KR102120479B1 - Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics - Google Patents

Abstract

The present invention provides a pharmaceutical composition and a food composition comprising Lactobacillus fermentum SRK414 strain with accession number KCTC13687BP and the strain or a culture thereof as an active ingredient. The strain of the present invention and its culture have excellent effects for preventing, improving, or treating osteoporosis, particularly secondary osteoporosis, and can be usefully used as a food composition and pharmaceutical composition for this purpose.

Description

Composition for preventing or treating secondary osteoporosis, including probiotics as an active ingredient {Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics}

The present invention relates to a composition for the prevention or treatment of secondary osteoporosis comprising probiotics as an active ingredient.

Osteoporosis is primary osteoporosis (primary) in which bone mass decreases due to normal physiological changes such as age and menopause, and secondary osteoporosis (secondary) caused by exposure to diseases or drugs regardless of age. It is divided into two categories. Osteoporosis is often thought to be a disease of old age, but secondary osteoporosis can develop even if you are not old, and in this case, because it has a great impact on the quality of life of patients in the future, developing a method for treating and preventing secondary osteoporosis It is very important.

The causes of secondary osteoporosis are very diverse. Osteoporosis caused by taking glucocorticoids is the most common. Glucocorticoid (Glucocorticoid: GC) has anti-inflammatory and immunosuppressive functions, and is a drug commonly used to treat various diseases such as rheumatoid arthritis, asthma, atopic dermatitis, and arthritis. Long-term administration of GC effectively suppresses these diseases, but inhibits the normal activity of osteoblasts and osteoclasts in most patients, resulting in osteoporosis. Statistically, it has been reported that in the case of GC-administered patients, 12% of bone density decreases rapidly within 1 year, and bone loss gradually progresses after that, and 2-5% of bone density gradually decreases every year. . In order to prevent secondary osteoporosis by GC administration as described above, it is the most ideal method to stop GC administration, but it is problematic in that even a small amount should be continued to treat the disease.

Eating foods that can prevent osteoporosis caused by long-term administration of GC may be a good alternative, but most commercially available products are health functional foods to improve'primary osteoporosis' such as postmenopausal women and senile osteoporosis. , And a product capable of preventing secondary osteoporosis has not been developed. Therefore, there is an urgent need to develop food or pharmaceutical products for the prevention, improvement or treatment of secondary osteoporosis.

Lee, C. B., (2008) Effect of weight reduction and calcitriol treatment for the obese patients with glucocorticoid induced osteoporosis. Endocrinology and Metabolism. 17(4):161-168. Jung, H. Y., (2007) Osteoporosis Diagnosis and Treatment. Endocrinology and Metabolism. 23(2):76-108. Kim, H. J., (2009) Pathological Mechanism of Glucocorticoid-induced Osteoporosis. Endocrinology and Metabolism. 24(4):231-235. Park Y. S., Seo W. S., Joe J. L., Kim T. H., (2009) Long Term Use of Glucocorticoid Induced Osteoporotic Multiple Compression Fractures in Autoimmune Disease. Journal of Rheumatic Diseases. 16(3)228-231.

The present inventors tried to develop a probiotic strain and fermented milk using the same, which can be applied as food and pharmaceutical products for the prevention, improvement or treatment of osteoporosis in the future. As a result, by preparing a fermented milk using a probiotic strain and then orally administering it to a glucocorticoid-induced secondary osteoporosis experimental animal, the bone density and bone rate are increased and the expression of genes related to calcium absorption and metabolism is increased. The present invention has been completed.

Accordingly, an object of the present invention is to provide a strain SRK414 of Lactobacillus fermentum with accession number KCTC13687BP.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of osteoporosis, including the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving osteoporosis, including the Lactobacillus fermentum SRK414 strain, or a culture thereof as an active ingredient.

According to one aspect of the present invention, the present invention provides Lactobacillus fermentum SRK414 strain having accession number KCTC13687BP.

The present inventors newly isolated the Lactobacillus fermentum SRK414 strain (Accession No.: KCTC13687BP). In addition, the present inventors prepared the fermented milk using the Lactobacillus fermentum SRK414 strain (Accession No.: KCTC13687BP). The fermented milk produced by the strain was administered to an experimental animal model inducing secondary osteoporosis, and it was confirmed that there is an effect of increasing bone density and bone proportion. Furthermore, the expression level of the calcium absorption-related protein expressed in the intestine of the experimental animal model administered with the fermented milk is increased, the blood calcium concentration is increased, and the expression level of genes related to suppressing osteoclast differentiation and promoting osteoblast differentiation is regulated. Was confirmed.

