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] This invention relates to novel lactic acid bacteria, Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or mixtures thereof, and their uses. [Background technology]

[0002] Under normal conditions, a woman's vagina is a slightly acidic environment due to the many lactic acid bacteria inside, which helps to suppress the invasion of pathogenic microorganisms and the growth of fungi. This slightly acidic environment inside the vagina can be disrupted by external factors, such as excessive vaginal douching, long-term use of antibiotics or contraceptives, and hormonal imbalances caused by pregnancy, childbirth, or menopause. This disruption of the amount of beneficial lactic acid bacteria normally present in the vagina can cause changes, which is known to be a cause of vaginitis, which induces discharge, itching, and pain.

[0003] Many methods have been studied to prevent and treat vaginitis as described above. Recently, the common treatment for bacterial vaginal infections involves oral or cream administration of vaginitis agents such as metronidazole. However, the use of broad-spectrum antibiotics like metronidazole is not only problematic due to the risk of antibiotic resistance, but is also considered undesirable because it can kill a wide range of normal bacteria in the vagina, including beneficial lactic acid bacteria. Furthermore, it has been reported that long-term use of antibiotics can cause systemic toxicity due to antibiotic absorption through the vagina. Therefore, there is an urgent need to develop safe vaginitis prevention or treatment compositions that can prevent and / or treat vaginal infections without causing significant side effects.

[0004] On the other hand, osteoporosis is a complex disease influenced by genetic factors and environmental factors such as diet and lifestyle. In women, bone loss accelerates rapidly during menopause due to hormonal changes. Currently available osteoporosis treatments include vitamin D, female hormones, bisphosphonates, selective estrogen receptor modulators, and calcitonin preparations, but most work by reducing the activity of osteoclasts and regulating bone resorption to prevent bone loss. However, osteoporosis treatments that suppress osteoclast activity are not fundamentally curative and have limitations in achieving a complete cure. Therefore, an increase in osteoblast activity, which can lead to a substantial increase in bone density and bone strengthening, is essential.

[0005] Most osteoporosis treatments currently in use are estrogen-based substances, and long-term administration of these substances presents problems such as side effects including cancer, gallstones, and thrombosis. Osteoporosis cannot be treated with short-term drug administration alone; long-term drug administration is essential. Therefore, there is a real need to develop new substances that do not have the aforementioned side effects when administered long-term and that possess superior efficacy to replace estrogen.

[0006] On the other hand, infectious diseases and hormonal imbalance (menopausal) disorders can be accompanied by conditions such as depression and anxiety disorders. Antidepressants and other drugs used to treat such mental illnesses can cause serious side effects such as cardiovascular disease and suicide, so their use is restricted. Furthermore, these mental illnesses cannot be treated with short-term drug administration alone and require long-term administration. Therefore, there is a need to develop new substances that have excellent efficacy without side effects when administered long-term. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The objective of the present invention is to provide the Lactococcus lactis P32 KCCM13222P strain.

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

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

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

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

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

[0013] One aspect of the present invention that aims to achieve the aforementioned objectives relates to Lactococcus lactis P32 KCCM13222P.

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

[0015] The 16S rDNA sequence for the identification and classification of Lactococcus lactis P32 of the present invention is as shown in Sequence ID No. 1 attached herein. Therefore, Lactococcus lactis P32 of the present invention may contain the 16S rDNA of Sequence ID No. 1.

[0016] Analysis of the 16S rDNA sequence of Sequence ID No. 1 showed 99% homology with known Lactococcus lactis strains and demonstrated 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 Center for Microbial Preservation on August 3, 2022 (KCCM13222P).

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

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

[0019] The 16S rDNA sequence for the identification and classification of Bifidobacterium bifidum P45 of the present invention is as shown in Sequence ID No. 2 attached herein. Therefore, Bifidobacterium bifidum P45 of the present invention may contain the 16S rDNA of Sequence ID No. 2.

[0020] The analysis result of the 16S rDNA nucleotide sequence of SEQ ID NO: 2 showed 99% homology with known Bifidobacterium bifidum strains and the highest molecular phylogenetic relationship with Bifidobacterium bifidum (Figure 3). Therefore, the lactic acid bacterium was identified as Bifidobacterium bifidum and named Bifidobacterium bifidum P45, which was deposited with the Korean Culture Center of Microorganisms on August 3, 2022 (KCCM13223P).

