16S rDNA base sequence of SEQ ID NO:

1.

3. Bifidobacterium bifidum P45 KCCM13223P.

4. The Bifidobacterium bifidum P45 KCCM13223P according to claim 3, wherein the Bifidobacterium bifidum P45 comprises the 16S rDNA base sequence of SEQ ID NO:

2.

5. An antibacterial composition comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

6. A pharmaceutical composition for the prevention or treatment of Gardnerella vaginalis or Staphylococcus aureus infections, comprising the composition of claim 5.

7. A pharmaceutical composition for preventing or treating inflammatory diseases or osteoporosis, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

8. The pharmaceutical composition of claim 7, wherein the inflammatory disease is vaginitis.

9. A pharmaceutical composition for preventing or treating depression or anxiety disorders, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

10. The pharmaceutical composition of claim 9, wherein the depression or anxiety disorder is caused by an inflammatory disease or an imbalance of female hormones.

11. The pharmaceutical composition according to any one of claims 6 to 10, wherein the mixture is a mixture of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P in a colony forming unit (CFU) ratio of 1:1 to 4:

1.

12. The pharmaceutical composition according to any one of claims 6 to 10, wherein the Lactococcus lactis P32 KCCM13222P or Bifidobacterium bifidum P45 KCCM13223P is in the form of live cells, killed cells, a culture, a disrupted product, or an extract.

13. A food composition for preventing or ameliorating inflammatory diseases or osteoporosis, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

14. The food composition of claim 13, wherein the inflammatory disease is vaginitis.

15. A food composition for preventing or ameliorating depression or anxiety disorders, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

16. The food composition according to claim 15, wherein the depression or anxiety disorder is caused by an inflammatory disease or an imbalance of female hormones.

17. 17. The food composition according to any one of claims 13 to 16, wherein the mixture is a mixture of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P in a colony forming unit (CFU) ratio of 1:1 to 4:

1.

18. The food composition according to any one of claims 13 to 16, wherein the Lactococcus lactis P32 KCCM13222P or Bifidobacterium bifidum P45 KCCM13223P is in the form of live cells, killed cells, a culture, a disrupted product, or an extract.

19. A method for preventing or treating inflammatory diseases, osteoporosis, depression, or anxiety disorders, comprising the step of administering to an individual Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

20. Use of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof for the prevention or treatment of inflammatory diseases, osteoporosis, depression, or anxiety disorders.

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