From the above results, it was confirmed that the fermented milk prepared using the Lactobacillus fermentum SRK414 strain (Accession No.: KCTC13687BP) of the present invention can be useful for the prevention, improvement, or treatment of secondary osteoporosis.

As used herein, the term "osteoporosis" refers to a "systemic skeletal disease characterized by a decrease in bone mass and abnormalities in microstructure, and as a result, a weakened bone and a condition prone to breakage". Bone strength is determined by the quantity and quality. Bone mass is mainly expressed by bone density (BMD), and bone quality consists of structure, bone replacement rate, mineralization, and micro-damage accumulation. Currently, osteoporosis is diagnosed by measuring bone density.

Osteoporosis is classified as primary and secondary osteoporosis. Primary osteoporosis or primary osteoporosis is classified as type 1 osteoporosis due to menopause and type 2 osteoporosis due to aging for convenience, but it is known to be difficult to accurately classify as it is merged and progressed at about the same time. Secondary osteoporosis refers to an increase in the risk of fracture due to changes in the microstructure of bones and decreased bone mass due to underlying diseases or drugs. As a cause of secondary osteoporosis, endocrine diseases (thyroidism, diabetes, hyperparathyroidism, etc.), gastrointestinal diseases (gastroresection, chronic liver disease, absorption disorder syndrome, inflammatory bowel disease, alcoholism, etc.), bone marrow disease, connective tissue disease ( Ankylosing spondylitis, rheumatoid arthritis, osteogenic insufficiency, etc.) and drugs. The most common causes in men are hypogonadism and glucocorticoid administration.

In one embodiment of the present invention, the osteoporosis is secondary osteoporosis, and more specifically, secondary osteoporosis by glucocorticoid drug administration. The glucocorticoid-based drug is, but is not limited to, cortisol, prednisone, prednisolone, methylprednisolone, cortisone, hydroxycortisone, triamcinolone, betamethasone, or dexamethasone.

In the present invention, the Lactobacillus performance SRK414 strain is deposited with the accession number "KCTC13687BP" to "Korean Microbiology Resource Center (KCTC)".

The microbial properties of the Lactobacillus performance SRK414 strain are as follows:

-Contains the 16s rRNA sequence of SEQ ID NO: 2

-Gram-positive rod-shaped or spherical bacilli

It is part of the normal microflora of the human mouth, stomach, and female sex organs and is generally considered safe for use in food (probiotics and fermented foods). L. fermentum is a member of the genus Lactobacillus , and species belonging to this genus are said to be versatile. These uses include food and feed fermentation. L. fermentum Several strains have been found to have natural resistance to antibiotics and chemotherapeutic agents.

Accordingly, according to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating osteoporosis comprising Lactobacillus permanent SRK414 strain having accession number KCTC13687BP, or a culture thereof as an active ingredient.

According to another aspect of the present invention, the present invention provides a food composition for preventing or improving osteoporosis comprising Lactobacillus permanent SRK414 strain having accession number KCTC13687BP, or a culture thereof as an active ingredient.

In one embodiment, the composition comprises as an active ingredient Lactobacillus Planta column (Lactobacillus plantarum), Lactobacillus casei (Lactobacillus casei), Lactobacillus ash FIG pillar's (Lactobacillus acidophilus), Lactobacillus Bulgaria kusu (Lactobacillus delbrueckii ssp bulgaricus ), Lactococcus lactis , Enterococcus faecium , Enterococcus fecalis , Streptococcus thermophilus , Bifidobacterium bifidum ), and Bifidobacterium lactis ), or one or more bacteria selected from the group consisting of, or a culture thereof.

In a specific embodiment of the present invention, the Lactobacillus plantarum may be Lactobacillus plantarum A41 strain (Accession No. KCTC13686BP).

In the present invention, the Lactobacillus plantarum A41 strain is deposited with the accession number "KCTC13686BP" to "Korean Collection for Type Culture (KCTC)", "Korea Research Institute of Bioscience and Biotechnology".

The microbial properties of the Lactobacillus plantarum A41 strain are as follows:

-Contains the 16s rRNA sequence of SEQ ID NO: 1

-Gram-positive rod-shaped or spherical bacilli

It is part of the normal microflora of the human mouth, stomach, and female sex organs and is generally considered safe for use in food (probiotics and fermented foods). L. plantarum is a member of the genus Lactobacillus , and is commonly found in many fermented foods and substances of anaerobic plants and is also present in saliva.