[0021] Bifidobacterium bifidum P45 of the present invention is a Gram-positive bacillus. More specific physiological characteristics of Bifidobacterium bifidum P45 can be analyzed by the usual 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] Another aspect of the present invention relates to an antibacterial composition containing Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

[0023] Still another aspect of the present invention relates to a pharmaceutical composition for preventing or treating Gardnerella vaginalis or Staphylococcus aureus infection, which contains the above 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), can be utilized for antibacterial purposes, and thus can also be used for preventing or treating Gardnerella vaginalis or Staphylococcus aureus infection.

[0025] A further 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] In this specification, "inflammatory disease" refers collectively to diseases in which inflammation is the main pathology. The inflammatory disease of the present invention may be one or more selected from the group including arthritis, gout, hepatitis, obesity, keratitis, gastritis, enteritis, nephritis, colitis, diabetes, tuberculosis, bronchitis, pleurisy, peritonitis, spondylitis, pancreatitis, inflammatory pain, urethritis, cystitis, vaginitis, arteriosclerosis, sepsis, and periodontitis. More specifically, the inflammatory disease may be vaginitis, but is not limited to vaginitis.

[0027] Vaginitis, as described above, is an inflammatory disease that occurs in the vagina of women and can be broadly divided into three main causes: bacterial vaginosis (BV), candidiasis (CV), and trichomonas vaginitis (Trichomonas vaginalis). Of these, bacterial vaginosis is now recognized as the most common type of vaginitis, and it has been reported that Atopobium vaginae, Gardnerella vaginalis, and Prevotella bivia have been found in vaginal secretions isolated from patients with bacterial vaginosis, with Gardnerella vaginalis being particularly known as the main causative agent of vaginitis. The novel lactic acid bacteria of the present invention shows an effective inhibitory effect against Gardnerella vaginalis and is 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 this can be utilized for therapeutic purposes against inflammatory diseases.

[0029] In this invention, "osteoporosis" is defined as a disease induced by an imbalance between osteoblasts and osteoclasts, where bone resorption outweighs bone formation. This leads to a decrease in calcium in bone tissue, thinning of the bone's compact layer, widening of the bone marrow cavity, and weakening of the bone as the condition progresses, making it more susceptible to fractures from even minor impacts. While factors such as old age, lack of exercise, low body weight, smoking, low-calcium diet, menopause, and oophorectomy are known to cause osteoporosis, in women, bone loss occurs continuously from age 30 onwards, and accelerates rapidly during menopause due to hormonal changes.

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

[0031] A further 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 aforementioned "depressive disorder" refers to a disorder characterized primarily by decreased motivation and feelings of sadness, causing a variety of cognitive and psychosomatic symptoms that impair daily functioning. It may be one or more of the following: 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 aforementioned "anxiety disorder" refers to a mental illness that causes impairment in daily life due to abnormal, pathological anxiety and fear in various forms, and may include, but is not limited to, one or more of the following: generalized anxiety disorder, specific phobia, agoraphobia, social anxiety disorder, panic disorder, separation anxiety disorder, and selective mutism.

[0034] The aforementioned “depression” or “anxiety disorder” may be caused by, but is not limited to, inflammatory diseases or female hormone imbalances.

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

[0036] More 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 embodiments of the present invention, mice of vaginitis and / or osteoporosis disease models were treated with a mixture of Lactococcus lactis P32 and Bifidobacterium bifidum P45, or it was confirmed that the mixture had excellent preventive or therapeutic effects on osteoporosis (Tables 8 to 10).

[0038] Furthermore, in more detail, Lactococcus lactis P32 KCCM13222P or Bifidobacterium bifidum P45 KCCM13223P may be their live cells, dead cells, cultures, lysates, or extracts, respectively, but can be applied without limitation as long as it is in a form that can achieve the desired effect.

[0039] In the present invention, "live bacteria" refers to the novel lactic acid bacteria of the present invention itself, "dead bacteria" refers to lactic acid bacteria that have been sterilized by heating, pressurizing, or chemical treatment, and "crushed material" refers to lactic acid bacteria that have been destroyed by enzymatic treatment, homogenization, or ultrasonic treatment.

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

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

[0042] The pharmaceutical compositions according to the present invention can be manufactured into pharmaceutical dosage forms using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. In manufacturing the dosage forms, the pharmaceutical compositions according to the present invention may further include pharmaceutically acceptable carriers, provided that they do not inhibit the activity of the novel lactic acid bacteria.

[0043] A further aspect of the present invention relates to a method for preventing or treating an inflammatory disease, osteoporosis, depression, or anxiety disorder, comprising the step of administering a pharmaceutical composition comprising the strain or a mixture thereof to a subject.

