Novel lactic acid bacteria and use thereof

Abstract

The invention relates to novel lactic acid bacteria, namely lactococcus lactis P32 (KCCM13222P), bifidobacterium bifidum P45 (KCCM13223P) or a mixture of the lactococcus lactis P32 and the bifidobacterium bifidum P45, and application of the novel lactic acid bacteria, the lactococcus lactis P32 and the bifidobacterium bifidum P45 and the mixture of the lactococcus lactis P32 and the bifidobacterium bifidum P45. The strains of the invention and mixtures thereof can be usefully used in the prevention or treatment of inflammatory diseases, osteoporosis, depression or anxiety.

Description

Technical Field

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

[0002] Under normal conditions, the female vagina forms a weakly acidic environment due to a large number of lactic acid bacteria in the vagina, which plays a role in inhibiting the invasion of pathogenic microorganisms or the proliferation of fungi. This weakly acidic environment in the vagina is disrupted when the number of beneficial bacteria normally present in the vagina, namely lactic acid bacteria, changes due to external factors such as excessive vaginal washing, long-term administration of antibiotics or contraceptives, and hormonal imbalances caused by pregnancy / childbirth / menopause, etc., which is considered to be the cause of vaginitis leading to secretions, itching and pain.

[0003] A large number of studies have been conducted on methods for preventing and treating the above-mentioned vaginitis. Recently, as a treatment method for vaginal bacterial infections, methods of orally administering or applying a vaginitis therapeutic agent such as Metronidazole in the form of a cream are generally used. However, the use of broad-spectrum antibiotics such as the aforementioned Metronidazole not only brings about the problem of antibiotic resistance, but also kills various normal flora in the vagina including beneficial lactic acid bacteria, and is thus considered undesirable. At the same time, there are also reports that long-term use of antibiotics can cause problems of systemic toxicity due to the absorption of antibiotics through the vagina. Therefore, there is an urgent need to develop a safe vaginitis prevention or treatment composition that can prevent and / or treat vaginitis without side effects.

[0004] On the other hand, osteoporosis is a complex disease that is also affected by environmental factors such as genetic factors, diet and lifestyle. Especially in women at menopause, due to hormonal changes, bone loss progresses rapidly. Currently marketed osteoporosis therapeutic agents include vitamin D, estrogen, bisphosphonate preparations, selective estrogen receptor modulators, calcitonin preparations, etc., but most methods rely on regulating bone resorption by reducing the activity of osteoclasts to prevent bone loss. However, the osteoporosis treatment method based on inhibiting osteoclast activation is not a therapy that can fundamentally treat osteoporosis, and thus there are limitations in complete cure. Increasing the activity of osteoblasts that can actually increase bone density and strengthen bones is the key.

[0005] Most of the currently used osteoporosis therapeutics are estrogenic substances, and there are problems of side effects such as cancer, gallstones, and thrombosis when administered for a long time. Osteoporosis is a disease that cannot be treated by short-term administration alone but requires long-term administration. Therefore, there is an urgent need to develop a new substance that does not cause the above side effects even when administered for a long time and has excellent drug efficacy sufficient to replace estrogen.

[0006] On the other hand, infectious diseases, hormonal imbalance (menopause) diseases, etc. may be accompanied by diseases such as depression and anxiety. Drugs such as antidepressants used to treat these mental diseases may cause serious side effects such as cardiovascular diseases and suicide, so their use is restricted. These mental diseases cannot be treated by short-term administration alone but require long-term administration. Therefore, there is an urgent need to develop a new substance that has no side effects when administered for a long time and has excellent drug efficacy. Summary of the Invention Technical Problem

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

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

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

[0010] Another 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] Another object of the present invention is to provide a method for preventing or treating an inflammatory disease, osteoporosis, depression or anxiety disorder, comprising the step of administering Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P or a mixture thereof to an individual. Technical solution

[0013] One aspect of the present invention aimed at achieving the above object relates to Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P.

[0014] Lactococcus lactis P32 of the present invention is characterized in that it is a novel lactic acid bacterium isolated and identified from the feces of healthy humans.

[0015] The 16S rDNA base sequence used for identifying and classifying Lactococcus lactis P32 of the present invention is as shown in SEQ ID NO: 1 attached to this specification. Therefore, Lactococcus lactis P32 of the present invention may include the 16S rDNA of SEQ ID NO: 1.

[0016] The analysis result of the 16S rDNA base sequence of SEQ ID NO: 1 shows that it has 99% homology with known Lactococcus lactis strains, showing the highest molecular phylogenetic relationship with Lactococcus lactis ( Figure 1 ). Therefore, the lactic acid bacterium was identified as Lactococcus lactis and named Lactococcus lactis P32 (Lactococcus lactis P32), and was deposited with the Korean Culture Center of Microorganisms (KCCM13222P) on August 3, 2022.

[0017] Lactococcus lactis P32 of the present invention is a Gram-positive coccus. The specific physiological characteristics of Lactococcus lactis P32 can be analyzed according to the common methods in the corresponding technical field. Specifically, Lactococcus lactis P32 can use 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, gentiobiose as carbon sources.

[0018] Another aspect of the present invention relates to Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P.

[0019] The Bifidobacterium bifidum P45 of the present invention is characterized in that it is a novel lactic acid bacterium isolated and identified from the feces of healthy humans.

[0020] The 16S rDNA base sequence used for the identification and classification of the Bifidobacterium bifidum P45 of the present invention is shown as SEQ ID NO: 2 attached to this specification. Therefore, the Bifidobacterium bifidum P45 of the present invention may include the 16S rDNA of SEQ ID NO: 2.

[0021] The analysis result of the 16S rDNA base sequence of SEQ ID NO: 2 indicates that it has 99% homology with known Bifidobacterium bifidum strains, showing 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, and was deposited at the Korean Culture Center of Microorganisms (KCCM13223P) on August 3, 2022.