The strain, or a culture thereof, which is an active ingredient of the compositions of the present invention, is a culture medium containing cells in addition to the cells isolated and/or purified from the strain, an extract of cells, a culture supernatant, a concentrate thereof, a concentrate, It is a dried product, and if necessary, a diluent, a diluent, and the like, and includes all of the state obtained by treating the culture medium and the culture.

The culture method, extraction method, separation method, concentration method, drying method, dilution method, etc. of the above-mentioned cell body are not particularly limited.

The medium for culturing the cells generally includes milk protein such as skim milk, whey, and casein, sugars, yeast extracts, etc., and various aerobic or anaerobic methods common to the culture method can be suitably used.

As the culture temperature, for example, 35 to 45°C is set, and during the culture, an alkali such as sodium hydroxide is used to use a neutralization culture method that maintains the pH of the medium from neutral to acidic, for example, about 5 to 6 pH. It might be. In addition to such a neutralization method, any culture method such as a batch culture method can be used. After cultivation, the culture or the supernatant may be concentrated, dried, or diluted, if necessary.

In addition, the supernatant and the cells of the culture may be separated using a centrifugation method or a membrane separation method, and the cells may be recovered in a concentrated state. In addition, the cells may be subjected to ultrasonic treatment or enzyme treatment to extract components in the cells, or the culture or supernatant, the cells or the extract may be dried. These can be used as an active ingredient of the composition of the present invention.

When the composition of the present invention is prepared as a food composition, as an active ingredient, as well as the lactic acid bacteria, it may include a component that is conventionally added in food production. The additive components include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. Examples of the carbohydrate include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, oligosaccharides, etc.) and polysaccharides (eg, dextrins, cyclodextrins, etc.). Sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Natural flavors (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizine, etc.)) and synthetic flavors (saccharin, aspartic acid) Tom).

For example, when the food composition of the present invention is prepared as a drink agent, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract or licorice extract may be additionally included in addition to the above-mentioned strain as an active ingredient of the present invention. have.

The food composition of the present invention includes processing forms of all natural materials such as food, functional food, nutritional supplement, health food and food additives. Food compositions of this type can be prepared in various forms according to conventional methods known in the art.

For example, as a healthy food, the lactic acid bacteria themselves may be prepared in the form of tea, juice, and drink to be consumed or granulated, encapsulated, and powdered. In addition, food products include beverages (including alcoholic beverages), fruits and processed foods thereof (eg, canned fruits, canned foods, jams, marmalades, etc.), fish, meat, and processed foods (eg ham, sausage corn beef, etc.) ), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), juice, various drinks, cookies, syrup, dairy products (e.g. yogurt, fermented milk, butter, cheese, etc.), edible vegetable oil, margarine, Vegetable protein, retort food, frozen food, various seasonings (eg, miso, soy sauce, sauce, etc.) can be prepared by adding the lactic acid bacteria of the present invention. In addition, in order to use the lactic acid bacteria of the present invention in the form of food additives, it may be prepared and used in the form of a powder or a concentrate.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto It does not work. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably applied by oral administration. The pharmaceutical composition of the present invention may be formulated in various oral or parenteral dosage forms, but is not limited thereto.

Formulations for oral administration include, for example, tablets, pills, hard/soft capsules, liquids, suspensions, emulsifiers, and syrups. There are granules, elixirs, and the like, and these formulations may use one or more diluents or excipients, such as fillers, extenders, wetting agents, disintegrating agents, lubricants, binders, and surfactants, which are commonly used in addition to the active ingredients. As a disintegrating agent, agar, starch, alginic acid or its sodium salt, calcium phosphate monohydrogen anhydride, etc. may be used, and as a lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. , As the binder, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, low-substituted hydroxypropylcellulose, and the like can be used. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, and cellulose. Glycine or the like can be used as a diluent, and in some cases, commonly known boiling mixtures, absorbents, colorants, flavoring agents, sweeteners and the like can be used together.

The composition may be sterilized or contain preservatives, stabilizers, hydration or emulsification accelerators, salts for osmotic pressure control, adjuvants such as buffers, and other therapeutically useful substances, conventional methods of mixing, granulating or coating methods It can be formulated according to.

Suitable dosages of the pharmaceutical compositions of the invention are variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. Can be.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of ex-agent, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides Lactobacillus fermentum SRK414 strain with accession number KCTC13687BP.

(b) The present invention provides a pharmaceutical composition and a food composition comprising Lactobacillus fermentum SRK414 strain having accession number KCTC13687BP, or a culture thereof as an active ingredient.

The strain of the present invention and cultures thereof can be usefully used as a food composition and pharmaceutical composition for the purpose of preventing, improving, or treating osteoporosis, particularly secondary osteoporosis.