[0044] The aforementioned "inflammatory diseases," "osteoporosis," "depression," and "anxiety disorders" are as described above, and in more detail, the aforementioned inflammatory disease may be vaginitis.

[0045] The subject organisms mentioned above are animals, and are typically mammals that can show beneficial effects from the treatment using the lactic acid bacteria of the present invention. Preferred examples of such subjects may include primates such as humans.

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

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

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

[0049] The aforementioned “depression” or “anxiety disorder” may be caused by, but is not limited to, inflammatory diseases or female hormone imbalances.

[0050] More specifically, the aforementioned mixture of bacterial strains may, 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 types of food products mentioned above are not particularly limited. Food products to which lactic acid bacteria may be added include sausages, meats, bread, chocolates, snacks, candies, confectionery, ramen, pizza, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes. When formulated into drinking water, the liquid components added in addition to the novel lactic acid bacteria are not limited to these, but may include various flavorings or natural carbohydrates as additional components, as is the case with ordinary beverages. The aforementioned natural carbohydrates may be monosaccharides (e.g., glucose, fructose), disaccharides (e.g., maltose, sucrose), polysaccharides (e.g., ordinary sugars such as dextrin and cyclodextrin), 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 live cells, dead cells, cultures, crushed products, or extracts thereof, but any form of lactic acid bacteria capable of achieving preventive or therapeutic effects for inflammatory diseases or osteoporosis can be used without limitation. The terms "live cells," "dead cells," "cultures," "crushed products," and "extracts" are as described above.

[0053] More specifically, the aforementioned food may be a health functional food. Such health functional foods are foods that emphasize the biological regulatory functions of food, and are foods to which added value has been added using physical, biochemical, and biotechnological methods to act and express specific purposes. The components of such health functional foods are related to biological defense and regulation of biorhythms, disease prevention, and recovery.

[0054] The product is designed and processed to fully exert its bodily regulatory functions on the living body, and may contain food additives, sweeteners, or functional ingredients that are acceptable as food.

[0055] When using Lactococcus lactis P32 or Bifidobacterium bifidum P45 of the present invention as a health functional food (or health functional beverage additive), the novel lactic acid bacteria can be added as is or used together with other foods or food components, and can be used appropriately by conventional methods. The mixing ratio of Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 can be appropriately determined depending on the intended use (prevention, health or improvement, therapeutic treatment).

[0056] A further 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 Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof, to an individual.

[0057] The aforementioned "inflammatory diseases," "osteoporosis," "depression," and "anxiety disorders" are as described above. More specifically, the aforementioned inflammatory disease may be vaginitis.

[0058] The aforementioned “individual” includes animals or humans having inflammatory diseases, osteoporosis, depression, or anxiety disorders whose symptoms can be improved by administration of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or mixtures thereof, according to the present invention. By administering the bacterial strains, strain mixtures, or compositions containing the same to an individual, inflammatory diseases, osteoporosis, depression, or anxiety disorders can be effectively prevented and treated.

[0059] The term "administration" means introducing a given substance into a person or animal in a suitable manner, and the administration route of the bacterial strain, strain mixture, or composition containing the same according to the present invention may be orally or parenterally via a common route, as long as it can reach the target tissue. Furthermore, the therapeutic composition according to the present invention may be administered by any device that allows the active ingredient to move into target cells.

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

[0061] Another aspect of the present invention relates to the preventive or therapeutic use of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or mixtures thereof, for inflammatory diseases, osteoporosis, depression, or anxiety disorders.

[0062] The aforementioned "inflammatory diseases," "osteoporosis," "depression," and "anxiety disorders" are as described above. More specifically, the aforementioned inflammatory disease may be vaginitis. [Effects of the Invention]

[0063] The present invention relates to Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or mixtures thereof, which are excellent in improving vaginitis, osteoporosis, depression, or anxiety disorders.

[0064] The effects of this invention should be understood to include not only those described above, but also all effects that can be inferred from the detailed description of the invention or the configuration of the invention as described in the claims. [Brief explanation of the drawing]

[0065] [Figure 1] This figure shows the homology of Lactococcus lactis P32 KCCM13222P to known Lactococcus lactis strains in terms of 16S rDNA. [Figure 2] This figure shows the phylogenetic characteristics of Lactococcus lactis P32 KCCM13222P. [Figure 3] This figure shows the homology of 16S rDNA between Bifidobacterium bifidum P45 KCCM13223P and known Bifidobacterium bifidum strains. [Figure 4] This figure shows the phylogenetic characteristics of Bifidobacterium bifidum P45 KCCM13223P. [Figure 5] This figure shows the effect of the lactic acid bacteria of the present invention on improving vaginitis, as confirmed by experimental example 6 of the present invention. [Figure 6] This figure shows the effect of the lactic acid bacteria of the present invention on improving vaginitis, as confirmed by experimental example 7 of the present invention. [Figure 7] This figure shows the effect of the lactic acid bacteria of the present invention on improving vaginitis, as confirmed by 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. However, the following examples are merely illustrative of the present invention and the present invention is not limited to the following examples.