[0022] The Bifidobacterium bifidum P45 of the present invention is a Gram-positive bacillus. The more specific physiological characteristics of Bifidobacterium bifidum P45 can be analyzed according to the common methods in the corresponding technical field. Specifically, Bifidobacterium bifidum P45 can use D-glucose, D-lactose, D-sucrose, D-maltose, L-arabinose, gelatin, esculin ferric citrate, D-cellobiose, D-mannose, D-raffinose as carbon sources.

[0023] Another aspect of the present invention relates to an antibacterial composition comprising Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P or a mixture thereof.

[0024] In addition, another aspect of the present invention relates to a pharmaceutical composition for preventing or treating Gardnerella vaginalis or Staphylococcus aureus infection, comprising the antibacterial composition.

[0025] In one embodiment of the present invention, it is confirmed that the Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 inhibits Gardnerella vaginalis and / or Staphylococcus aureus (Table 6), and can be used for antibacterial purposes, and further can be used for the prevention or treatment of Gardnerella vaginalis or Staphylococcus aureus infections.

[0026] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases or osteoporosis, which comprises Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P or a mixture thereof.

[0027] In the present invention, "inflammatory disease" means the general term for diseases mainly characterized by inflammation. The inflammatory diseases of the present invention can be any 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 can be, but is not limited to, vaginitis.

[0028] The vaginitis is an inflammatory disease that occurs in the female vagina and is roughly divided into three main causes: bacterial vaginosis (BV), candidal vaginitis (CV), and trichomonas vaginitis. Among them, bacterial vaginosis is considered to be the most common vaginitis recently. It has been reported that Atopobium vaginae, Gardnerella vaginalis, Prevotella bivia, etc. are found in the vaginal secretions isolated from patients with bacterial vaginosis. In particular, Gardnerella vaginalis is considered to be the main pathogenic bacterium of vaginitis. The novel lactic acid bacteria of the present invention show an effective inhibitory effect on Gardnerella vaginalis and are effective for the treatment of vaginitis.

[0029] In one embodiment of the present invention, it is confirmed that the Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 reduces the expression of inflammatory cytokines (Table 6), and can be used for the treatment of inflammatory diseases.

[0030] In the present invention, "osteoporosis" is a disease induced by the imbalance between osteoblasts and osteoclasts, resulting in more bone resorption than bone formation. The calcium in bone tissue decreases, the compact bone becomes thinner, and thus the bone marrow cavity expands. As the disease progresses, the bones become increasingly fragile and are prone to fracture with minor impacts. Known causes of osteoporosis include advanced age, lack of exercise, low body weight, smoking, low-calcium diet, menopause, and ovarian resection. In particular, in women, bone loss progresses continuously after the age of 30, and after entering menopause, bone loss progresses rapidly due to hormonal changes.

[0031] In one embodiment of the present invention, it was confirmed that the Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 exhibited the effect of improving osteoporosis indicators (Tables 9 and 10).

[0032] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating depression or anxiety disorder, which comprises Lactococcus lactis P32 (KCCM13222P), Bifidobacterium bifidum P45 (KCCM13223P) or a mixture thereof.

[0033] The "depression" is a disease mainly characterized by low desire and melancholy, causing various cognitive and physical and mental symptoms, resulting in low daily function, including but not limited to any one or more selected from the group consisting of 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.

[0034] The "anxiety disorder" is a mental disorder that causes daily life disorders due to various forms of abnormal and pathological anxiety and fear, including but not limited to any one or more selected from the group consisting of generalized anxiety disorder, specific phobia, agoraphobia, social anxiety disorder, panic disorder, separation anxiety, and selective mutism.

[0035] The "depression" or "anxiety disorder" may be induced by inflammatory diseases or estrogen imbalance, but is not limited thereto.

[0036] In an embodiment of the present invention, through behavioral experiments and measurement of BDNF and Claudin-5 values, it was confirmed that the Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 significantly improved depressive and anxiety behaviors in a mouse model induced by inflammation and / or ovariectomy (Table 8 to Table 10).

[0037] 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.

[0038] In an embodiment of the present invention, it was confirmed that treating a mouse model of vaginitis and / or osteoporosis disease with a mixture of Lactococcus lactis P32 and Bifidobacterium bifidum P45 had excellent osteoporosis prevention or treatment effects (Table 8 to Table 10).

[0039] In addition, specifically, the Lactococcus lactis P32 KCCM13222P or Bifidobacterium bifidum P45 KCCM13223P may be their live bacteria, their dead bacteria, their cultures, their lysates, or their extracts, and as long as it is a form that can achieve the target effect, it can be applied without limitation.

[0040] 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 heat treatment, pressure treatment, or drug treatment, etc., and "lysate" refers to a product obtained by lysing lactic acid bacteria by enzymatic treatment, homogenization, or ultrasonic treatment, etc.

[0041] In the present invention, "extract" refers to a product obtained by extracting lactic acid bacteria using a known extraction solvent.

[0042] In the present invention, "culture" or "culture solution" means a product obtained by culturing lactic acid bacteria in a well-known culture medium, and the product may contain novel lactic acid bacteria. The culture medium may be selected from well-known liquid culture media or solid culture media, and for example, may be but is not limited to MRS liquid culture medium, GAM liquid culture medium, MRS agar culture medium, GAM agar culture medium, BL agar culture medium.

[0043] The pharmaceutical composition of the present invention can be prepared into a pharmaceutical dosage form by methods well-known in the industry, so as to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the dosage form, the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier within the range that does not damage the activity of the novel lactic acid bacteria.

[0044] Another 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 to an individual a pharmaceutical composition comprising the strain or a mixture of the strains.

[0045] The "inflammatory disease", "osteoporosis", "depression" and "anxiety disorder" are the same as those described above. Specifically, the inflammatory disease may be vaginitis.

[0046] The individual may refer to an animal, typically a mammal that can exhibit beneficial effects by being treated with the lactic acid bacteria of the present invention. As a preferred example of such an individual, it may include primates, such as humans.

[0047] One aspect of the present invention relates to a food composition comprising Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P or a mixture thereof for preventing or treating an inflammatory disease or osteoporosis.

[0048] The "inflammatory disease" and "osteoporosis" are the same as those described above. Specifically, the inflammatory disease may be vaginitis.