1 is a diagram showing bone mineral density (mg/cm ³ ) for each experimental group.
2 is a view showing the percentage of bone (percent bone volume, %) for each experimental group.
3 is a diagram showing the mRNA expression level of the intestinal calcium-accepting protein TRPV6 by experimental group.
4 is a diagram showing the mRNA expression level of Calbindin-D9k, an intestinal calcium-accepting protein, for each experimental group.
5 is a diagram showing the mRNA expression level of the intestinal calcium-accepting protein PMCA1 for each experimental group.
6 is a diagram showing the blood calcium concentration for each experimental group.
7 is a diagram showing the expression level of RANKL mRNA for each experimental group.
8 is a diagram showing the expression level of OPG mRNA for each experimental group.
9 is a diagram showing the expression level of BSP mRNA for each experimental group.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention more specifically, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance, unless otherwise specified, solids/solids (weight/weight)%, solids/liquids (weight/volume)%, and The liquid/liquid is (volume/volume) %.

Example One: Probiotics Preparation of strains and fermented milk

Probiotic strains used in this experiment were isolated from feces and traditional fermented foods of infants and young children (Table 2). In order to prepare the fermented milk of the present invention, skim milk medium was inoculated with probiotic strains isolated from the above, and cultured at 37°C for 48 hours. After the fermentation was completed, the fermented milk was lyophilized and powdered, suspended in PBS at a concentration of 1×10 ¹⁰ CFU/ml, and fed to the experimental animals. The composition of the skim milk medium used in the present invention and the list of probiotic strains are shown in Table 1 and Table 2.

Composition of skim milk medium of the present invention Serial number Raw material name Capacity (g/L) One Skim milk powder 100 2 Glucose 20 3 Yeast extract 3 4 Bacto peptone 3

List of probiotic strains of the present invention Serial number Strain name Abbreviation One Lactobacillus plantarum A41 A41 2 Lactobacillus fermentum SRK414 SRK414 3 Lactobacillus plantarum B719 B719 4 Lactobacillus casei 393 393 5 Lactobacillus plantarum KSK411 KSK411

Example 2: Production of secondary osteoporosis animal model and Experimental group Set

2.1 Production of secondary osteoporosis animal model

Animal models of secondary osteoporosis were constructed as follows. Eight week old Wistar female rats (Samtako, South Korea), weighing around 175 to 185 g, were sufficiently supplied with pellet-type feed and water to adapt to individual cages for one week before being used in the experiment. The establishment of a secondary osteoporosis model is an intramuscular injection method according to Lucinda et al. (PHYTOTHERAPY RESEARCH 27(4), (2012), 515-520) and Sousa et al. (Archives of Oral Biology 77 (2017) 55-61). As a type of glucocorticoid, dexamethasone (DEX) was administered at a concentration of 7 mg/kg BW once a week for 5 weeks.

2.2 Experimental Setting and feeding of fermented milk

In order to confirm the therapeutic effect of the probiotic strain and fermented milk of the present invention on secondary osteoporosis, the experimental groups were classified into a total of 9 groups as shown in Table 3 below. 7 experimental animals were assigned to each group, and all groups except the normal group were administered dexamethasone (DEX) once a week at a concentration of 7 mg/kg (BW) for 5 weeks as described in the above 2.1.

All fermented milk was diluted or diluted in PBS (Phosphate buffer saline) at a concentration of 1×10 ¹⁰ CFU/ml and orally administered by oral gavage. On the other hand, since the purpose of this experiment is to confirm the anti-osteoporosis effect of milk fermentation using probiotics, a Skim milk administration group (SMP) that did not undergo fermentation was used as a control.

Experimental group setting of the present invention Serial number Experiment group density Abbreviation One Normal-PBS administration group - Normal 2 Dexamethasone group 7 mg/kg Dex 3 Skim milk administration group SMP 4 Fermented milk administration group ( L. plantarum A41) 1×10 ¹⁰ CFU/ml A41 5 Fermented milk administration group ( L. fermemtum SKR414) SKR414 6 Fermented milk administration group ( L. plantarum B719) B719 7 Fermented milk administration group ( L. casei 393) 393 8 Fermented milk administration group ( L. plantarum KSK411) KSK411 9 Secondary osteoporosis drug (alendronate) group 0.16 mg/kg Drug

Example 3: Morphological analysis of bone according to the administration of fermented milk of the present invention

Bone mineral density and percent bone volume are the most fundamental indicators when measuring bone strength. In order to measure the effect of improving the bone density of the probiotic strain and fermented milk of the present invention, a Micro Ct image analyzer (Sky Scan 1176) and a program to reconstruct it (NRecon), and a program to analyze it (CTAn) were used.