[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 liquid medium (GAM broth; Nissui Pharmaceutical, Japan). The supernatant was then taken and transferred to GAM agar medium (GAM agar medium; Nissui Pharmaceutical, Japan), MRS agar medium (BD, USA), or BL agar medium (BL agar medium; Nissui Pharmaceutical, Japan). After anaerobic incubation at 37°C for approximately 48 hours, strains that formed colonies 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 given lactic acid bacteria strain names are shown in Table 1 below. For details, see: 6 species of Lactobacillus plantarum (numbers 1 to 6 in Table 1), 5 species of Lactobacillus sakei (numbers 6 to 10 in Table 1), 2 species of Lactobacillus reuteri (numbers 11 and 12 in Table 1), 3 species of Lactobacillus rhamnosus (numbers 13 to 15 in Table 1), 5 species of Lactococcus lactis (numbers 16 to 20 in Table 1), 3 species of Bifidobacterium adolescentis (numbers 21 to 23 in Table 1), Bifidobacterium bifidum These included 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).

[0072] [Table 1] JPEG0007864254000002.jpg98159

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

[0074] Of the bacterial 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 Sequence ID No. 1. A BLAST search of the 16S rDNA sequences revealed that no other Lactococcus lactis strains with the same 16S rDNA sequence were found, confirming 99% homology with the 16S rDNA sequences of known Lactococcus lactis strains.

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

[0076] [Table 2] JPEG0007864254000004.jpg126159

[0077] The aforementioned novel strain, Lactococcus lactis P32, has been deposited with the Korea Center for Microbial Preservation (address: Yu-lim Bldg. 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an authorized depositary institution, and has been assigned accession number KCCM13222P.

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

[0079] Bifidobacterium bifidum P45 is a Gram-positive rod-shaped bacterium that did not exhibit hemolysis in blood culture media. The 16S rDNA of Bifidobacterium bifidum P45 has been shown to have the nucleotide sequence of Sequence ID No. 2. Comparison of the 16S rDNA sequences using BLAST search revealed that no other Bifidobacterium bifidum strains with the same 16S rDNA sequence were found, confirming 99% homology with the 16S rDNA sequences of known Bifidobacterium bifidum strains.

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

[0081] [Table 3] JPEG0007864254000006.jpg212159

[0082] The aforementioned novel strain, Bifidobacterium P45, was deposited with the Korea Center for Microbial Preservation (address: Yu-lim Bldg. 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Republic of Korea), an authorized depositary institution, and was given accession number KCCM13223P.

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

[0084] The minimum inhibitory concentration (MIC) for antibiotics is determined by adding 2 × 10⁶ Lactococcus lactis P32 and Bifidobacterium bifidum P45 to 2 ml of antibiotic-containing BHI (Brain Heart Infusion) medium (BD Biosciences Korea, Seoul, South Korea). 5 The cells were transplanted at CFU / ml and cultured anaerobically at 37°C for 24 hours for evaluation. 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 that cause vaginitis.

[0088] BHIS medium was prepared by adding 1% yeast extract (BD), 0.1% maltose, 0.1% glucose, and 10% horse serum to BHI (Brain Heart Infusion) medium (BD Biosciences Korea, Seoul, South Korea). Lactobacillus (1 × 10⁶) isolated from BHIS medium was added. 6 CFU / ml) and Gardnerella vicinalis or Staphylococcus aureus (1 x 10) 6 CFU / ml was transplanted and cultured at 37°C under anaerobic conditions for 24 hours. Then, the cells were collected and bacterial DNA was extracted using the Qiagen DNA purification kit (Qiagen, Germany), and Gardnerella vaginalis and Staphylococcus aureus were quantified by qPCR.

[0089] For details, qPCR was performed using a thermal cycler (QIAGEN NV, Germany) with extracted DNA (100 ng), Sybr premix (Takara Bio Inc., Japan), and primers. The primers listed in Table 5 below were used, and the assay was performed over 40 cycles, alternating between 95°C for 5 seconds and 72°C for 30 seconds. The results of the antibacterial efficacy measurement are shown in Table 5 below.