[0049] Another aspect of the present invention relates to a food composition comprising Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P or a mixture thereof for preventing or improving depression or anxiety disorder.

[0050] The "depression" or "anxiety disorder" may be induced by an inflammatory disease or estrogen imbalance, but is not limited thereto.

[0051] Specifically, the mixture of the strains can, but is not limited to, be formed by mixing Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P in a colony forming unit (CFU) ratio of 1:1 to 4:1.

[0052] There is no particular limitation on the type of the food. The food to which lactic acid bacteria can be added can be sausage, meat, bread, chocolate, snacks, candies, pastries, instant noodles, pizza, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, rehydration fluids, alcoholic beverages, and vitamin complexes, etc. When the preparation is a beverage, the liquid components added in addition to the novel lactic acid bacteria can, like ordinary beverages, contain various flavoring agents or natural carbohydrates, etc. as supplementary components, but are not limited thereto. The above natural carbohydrates can be monosaccharides (such as glucose, fructose, etc.), disaccharides (such as maltose, sucrose, etc.), and polysaccharides (such as common sugars like dextrin, cyclodextrin, etc.), as well as sugar alcohols such as xylitol, sorbitol, erythritol, etc.

[0053] In addition, specifically, the lactic acid bacteria contained in the pharmaceutical composition of the present invention can be its live bacteria, its dead bacteria, its culture, its lysate or its extract. However, as long as it is in a form of lactic acid bacteria that can achieve the preventive or therapeutic effect on inflammatory diseases or osteoporosis, it can be used without limitation. The "live bacteria", "dead bacteria", "culture", "lysate" and "extract" are the same as the foregoing content.

[0054] Specifically, the food can be a health functional food. As a food that emphasizes the biological regulatory function of food, the health functional food is a food that uses physical, biochemical, and bioengineering methods to add value for a specific purpose to achieve a specific purpose. The components of such a health functional food are designed and processed so as to fully exert the body regulatory functions related to biological defense, body rhythm regulation, disease prevention and rehabilitation on organisms, and can contain food acceptable food auxiliary additives, sweeteners or functional raw materials.

[0055] When using Lactococcus lactis P32 or Bifidobacterium bifidum P45 of the present invention as a health functional food (or an additive for a health functional beverage), the novel lactic acid bacteria can be directly added, or used together with other foods or food ingredients, and used appropriately according to conventional methods. The mixing amount of Lactococcus lactis P32 and / or Bifidobacterium bifidum P45 can be appropriately determined according to its use purpose (prevention, health care or improvement, therapeutic treatment).

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

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

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

[0059] The "administration" means introducing a given substance into a human or an animal by a suitable method. The administration route of the strain, strain mixture or composition containing the same of the present invention can be administered orally or parenterally by any common route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention can be administered by means of any device that enables the active ingredient to move to the target cell.

[0060] The preferred dosage of the strain, strain mixture or composition containing the same of the present invention varies depending on the condition and body weight of the patient, the degree of the disease, the drug form, the administration route and the period, and can be appropriately selected by a practitioner.

[0061] Another aspect of the present invention relates to the use of Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P or a mixture thereof for preventing or treating an inflammatory disease, osteoporosis, depression or anxiety disorder.

[0062] The "inflammatory disease", "osteoporosis", "depression" and "anxiety disorder" are the same as those described above. Specifically, the inflammatory disease may be vaginitis. Advantageous effects

[0063] The Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P, Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P of the present invention, or a mixture thereof have excellent effects in improving vaginitis, osteoporosis, depression or anxiety disorder.

[0064] The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the invention configuration described in the detailed description or claims of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The 16S rDNA homology between Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P and known Lactococcus lactis strains was confirmed.

[0066] Figure 2 The taxonomic characteristics of Lactococcus lactis P32 (Lactococcus lactis P32) KCCM13222P are shown.

[0067] Figure 3 The 16S rDNA homology between Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P and known Bifidobacterium bifidum strains was confirmed.

[0068] Figure 4 The taxonomic characteristics of Bifidobacterium bifidum P45 (Bifidobacterium bifidum P45) KCCM13223P are shown.

[0069] Figures 5 to 7 The effect of the lactic acid bacteria of the present invention in improving vaginitis based on Experimental Examples 6 to 8 of the present invention was confirmed. BEST MODE

[0070] The present invention will be described in detail below based on examples. However, the following examples are only examples of the present invention, and the present invention is not limited by the following examples.

[0071] Example 1. Isolation and identification of lactic acid bacteria

[0072] 1-1. Isolation of lactic acid bacteria from human feces

[0073] Healthy human feces were suspended in GAM liquid medium (GAM broth, Nissui Pharmaceutical, Japan). Then, the supernatant was transplanted onto GAM agar medium (GAM agar medium; Nissui Pharmaceutical, Japan), MRS agar medium (BD, USA), or BL agar medium (BL agar medium; Nissui Pharmaceutical, Japan), and anaerobic culture was carried out at 37°C for approximately 48 hours. After that, the strains forming colonies were isolated.

[0074] 1-2. Identification of isolated lactic acid bacteria

[0075] For the strains isolated from human feces, the strains were identified by analyzing Gram staining, physiological characteristics, 16S rDNA gene sequences, etc., and strain names were assigned. The strain names of the lactic acid bacteria assigned are shown in Table 1 below. Specifically, there are 6 Lactobacillus plantarum (numbers 1 to 6 in Table 1), 5 Lactobacillus sakei (numbers 6 to 10 in Table 1), 2 Lactobacillus reuteri (numbers 11 and 12 in Table 1), 3 Lactobacillus rhamnosus (numbers 13 to 15 in Table 1), 5 Lactococcus lactis (numbers 16 to 20 in Table 1), 3 Bifidobacterium adolescentis (numbers 21 to 23 in Table 1), 3 Bifidobacterium bifidum (numbers 24 to 26 in Table 1), and 4 Bifidobacterium longum (numbers 27 to 30 in Table 1).