[0090] [Table 5]

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

[0092] Ten C57BL / 6 mice (male, 6 weeks old, 19-22g) were used per group. Two mL of sterile 4% thioglycolate was administered intraperitoneally to the mice. Four days later, the mice were anesthetized, and eight mL of RPMI1640 medium was administered intraperitoneally. After 5-10 minutes, the RPMI medium (containing macrophages) was taken from the mouse peritoneal cavity, centrifuged at 1,000 rpm for 10 minutes, and then washed twice with RPMI1640 medium. Macrophages were separated into 0.5 × 10⁶ wells. 6 The bacteria were spread in a 24-well plate in the specified number, along with heat-treated inactivated G. vaginalis (1 × 10⁶), which is an inflammatory response inducer. 5 The culture was performed in a CO2 / air incubator with CFU / ml added. The supernatant was obtained, and the expression levels of TNF-α, IL-1β, and IL-10 cytokines were measured using an ELISA (Enzyme-Linked ImmunoSorbent Assay) kit (eBioscience, San Diego, CA, USA).

[0093] For details, first, 0.05 mL of the supernatant was placed in a 96-well plate coated with TNF-α, IL-1β, and IL-10 antibodies, and reacted at room temperature for 2 hours. Then, it was washed with phosphate-buffered saline-Tween (PBS-Tween), a chromogenic agent was added and allowed to develop for 20 minutes, and the absorbance was measured and calculated at a wavelength of 450 nm. The amount of cells attached to the well plate was then measured by the MTT method to determine the amount of viable cells. The results of the lactic acid bacteria anti-inflammatory efficacy test are shown in Table 6 below.

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

[0095] Caco2 cells were obtained from the Korea Cell Line Bank and cultured in RPMI1640 medium for 48 hours. Then, 2 × 10⁶ Caco2 cells were cultured per well. 6 The cells were dispensed according to the cell count. Next, the isolated lactic acid bacteria (1 × 10) were placed in each well. 4 Add CFU / ml, along with G. vaginalis (1 × 10) 5 20 ng / ml of CFU (Cholesterol Fusate) was added, and the cells were cultured in a CO2 / air incubator for 24 hours. Next, the cells were collected, lysed with RIPA buffer, and then 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 described in Experimental Example 3 above was followed. The expression levels of claudin-1 for each type of lactic acid bacterium are shown in Table 6 below.

[0096] [Table 6] JPEG0007864254000010.jpg220159JPEG0007864254000011.jpg221159JPEG0007864254000012.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 only G. vaginalis or S. aureus - Group treated with lactic acid bacteria and G. vaginalis or S. aureus] / [Group treated with only G. vaginalis or S. aureus - Group treated with only vehicle])

[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 lactic acid bacteria and G. vaginalis - Group treated with only G. vaginalis] / [Group treated with only vehicle - Group treated with only G. vaginalis])

[0099] As shown in Table 6 above, Lactococcus lactis P32 and Bifidobacterium bifidum P45 among the isolated lactic acid bacteria are excellent in antibacterial effects against Gardnerella vaginalis or Staphylococcus aureus. Also, in mice with induced inflammatory responses, the numerical values of inflammatory cytokines (TNF-α, IL-1β) compared to the anti-inflammatory cytokine (IL-10) were decreased. 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 Lactobacillus for Improving Vaginitis

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

[0102] After adapting C57BL / 6 mice (female, 18 - 21 g, 6 weeks old) in the laboratory for one week, they were divided into 7 groups of 5 mice each for the experiment. The mice were subcutaneously injected with 0.125 mg of β-estradiol 17-benzoate (manufactured by Sigma, MO, USA) dissolved in olive oil. 72 hours later, Gardnerella vaginalis (1×10 8The vagina was infected with CFU / mouse / 20μL. From day 8 after infection, for 14 days, one dose each of the following lactic acid bacteria was administered daily: Lactobacillus plantarum P4, Lactobacillus sakei P11, Lactococcus lactis P32, Bifidobacterium bifidum P45, or Bifidobacterium longum P51 (1 x 10⁶). 9 CFU / mouse was administered orally. Lactobacillus was administered for 14 days, followed by behavioral experiments for 2 days 24 hours later. The mice were sacrificed the following day to separate their blood, and their vaginas were washed to obtain vaginal douche. Next, vaginal, colon, and brain tissue were 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 examined. DNA was isolated from the washing fluid obtained from mice using the Qiagen DNA purification kit (Qiagen, Germany), and Gardnerella vaginalis was quantified by PCR. PCR was performed in the same manner as in Experimental Example 2. The measurement results are shown in Table 7 below.

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

[0106] Vagina, colon, and hippocampus isolated from mice were homogenized with RIPA lysis buffer, centrifuged, and the supernatant was obtained. The amounts of TNF-α, IL-1β, IL-6, IL-10, and BDNF were measured using an ELISA kit. Protein levels were measured using the Bradford assay. Corticosterone and IL-6 levels were also measured from blood using an ELISA kit. The measurement methods described in Experimental Example 3 were followed. 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 cross maze experimental apparatus consists of two open arms (30 x 7 cm) and two enclosed arms (30 x 7 cm) with walls 20 cm high, each extending 7 cm (7 x 7 cm) from a central platform. It is made of black plexiglass and the walls are stacked 50 cm higher than the base. The movement of mice placed in the elevated cross maze was recorded in a room with a brightness of 20 lux and a video camera mounted above.

[0110] Mice were placed in an elevated cross maze (EPM) with their heads facing the open passages, and the time and number of times they spent in the open and closed passages were measured over a 5-minute period. Arm entry was defined as all four paws entering a passage. The time spent in the open passages during the total experimental time (Time spent in open arms) was calculated as (Time spent in open arms + Time spent in enclosed arms) × 100 for each mouse. 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] A mouse's tail, 1 cm from the tip, was suspended from the center of a cylinder 35 cm in diameter and 50 cm in height using a fixing device. The mouse's immobility time was measured for a total of 6 minutes. Immobility refers to a state in which the mouse does not move and only performs minimal actions. The measurement results are shown in Table 7 below.

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

[0114] A cylindrical tank, 40 cm high and 20 cm in diameter, was filled with water at a temperature of 25 ± 1°C to a height of 30 cm. One mouse was placed in each tank. Of the total 6 minutes, the first 2 minutes were not measured as adaptation time, and the last 4 minutes were measured for the time the experimental animals remained motionless. Motionlessness refers to a state in which the animal floats upright without moving, 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] JPEG0007864254000014.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 only with G. vaginalis or S. aureus - Group treated with lactic acid bacteria and G. vaginalis or S. aureus] / [Group treated only with G. vaginalis or S. aureus - Group treated only with vehicle])

[0117] As shown in Table 7 above, among the isolated lactic acid bacteria, Lactococcus lactis P32 and Bifidobacterium bifidum P45 were excellent at inhibiting the growth of Gardnerella vaginalis in the vagina and significantly reduced inflammatory cytokine levels in the vagina, blood, large intestine, and hippocampus.

[0118] Furthermore, in the lactic acid bacteria administration group, the time and frequency spent in the open passage of the elevated cross maze test increased, and the time spent immobile in the forced swimming test and tail suspension test decreased significantly.

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

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

[0121] 6-1. Production of vaginitis-inducing mice and administration of lactic acid bacteria

[0122] C57BL / 6 mice (female, 18-21g, 6 weeks old) were divided into 5 groups of 5 mice each and allowed to acclimate to the laboratory for 1 week. 0.125 mg of β-estradiol 17-benzoate (Sigma, MO, USA) dissolved in olive oil was subcutaneously injected into the mice, and 72 hours later, Gardnerella vaginalis (1 x 10⁶) was administered. 8 CFU / mouse / 20μL) was administered to the vagina once daily for 7 days. The negative control group (NC) was treated only with the saline solution used to suspend Gardnerella vaginalis, while the positive control group (GV) was infected with Gardnerella vaginalis but no lactic acid bacteria were administered afterward.

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

[0124] Lactic acid bacteria were administered for 14 days, followed by behavioral experiments for 2 days 24 hours later. The next day, the mice were sacrificed to separate their blood, and their vaginas were washed to obtain vaginal douche. Next, the blood, vagina, large intestine, and hippocampus were separated.

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

[0126] We confirmed the occurrence of inflammation and its improvement with lactic acid bacteria administration from vaginas isolated from mice. We also quantified Gardnerella vaginalis strains from the vaginas. The procedure was the same as in Experimental Example 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 the inflammation improved when the lactic acid bacteria of the present invention were administered. Furthermore, the number of Gardnerella vaginalis strains in the vagina decreased when the lactic acid bacteria of the present invention were administered. This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains thereof are effective strains for preventing or improving inflammation and preventing or improving Gardnerella vaginalis infection.