[0076]

Table 1

[0077] 1 - 3. Physiology of the novel lactic acid bacterium Lactococcus lactis P32 Characteristics

[0078] Among the strains listed in Table 1 above, Lactococcus lactis P32 is a Gram-positive coccus and does not show hemolysis in blood culture medium. The 16S rDNA of Lactococcus lactis P32 shows a base sequence of SEQ ID NO: 1. As a result of comparing the 16S rDNA base sequence by BLAST search, no Lactococcus lactis strain with the same 16S rDNA base sequence was retrieved, and it was confirmed that the 16S rDNA sequence has 99% homology with the known Lactococcus lactis strains.

[0079] Using the API 50CHL kit (BioMerieux's, USA), the carbon source availability in the physiological characteristics of Lactococcus lactis P32 was analyzed by sugar fermentation test. The results are shown in Table 2 below. In Table 2 below, "+" indicates the case where the carbon source availability is positive, and "-" indicates the case where the carbon source availability is negative.

[0080]

Table 2

[0081] The novel strain Lactococcus lactis P32 was applied for preservation to the authenticated depositary institution, the Korean Collection for Microorganisms (Address: Yulin Building, 45, Byeolji-ro 2-gil, Hongje-dong, Seodaemun-gu, Seoul, Korea), and was given the deposit number KCCM13222P.

[0082] 1-4. Novel lactic acid bacterium Bifidobacterium bifidum P45 Physiological characteristics

[0083] Bifidobacterium bifidum P45 is a Gram-positive bacillus and does not show hemolysis in blood culture medium. The 16S rDNA of Bifidobacterium bifidum P45 shows a base sequence of SEQ ID NO: 2. As a result of comparing the 16S rDNA base sequence by BLAST search, no Bifidobacterium bifidum strain with the same 16S rDNA base sequence was retrieved, and it was confirmed that the 16S rDNA sequence has 99% homology with the known Bifidobacterium bifidum strains.

[0084] Using an API 20CHL kit (BioMerieux's, USA), the carbon source availability in the physiological characteristics of Bifidobacterium bifidum P45 was analyzed through sugar fermentation tests. 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.

[0085]

Table 3

[0086] The novel strain Bifidobacterium bifidum P45 was applied for deposit to the authenticated deposit institution, the Korean Collection for Type Cultures (Address: Yulin Building, 45, Byeolji-ro 2-gil, Hongje-dong, Seodaemun-gu, Seoul, Korea), and was assigned the deposit number KCCM13223P.

[0087] Experimental example 1. Confirmation of antibiotic sensitivity of Lactococcus lactis P32 and Bifidobacterium bifidum P45 strains

[0088] The MIC (minimum inhibitory concentration) of antibiotics was judged as follows. In 2 ml of BHI (Brain Heart Infusion) medium containing antibiotics (BD Biosciences Korea, Seoul, Korea), Lactococcus lactis P32 and Bifidobacterium bifidum P45 were transplanted at 2×10 5 CFU / ml, anaerobically cultured at 37°C for 24 hours and judged. The results are shown in Table 4 below.

[0089]

Table 4

[0090] *AM: ampicillin; VM: vancomycin; GM: gentamicin; KM: kanamycin; SM: streptomycin; EM: erythromycin; ClM: clindamycin; TC: tetracycline; CM: chloramphenicol

[0091] Experimental example 2. Confirmation of antibacterial effect of the lactic acid bacteria of the present invention against pathogenic bacteria causing vaginitis

[0092] Yeast extract (BD), 1%, maltose, 0.1%, glucose, 0.1%, and horse serum, 10%, were added to BHI (Brain Heart Infusion) medium (BD Biosciences, Seoul, Korea) 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 transplanted into the BHIS medium and cultured under anaerobic conditions at 37°C for 24 hours. Then, the bacteria were collected, and bacterial DNA was extracted using a Qiagen DNA purification kit (Qiagen, Germany). The Gardnerella vaginalis and Staphylococcus strains were quantified by qPCR.

[0093] Specifically, the extracted DNA (100 ng), Sybr premix (Takara Bio Inc., Japan), and primers were added, and qPCR was performed in a thermal cycler (Qiagen). Using the primers shown in Table 5 below, the samples were treated at 95°C for 30 seconds, followed by 40 cycles of 5 seconds at 95°C and 30 seconds at 72°C. The antibacterial effect measurement results are shown in Table 5 below.

[0094]

Table 5

[0095] Experimental example 3. Confirmation of anti-inflammatory effect of the lactic acid bacteria of the present invention

[0096] C57BL / 6 mice (male, 6 weeks old, 19 - 22 g), 10 mice per group, were used. 2 ml of sterile 4% thioglycolate was injected into the abdominal cavity of the mice. Four days later, the mice were anesthetized, and 8 ml of RPMI 1640 medium was injected into the abdominal cavity of the mice. After 5 - 10 minutes, the RPMI medium (including macrophages) in the abdominal cavity of the mice was extracted again, centrifuged at 1000 rpm for 10 minutes, and washed twice with RPMI 1640 medium. The macrophages were plated in 24-well plates at a density of 0.5×10 6 per well, and lactic acid bacteria and heat-treated inactivated G. vaginalis (1×10 5(CFU / ml), and cultured for 24 hours in a CO2 / air incubator. The supernatant was obtained, and an ELISA (Enzyme-Linked ImmunoSorbent Assay) kit (eBioscience, San Diego, California, USA) was used to measure the expression levels of TNF-α, IL-1β, and IL-10 cytokines.

[0097] Specifically, first, 0.05 ml of the supernatant was placed in a 96-well plate coated with antibodies against TNF-α, IL-1β, and IL-10, reacted at room temperature for 2 hours, then washed with phosphate buffered saline-Tween (PBS-Tween), a chromogenic agent was added, and color development was carried out for 20 minutes, and the absorbance was measured and calculated at a wavelength of 450 nm. Also, the amount of viable cells was measured by the MTT method to obtain the amount of cells attached to the well plate. The results of the inflammatory inhibitory efficacy test of lactic acid bacteria are shown in Table 6 below.

[0098] Experimental example 4. Expression effect of claudin-1 protein in Caco2 cells

[0099] Caco2 cells were purchased from the Korean Cell Line Bank, cultured in RPMI 1640 medium for 48 hours, and then the Caco2 cells were dispensed at 2×10 6 cells / well. Then, the isolated lactic acid bacteria (1×10 4 CFU / ml) were added to each well, and simultaneously 20 ng / ml of G. vaginalis (1×10 5 CFU / ml) was added, and cultured for 24 hours in a CO2 / air incubator. Then, the cells were collected, RIPA buffer was added, the cells were lysed, and centrifuged (10,000 g, 10 minutes). The supernatant was separated, and an ELISA (Enzyme-Linked ImmunoSorbent Assay) kit (eBioscience, San Diego, California, USA) was used to measure the amount of claudin-1 protein. Refer to the method of Experimental Example 3 above. The expression levels of claudin-1 of each lactic acid bacterium are shown in Table 6 below.

[0100]

Table 6

[0101] 1) Bacteriostatic rate: -, inhibition < 5%; +, inhibition 5 - 20%; ++, inhibition 21 - 40%; +++, inhibition 41 - 60%; ++++, inhibition 61 - 80%; +++++, inhibition 81 - 100%. Bacteriostatic rate (%) = 100 × ([Group treated only with Gardnerella vaginalis or Staphylococcus aureus - Group treated with Lactobacillus and Gardnerella vaginalis or Staphylococcus aureus] / [Group treated only with Gardnerella vaginalis or Staphylococcus aureus - Group treated only with vehicle])

[0102] 2) Ascent rate: -, increase < 5%; +, increase 5 - 20%; ++, increase 21 - 40%; +++, increase 41 - 60%; ++++, increase 61 - 80%; +++++, increase 81 - 100%. Induction / Ascent (%) = 100 × ([Group treated with Lactobacillus and Gardnerella vaginalis - Group treated only with Gardnerella vaginalis] / [Group treated only with vehicle - Group treated only with Gardnerella vaginalis])

[0103] As shown in Table 6 above, Lactococcus lactis P32 and Bifidobacterium bifidum P45 among the isolated Lactobacilli have excellent antibacterial effects against Gardnerella vaginalis or Staphylococcus aureus. Additionally, in mice induced with an inflammatory response, the values of inflammatory cytokines (TNF-α, IL-1β) are decreased compared to the anti-inflammatory cytokine (IL-10). This indicates that Lactococcus lactis P32 and Bifidobacterium bifidum P45 are Lactobacilli with excellent antibacterial and anti-inflammatory effects.

[0104] Experimental example 5. Screening of lactic acid bacteria for improving vaginitis

[0105] 5-1. Preparation of vaginitis model mice and administration of lactic acid bacteria

[0106] C57BL / 6 mice (female, 18 - 21 g, 6 weeks old) were acclimated in the laboratory for one week and then divided into 7 groups of 5 mice each for the experiment. 0.125 mg of 17-β-estradiol benzoate (Sigma, Missouri, USA) was dissolved in olive oil and injected subcutaneously into the mice. 72 hours later, Gardnerella vaginalis (1×10 8 CFU / mouse / 20 μL) was infected into the vagina. Starting from the 8th day after infection, Lactobacillus plantarum P4, Lactobacillus sakei P11, Lactococcus lactis P32, Bifidobacterium bifidum P45, or Bifidobacterium longum P51 was administered orally at 1×10 9 CFU / mouse once a day for 14 consecutive days. After administering the Lactobacilli for 14 days, a 2-day behavioral test was conducted 24 hours later. The mice were sacrificed on the second day, blood was separated, the vagina was rinsed, and vaginal lavage fluid was obtained. Then, the vagina, colon, and brain tissues were separated.

[0107] 5-2. Confirmation of occurrence of vaginitis and quantification of Gardnerella vaginalis strains

[0108] Confirm the degree of inflammation accompanied by swelling in the vagina and uterus infected with Gardnerella vaginalis from the appearance. Isolate DNA from the washings obtained from mice using the Qiagen DNA purification kit (Qiagen, Germany), and quantify Gardnerella vaginalis by PCR. PCR was performed in the same manner as in Experimental Example 2 above. The measurement results are shown in Table 7 below.

[0109] 5-3. Cytokine analysis of vagina, colon and hippocampus

[0110] Add RIPA lysis buffer to the vagina, colon, and hippocampus isolated from mice, homogenize and centrifuge to obtain the supernatant, and measure the amounts of TNF-α, IL-1β, IL-6, IL-10, and BDNF using an ELISA kit. The amount of protein was determined by the Bradford assay. In addition, the amounts of corticosterone and IL-6 in the blood were measured using an ELISA kit. The measurement method refers to the method of Experimental Example 3. The measurement results are shown in Table 7 below.

[0111] 5-4. Behavioral experiments

[0112] 5-4-1. Elevated plus maze (EPM) test

[0113] The elevated plus-maze experimental apparatus is a device in which two open channels (Open arm, 30×7 cm) and two enclosed channels (Enclosed arm, 30×7 cm) with a wall height of 20 cm each extend 7 cm (7×7 cm) from the central platform. It is made of black plexiglass, and the walls are 50 cm above the ground. The room brightness is 20 lux, and a camera is installed above to record the movement of mice in the elevated plus-maze.

[0114] Place the mouse's head at the center of the elevated plus-maze (EPM), facing the open channel, and measure the time and number of times it spends in the open channel and the enclosed channel during a 5-minute period. When all four feet enter, it is considered to enter the channel (Arm entry). Calculate the time spent in the open arms in the total experimental time of each mouse, that is, (Time spent in open arms) / (Time spent in open arms + Time spent in enclosed arms)×100. After each behavioral experiment is completed, remove the residual odor with 70% ethanol. The measurement results are shown in Table 7 below.

[0115] 5-4-2. Tail Suspension Test (TST)

[0116] The end 1 cm of the mouse tail was suspended in the center of a bucket with a diameter of 35 cm and a height of 50 cm using a fixing device. The immobility time of the mouse was measured during a 6-minute period. Immobility refers to a state of not moving and only performing minimal movements. The measurement results are shown in Table 7 below.