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

[0129] Vagina, colon, and hippocampus isolated from mice were homogenized with RIPA lysis buffer, centrifuged, and the supernatant was obtained for the analysis of cytokines and other substances. The expression levels of TNF-α, IL-1β, IL-6, and IL-10 were measured from the vaginal supernatant, the colon supernatant, and the hippocampal supernatant using an ELISA kit. The levels of corticosterone and IL-6 in the blood were also measured using an ELISA kit. The measurement methods described above refer to those in Experimental Example 3.

[0130] Furthermore, the expression levels of claudin-1 were measured from the supernatant of the vagina and large intestine, and the expression levels of claudin-5 were measured from the supernatant of the hippocampus using immunoblotting. Protein levels were measured using the Bradford assay.

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

[0132] 6-4. Behavioral Experiments

[0133] Similar to Experimental Example 5, the elevated cross maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were conducted. The measurement results are shown in Table 8 below.

[0134] [Table 8] JPEG0007864254000016.jpg167159

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

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

[0137] Furthermore, when the aforementioned lactic acid bacteria and mixed strains thereof were administered, the time and frequency spent in the open passage of the elevated cruciform maze test increased, the time spent in a stationary state in the forced swimming test and tail suspension test decreased significantly, and BDNF and Claudin-5 levels in the hippocampus were elevated.

[0138] This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains of these bacteria are not only highly effective in preventing or treating vaginitis, but are also effective in improving depressive 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. Production of osteoporosis mice and administration of lactic acid bacteria

[0141] C57BL / 6 mice (female, 18-21g, 6 weeks old) were divided into three groups of seven mice each 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) did not have their ovaries removed, while the positive control group (OV) did not receive lactic acid bacteria after their ovaries were removed.

[0143] The experimental group underwent 1 x 10⁶ tests once daily for 4 weeks (28 days). 9 A mixed strain of Lactococcus lactis P32 and Bifidobacterium bifidum P45 (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) isolated from CFU / mouse was orally administered. Behavioral experiments were conducted for two days starting 24 hours after the last dose of lactic acid bacteria was administered, and the mice were sacrificed the following day to isolate their blood. Next, the vagina, brain, and femur were isolated.

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

[0145] We examined the onset of inflammation and its improvement through lactic acid bacteria administration from vaginal tissue isolated from mice. The procedure was the same as in Experimental Example 5, and the results are shown in Figure 6 below.

[0146] As shown in Figure 6 below, inflammation occurred in mice from which the ovaries had been removed, and the inflammation improved when the lactic acid bacteria of the present invention were administered. 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 the vagina, large intestine, blood, and hippocampus, and measurement of bone weight.

[0148] Vagina, colon, and hippocampus isolated from mice were homogenized with RIPA lysis buffer, centrifuged, and the supernatant was obtained for the analysis of cytokines and other substances. The expression levels of TNF-α and IL-6 were measured in the vaginal supernatant, and the expression levels of TNF-α, IL-1β, and BDNF were measured in the hippocampal supernatant using an ELISA kit. The measurement method described in Experimental Example 3 was used as a reference. Blood corticosterone and IL-6 were measured using an ELISA kit, and osteocalcin was measured using the R&D system ELISA Kit (Minneapolis, MN, USA). Calcium (Ca) was measured using ASAN Ca-Lq Reagents (ASAN PHARM CO., LTD.). The weight of the femur bone was also measured. The measurement results are shown in Table 9 below.

[0149] 7-4. Behavioral Experiments

[0150] Similar to Experiment Example 5, the elevated cross maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were conducted. The measurement results are shown in Table 9 below.

[0151] [Table 9] JPEG0007864254000018.jpg20159

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

[0153] Furthermore, when the mixed strain of lactic acid bacteria was administered, the time and frequency spent in the open passage of the elevated cruciform maze test increased, the time spent in a stationary state in the forced swimming test and tail suspension test decreased significantly, and BDNF levels in the hippocampus increased.

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

[0155] Experimental Example 8. Confirmation of the effects of Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains on improving vaginitis and osteoporosis in mice with co-induced vaginitis and osteoporosis.

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

[0157] Seven C57BL / 6 mice (female, 21-23g, 6 weeks old) were adapted to laboratory conditions for one week. The mice were anesthetized with isoflurane, their ovaries were removed to induce osteoporosis, and the following day, 0.125 mg of β-estradiol 17-benzoate (Sigma, MO, USA) dissolved in olive oil was subcutaneously injected into the mice. 72 hours later, Gardnerella vaginalis (1 x 10⁻¹⁶) was administered. 8 CFU / mouse / 20μL was administered to the vagina once daily for 7 days.