[0117] 5-4-3. Forced Swimming Test (FST)

[0118] Water at a temperature of 25 ± 1 °C was poured into a cylindrical water tank with a height of 40 cm and a diameter of 20 cm to a height of 30 cm. The mice were placed in the water tank one by one. During a total of 6 minutes, the first 2 minutes were for adaptation time and not measured, and the immobility time of the experimental animals was measured in the next 4 minutes. Immobility refers to a state of standing still and only performing the minimal movements required to keep the head above the water surface. The measurement results are shown in Table 7 below.

[0119]

Table 7

[0120] 1) Bacteriostatic rate: -, inhibition < 5%; +, inhibition 5 - 20%; ++, inhibition 21 - 40%; +++, inhibition 41 - 60%; ++++, inhibition 61 - 80%; +++++, inhibition 81 - 100%. Bacteriostatic rate (%) = 100 × ([Group treated with only Gardnerella vaginalis or Staphylococcus aureus - Group treated with lactic acid bacteria and Gardnerella vaginalis or Staphylococcus aureus] / [Group treated with only Gardnerella vaginalis or Staphylococcus aureus - Group treated with only vehicle])

[0121] As shown in Table 7 above, Lactococcus lactis P32 and Bifidobacterium bifidum P45 among the isolated lactic acid bacteria have excellent effects in inhibiting the growth of Gardnerella vaginalis in the vagina, and significantly reduce the values of inflammatory cytokines in the vagina, blood, colon, and hippocampus.

[0122] In addition, the time and frequency of the lactic acid bacteria administration group spent in the open arms of the elevated plus maze test increased, and the immobility time in the forced swimming test and tail suspension test decreased significantly.

[0123] This indicates that Lactococcus lactis P32 and Bifidobacterium bifidum P45 can not only effectively prevent and treat inflammatory diseases, but also are effective strains for improving depression and anxiety disorders.

[0124] Experimental example 6. Confirmation of improvement effect of Lactococcus lactis P32, Bifidobacterium bifidum P45 and mixed strains on vagina of vaginitis-induced mice of vaginitis

[0125] 6-1. Preparation of mice with induced vaginitis and administration of lactic acid bacteria

[0126] C57BL / 6 mice (female, 18 - 21 g, 6 weeks old) were divided into 5 groups of 5 mice each and acclimated in the laboratory for 1 week. 0.125 mg of β - estradiol 17 - benzoate (Sigma, Missouri, USA) was dissolved in olive oil and injected subcutaneously into the mice. 72 hours later, Gardnerella vaginalis (1×10 8 CFU / mouse / 20 μL) was inoculated into the vagina once daily for 7 consecutive days. The negative control group (NC) was treated only with physiological saline used to suspend Gardnerella vaginalis, and the positive control group (GV) was not administered lactic acid bacteria after infecting with Gardnerella vaginalis.

[0127] From the 8th day after infection, the experimental groups were orally administered the isolated lactic acid bacteria Lactococcus lactis P32, Bifidobacterium bifidum P45, and a mixed strain (Lactococcus lactis P32: Bifidobacterium bifidum P45 = 4:1) at 1×10 9 CFU / mouse once daily for 14 consecutive days.

[0128] After administering the lactic acid bacteria for 14 days, 24 hours later, a 2 - day behavioral experiment was conducted. The mice were sacrificed on the second day, blood was separated, the vagina was washed to obtain vaginal lavage fluid. Then, blood, vagina, colon, and hippocampus were isolated.

[0129] 6-2. Confirmation of improvement effect of vaginitis and quantification of Gardnerella vaginalis strains

[0130] It was confirmed that vaginitis was developed in the vagina isolated from the mice and the improvement effect after administering lactic acid bacteria. In addition, the Gardnerella vaginalis strains in the vagina were quantified. It was carried out in the same method as in Experimental Example 5, and the measurement results are shown below Figure 5 and Table 8 below.

[0131] As follows Figure 5 and as shown in Table 8 below, inflammation occurred in the positive control group (GV) infected with Gardnerella vaginalis, and the inflammation was improved when the lactic acid bacteria of the present invention were administered. In addition, when the lactic acid bacteria of the present invention were administered, the Gardnerella vaginalis strains in the vagina decreased. This indicates that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are effective strains for preventing or improving inflammation and preventing or treating Gardnerella vaginalis infection.

[0132] 6-3. Cytokine analysis of vagina, colon, blood and hippocampus

[0133] RIPA lysis buffer was added to the vagina, colon, and hippocampus isolated from mice, homogenized, and centrifuged to obtain the supernatant for the analysis of cytokines and the like. Using an ELISA kit, the expression levels of TNF-α, IL-1β, IL-6, and IL-10 in the vaginal supernatant were measured, the expression levels of TNF-α, IL-1β, IL-6, and IL-10 in the colon supernatant were measured, and the expression levels of TNF-α, IL-1β, IL-6, IL-10, and BDNF in the hippocampal supernatant were measured. An ELISA kit was used to measure the levels of corticosterone and IL-6 in the blood. The measurement method is referred to the method in Experimental Example 3.

[0134] In addition, the expression levels of claudin-1 in the vaginal and colon supernatants and claudin-5 in the hippocampal supernatant were determined by immunoblotting, and the amount of protein was determined by Bradford quantification.

[0135] Specifically, 50 μg of the supernatant was electrophoresed in a 10% (w / v) SDS polyacrylamide gel for 1 hour and 30 minutes. The electrophoresed sample was transferred to a nitrocellulose paper and transferred at a voltage of 100 V and a current of 400 mA for 1 hour and 10 minutes. The nitrocellulose paper with the transferred sample was blocked with 5% skim milk for 30 minutes, then washed 3 times with phosphate buffer-Tween for 5 minutes each time, and claudin-1 or claudin-5 antibody (Santa Cruz Biotechnology, Inc., USA) was added at a ratio of 1:100 and reacted overnight. Then it was washed 3 times for 10 minutes each time, secondary antibody (Santa Cruz Biotechnology, Inc., USA) was added at a ratio of 1:1000, and reacted for 1 hour and 20 minutes. Then it was washed 3 times for 15 minutes each time, developed after fluorescence staining, and the intensity of the developed band was measured. The measurement results are shown in Table 8 below.

[0136] 6-4. Behavioral experiments

[0137] In the same manner as in Experimental Example 5, the elevated plus maze test (EPM), tail suspension test (TST), and forced swimming test (FST) were performed. The measurement results are shown in Table 8 below.

[0138]

Table 8

[0139] 1) The increase rate was calculated relatively with the negative control group (NC) treated only with the vehicle as 1.

[0140] As shown in Table 8 above, Lactococcus lactis P32, Bifidobacterium bifidum P45 or their mixed strains decreased the values of inflammatory cytokines in the vagina, colon, blood and hippocampus, and increased the values of anti-inflammatory cytokines.

[0141] In addition, when the lactic acid bacteria and their mixed strains were administered, the time spent and frequency in the open arms in the elevated plus maze test increased, the time in the immobile state in the forced swimming test and tail suspension test decreased significantly, and the amounts of BDNF and Claudin-5 in the hippocampus increased.

[0142] This indicates that Lactococcus lactis P32, Bifidobacterium bifidum P45 and their mixed strains not only have excellent effects in preventing or treating vaginitis, but also are effective strains for improving depression, anxiety and the like caused by inflammatory diseases.

[0143] Experimental example 7. Confirmation of improvement effect of Lactococcus lactis P32 and Bifidobacterium bifidum P45 mixed strains on osteoporosis in osteoporosis-induced mice of osteoporosis

[0144] 7-1. Preparation of osteoporosis mice and administration of lactic acid bacteria

[0145] C57BL / 6 mice (female, 18 - 21 g, 6 weeks old) were divided into 3 groups with 7 mice in each group and acclimated in the laboratory for 1 week. After anesthesia with isoflurane, ovariectomy was performed to induce osteoporosis.

[0146] The negative control group (NC) did not have ovaries removed, and the positive control group (OV) did not administer lactic acid bacteria after ovariectomy.

[0147] The experimental group was orally administered a mixed strain of the isolated lactic acid bacteria Lactococcus lactis P32 and Bifidobacterium bifidum P45 (Lactococcus lactis P32:Bifidobacterium bifidum P45 = 4:1) at 1×10 9 CFU / mouse once a day for 4 weeks (28 days). Behavioral tests were started 24 hours after the last administration of lactic acid bacteria, and the mice were sacrificed on the second day and blood was isolated. Then the vagina, brain and femur were isolated.

[0148] 7-2. Confirmation of occurrence and improvement effect of vaginitis

[0149] The inflammatory conditions of the vagina isolated from the mice and the improvement effects after administration of lactic acid bacteria were confirmed. It was carried out in the same manner as in Experimental Example 5, and the results are shown in Figure 6 as follows

[0150] as follows Figure 6As shown, inflammation occurred in ovariectomized mice, and when the lactic acid bacteria of the present invention were administered, the inflammation was improved. This indicates that the mixed strains of Lactococcus lactis P32 and Bifidobacterium bifidum P45 are effective strains for preventing or improving inflammation.

[0151] 7-3. Cytokine analysis and bone weight measurement of vagina, colon, blood and hippocampus

[0152] RIPA lysis buffer was added to the vagina, colon, and hippocampus isolated from mice, homogenized, centrifuged, and the supernatant was taken for analysis of cytokines and the like. The expression levels of TNF-α and IL-6 in the vaginal supernatant were measured using an ELISA kit, and the expression levels of TNF-α, IL-1β, and BDNF in the hippocampal supernatant were measured. The measurement method is referred to the method in Experimental Example 3. Corticosterone and IL-6 in the blood were measured using an ELISA kit, and osteocalcin was measured using an R&D Systems ELISA kit (Minneapolis, Minnesota, USA). Calcium (Ca) was measured using ASAN Ca-Lq Reagents (Asan Pharmaceutical). In addition, the bone weight of the femur was also measured. The measurement results are shown in Table 9 below.

[0153] 7-4. Behavioral experiments

[0154] 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 9 below.

[0155]

Table 9

[0156] As shown in Table 9 above, when the mixed strains of Lactococcus lactis P32 and Bifidobacterium bifidum P45 were administered, the values of calcium and osteocalcin in the blood increased, and the bone weight increased compared with the positive control group without the administration of lactic acid bacteria.

[0157] In addition, when the mixed strains of the lactic acid bacteria were administered, the time and frequency of staying in the open channels in the elevated plus-maze test increased, the time in the immobile state in the forced swimming test and tail suspension test decreased significantly, and the value of BDNF in the hippocampus increased.

[0158] This indicates that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are excellent in preventing or treating osteoporosis and are effective strains for improving depression and anxiety caused by estrogen imbalance.

[0159] Experimental example 8. Confirmation of improvement effect of Lactococcus lactis P32, Bifidobacterium bifidum P45 and mixed strains on vaginitis and osteoporosis in mice accompanied by induction of vaginitis and osteoporosis

[0160] 8-1. Preparation of mice with vaginitis and osteoporosis and administration of lactic acid bacteria

[0161] Seven C57BL / 6 mice (female, 21 - 23 g, 6 weeks old) were acclimated in the laboratory for 1 week. After anesthetizing the mice with isoflurane, the ovaries were removed to induce osteoporosis. The next day, 0.125 mg of β-estradiol 17-benzoate (Sigma, Missouri, USA) was dissolved in olive oil and injected subcutaneously into the mice. 72 hours later, Gardnerella vaginalis (1×10 8 CFU / mouse / 20 μL) was infected into the vagina for 7 days, once a day.

[0162] The negative control group (NC) was treated only with normal saline used to suspend Gardnerella vaginalis, and the positive control group (GV / OV) was not administered lactic acid bacteria after infecting with Gardnerella vaginalis.