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

[0159] The experimental group received 1x10⁶ treatments once daily for 4 weeks (28 days) starting from day 8 after infection. 9CFU / mouse isolated lactic acid bacteria Lactococcus lactis P32, Bifidobacterium bifidum P45, and a mixed strain (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) were orally administered. Behavioral experiments were conducted for 2 days starting 24 hours after the last dose of lactic acid bacteria was administered. The following day, the mice were sacrificed to separate their blood, and their vaginas were washed to obtain vaginal douche. Next, the blood, vagina, large intestine, and brain were isolated.

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

[0161] We confirmed the occurrence of inflammation and its improvement with lactic acid bacteria administration from vaginas isolated from mice. We also quantified Gardnerella vaginalis strains from the vaginas. The procedure was the same as in Experimental Example 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 the ovaries were removed after infection with Gardnerella vaginalis, and the inflammation improved when the lactic acid bacteria of the present invention were administered. Furthermore, the number of Gardnerella vaginalis strains in the vagina decreased when the lactic acid bacteria of the present invention were administered. This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains thereof are effective strains for preventing or improving inflammation and preventing or improving Gardnerella vaginalis infection.

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

[0164] The vagina, colon, and hippocampus isolated from mice were homogenized with RIPA lysis buffer, centrifuged, and the supernatant was obtained for the analysis of cytokines and other substances. The expression levels of TNF-α, IL-1β, IL-6, and IL-10 were measured from the vaginal supernatant, the colonal supernatant, and the hippocampal supernatant, along with BDNF, using an ELISA kit. The measurement method described in Experimental Example 3 was used as a reference.

[0165] Furthermore, the expression levels of claudin-1 were measured from the supernatant of the vagina and large intestine, and the expression levels of claudin-5 were measured from the supernatant of the hippocampus by immunoblotting, while protein levels were measured by the Bradford assay. The measurement methods described above are based on those in Experimental Example 6.

[0166] Blood corticosterone and IL-6 were measured using an ELISA kit, and osteocalcin was measured using the 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 levels were measured using the Bradford assay. The weight of the femur bone was also measured. The measurement results are shown in Table 10.

[0167] 8-4. Behavioral Experiments

[0168] Similar to Experiment Example 5, the elevated cross maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were conducted. The measurement results are shown in Table 10 below.

[0169] [Table 10] JPEG0007864254000020.jpg196159JPEG0007864254000021.jpg33159

[0170] 1) The rate of increase was calculated relatively, with the negative control group (NC) treated only with vehicles set to 1.

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

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

[0173] This suggests that Lactococcus lactis P32, Bifidobacterium bifidum P45, and mixed strains thereof 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 imbalances.

[0174] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains should understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the above-described embodiments should be understood to be illustrative and not limiting in all respects. For example, each component described as a single form may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0175] The scope of the present invention is defined by the claims described below, and all modifications or alterations derived from the meaning and scope of the claims and the concept of equivalents thereto should be interpreted as being included within the scope of the present invention.

[0176] [Accession Number] Depository name: Korea Microbial Conservation Center (overseas) Accession number: KCCM13222P Date of acceptance: 20220803 Depository name: Korea Microbial Conservation Center (overseas) Accession number: KCCM13223P Date of acceptance: 20220803

Claims

1. Lactococcus lactis P32 (KCCM13222P).

2. The Lactococcus lactis P32 according to claim 1, wherein the Lactococcus lactis P32 comprises the 16S rDNA base sequence of Sequence ID No.

1.

3. Bifidobacterium bifidum P45 (KCCM13223P).

4. The Bifidobacterium bifidum P45 described in claim 3 comprises the 16S rDNA base sequence of Sequence ID No.

2.

5. An antimicrobial 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 infection, comprising the composition described in claim 5.

7. 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.

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

9. 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.

10. The pharmaceutical composition according to claim 9, wherein the depression or anxiety disorder is caused by an inflammatory disease or a female hormone imbalance.

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 its live cells, its dead cells, its culture, its crushed product, or its extract.

13. A food composition for the prevention or improvement of inflammatory diseases or osteoporosis, comprising Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof.

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

15. A food composition for the prevention or improvement of 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 a female hormone imbalance.

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 its live cells, its dead cells, its culture, its crushed product, or its extract.

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