[0163] From the 8th day after infection, the experimental group was orally administered the isolated lactic acid bacteria Lactococcus lactis P32, Bifidobacterium bifidum P45, and the mixed strain (Lactococcus lactis P32: Bifidobacterium bifidum P45 = 4:1) at 1×109 CFU / mouse once a day for 4 weeks (28 days). A 2-day behavioral experiment was started 24 hours after the last administration of lactic acid bacteria. The mice were sacrificed on the second day, blood was separated, and the vagina was washed to obtain vaginal lavage fluid. Then, blood, vagina, colon, and brain were separated.

[0164] 8-2. Confirmation of improvement effect of vaginitis and quantification of Gardnerella vaginalis strains

[0165] The inflammation of the vagina isolated from the mice and the improvement effect after administration of lactic acid bacteria were confirmed. In addition, the Gardnerella vaginalis strains in the vagina were quantified. It was carried out in the same manner as in Experimental Example 5, and the measurement results are shown below Figure 7 and in Table 10 below.

[0166] As follows Figure 7 As shown in Table 10 below, inflammation occurred in the positive control group (GV / OV) infected with Gardnerella vaginalis and with ovaries removed. When the lactic acid bacteria of the present invention were administered, the inflammation was improved. In addition, when the lactic acid bacteria of the present invention were administered, the number of Gardnerella vaginalis strains in the vagina decreased. This indicates that Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains are effective strains for preventing or improving inflammation and preventing or treating Gardnerella vaginalis infection.

[0167] 8-3. Cytokine analysis and bone weight measurement of vagina, colon, blood and hippocampus

[0168] RIPA lysis buffer was added to the vagina, colon, and hippocampus isolated from mice, homogenized, centrifuged, and the supernatant was taken for cytokine analysis. The expression levels of TNF-α, IL-1β, IL-6, and IL-10 in the vaginal supernatant were measured using an ELISA kit, the expression levels of TNF-α, IL-1β, IL-6, and IL-10 in the vaginal supernatant were measured, and the expression levels of TNF-α, IL-1β, IL-6, IL-10, and BDNF in the colon supernatant were measured. The measurement method is referred to the method in Experimental Example 3.

[0169] In addition, the expression levels of claudin-1 in the vaginal and colon supernatants and claudin-5 in the hippocampal supernatant were measured by immunoblotting, and the amount of protein was measured by Bradford quantification. The measurement method is referred to the method in Experimental Example 6.

[0170] Corticosterone and IL-6 in the blood were measured using an ELISA kit, and osteocalcin was measured using an R&D Systems ELISA kit (Minneapolis, MN, USA). Calcium (Ca) and phosphorus (P) were measured using ASAN Ca-Lq reagent (Asan Pharmaceutical) and ASAN Pi-Lq reagent (Asan Pharmaceutical, Korea), respectively. The amount of protein was measured by Bradford quantification. In addition, the bone weight of the femur was also measured. The measurement results are shown in Table 10.

[0171] 8-4. Behavioral experiments

[0172] 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 10 below.

[0173]

Table 10

[0174] 1) The increase rate was relatively calculated with the negative control group (NC) treated only with the vehicle as 1.

[0175] As shown in Table 10 above, Lactococcus lactis P32, Bifidobacterium bifidum P45, and their mixed strains decreased the values of inflammatory cytokines in the vagina, colon, blood, and hippocampus and increased the values of anti-inflammatory cytokines. In addition, the values of calcium, phosphorus, and osteocalcin in the blood increased, and the bone weight increased.

[0176] On the other hand, when the lactic acid bacteria and their mixed strains are administered, the time spent and frequency in the open channels during the elevated plus maze test increase, the time in the immobile state during the forced swimming test and tail suspension test significantly decreases, and the amounts of BDNF and Claudin-5 in the hippocampus increase.

[0177] This indicates that Lactococcus lactis P32, Bifidobacterium bifidum P45 and their mixed strains have excellent effects in preventing or treating vaginitis, and are also effective strains for improving inflammatory diseases or depression or anxiety caused by estrogen imbalance.

[0178] The description of the present invention as described above is for illustration. Those skilled in the technical field to which the present invention pertains can understand that without changing the technical idea or essential features of the present invention, it can be easily deformed into other specific forms. Therefore, the embodiments described above should be understood as exemplary in all aspects and not restrictive. For example, each component described in a single form can also be implemented dispersedly, and similarly, the components described in a dispersed form can also be implemented in a combined form.

[0179] The scope of the present invention is represented by the claims described hereinafter, and the meaning and scope of the claims and all changes or deformed forms derived from their equivalent concepts should be construed as being included in the scope of the present invention.

[0180] [Deposit number]

[0181] Name of depositary institution: Korean Collection for Type Cultures (abroad)

[0182] Deposit number: KCCM13222P

[0183] Deposit date: 20220803

[0184] Name of depositary institution: Korean Collection for Type Cultures (abroad)

[0185] Deposit number: KCCM13223P

[0186] Deposit date: 20220803

[0187]

[0188]

Claims

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

2. The Lactococcus lactis P32 (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 (Bifidobacterium bifidum P45) KCCM13223P.

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

2.

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

6. A pharmaceutical composition for preventing or treating Gardnerella vaginalis or Staphylococcus aureus infection, comprising the composition of claim 5.

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

10. The pharmaceutical composition according to claim 9, wherein, The depression or anxiety disorder is induced by an inflammatory disease or estrogen imbalance.

11. The pharmaceutical composition according to any one of claims 6 to 10, wherein, The mixture is composed of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P mixed at a colony-forming unit 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 bacteria, its dead bacteria, its culture, its lysate or its extract.

13. A food composition for preventing or improving 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 preventing or improving depression or anxiety, 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 induced by an inflammatory disease or estrogen imbalance.

17. The food composition according to any one of claims 13 to 16, wherein, The mixture is composed of Lactococcus lactis P32 KCCM13222P and Bifidobacterium bifidum P45 KCCM13223P mixed at a colony-forming unit 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 includes its live bacteria, its dead bacteria, its culture, its lysate or its extract.

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

20. Use of Lactococcus lactis P32 KCCM13222P, Bifidobacterium bifidum P45 KCCM13223P, or a mixture thereof for preventing or treating inflammatory diseases, osteoporosis, depression or anxiety.

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