As a result of the experiment, it was confirmed that secondary osteoporosis was induced in the DEX group administered with dexamethasone, as the bone density and bone ratio were significantly decreased compared to the normal group. In the drug group, in which the secondary osteoporosis drug (alendronate) was administered together with dexamethasone, it was shown that the reduced bone density and bone fraction were restored by administration of dexamethasone. The A41 group, the SRK414 group, the B719 group, and the 393 group to which the fermented milk fermented with the probiotic strain of the present invention were administered also exhibited similar bone mineral density and bone rate recovery effects. (FIGS. 1 and 2)

From the above results, the fermented milk fermented with the probiotic strains L. plantarum A41, L. fermentum SRK414, L. plantarum B719, L. casei 393 of the present invention is a symptom of secondary osteoporosis caused by administration of dexamethasone, which decreases bone density and bone rate It was confirmed that the recovery.

Example 4. Analysis of indicators related to calcium absorption by administration of fermented milk of the present invention

When the calcium concentration in the blood is insufficient, the calcium in the bone is released into the blood, thereby increasing bone loss. Therefore, increasing calcium concentration in the blood by promoting calcium absorption in the intestine may be an important preventive measure to lower bone loss. Calcium is introduced into the blood vessels from the small intestine by TRPV6, Calbindin-D9k, PMCA1, etc., proteins that play a role in moving calcium from the intestine. Therefore, the present inventors performed qRT-PCR analysis to determine the mRNA expression level of the intestinal calcium-accepting protein according to the administration of fermented milk of the present invention, and confirmed the relationship with the blood calcium concentration.

4.1 Confirmation of intestinal calcium receptor gene expression

To investigate the effect of fermented milk on the expression level of intestinal calcium-accepting proteins (TRPV6, Calbindin-D9k, PMCA1), qRT-PCR analysis was performed. Specifically, after the end of the experimental period, the ileum of each experimental animal was extracted, subjected to a homogenized process, and then RNA contained in the tissue was extracted using TRIzol (Invitrogne). The purified RNA was used for cDNA synthesis together with reverse transcriptase oligo (dT) primer. PCR conditions were repeated for 3 cycles at 95° C., 3 seconds at 94° C., 10 seconds at 60° C., and 30 seconds at 60° C. for a total of 44 cycles. Primers for qRT-PCT analysis are shown in Table 4. The degree of total transcription was graphically normalized from the β-actin transcription level (FIGS. 3 to 5 ).

Genetic name Forward primer (5' to 3') Reverse primer (5' to 3') TRPV6 GAT GGC ACG ACC CTT TGG CTT CGG GAG GTA CTT CGA GAC A Calbindin-D9k CGC TAA GAA ATC TCC CGA AG CTC CAT CAC CGT TCT TAT CCA PMCA1 ACT CTG GGG CCA GCT TAT TT GTT CGG CAT GGT CAA TCT CT

3 to 5, the A41 group and the SRK414 group in the fermented milk administration group of the present invention showed a relatively high expression level of intestinal calcium-accepting proteins (TRPV6, Calbindin-D9k, PMCA1) compared to other groups (FIG. 3 to 5).

4.2 Effect of regulating blood calcium concentration

In order to confirm the effect of the fermented milk administration of the present invention on the blood calcium concentration, the blood calcium concentration was confirmed. Specifically, blood was collected by a heart puncture method after a period of animal testing, and serum collected by centrifugation (2500 g, 15 minutes, 4°C) was stored at -70°C until analysis. Calcium (Ca) concentration in the serum was analyzed using a Calcium activity assay kit (MBL, Korea). The results are shown in FIG. 6.

As shown in FIG. 6, it was confirmed that the A41 group and the SRK414 group in the fermented milk administration group of the present invention showed a relatively high concentration of calcium in the blood as compared with the other groups, similar to the expression level of the intestinal calcium-accepting protein. Therefore, it is considered that the fermented milk fermented by the L. plantarum A41 and L. fermentum SRK414 strains of the present invention can effectively prevent bone loss by maintaining high blood calcium concentration by promoting calcium absorption in the small intestine of animals to which fermented milk is administered. .

Example 5. Analysis of bone metabolism related indicators according to fermented milk administration of the present invention

When the Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL) is expressed in osteoblasts or activated immune cells, osteoclast differentiation occurs by binding to Receptor Activator of Nuclear Factor Kappa B (RANK) of the osteoclast cell membrane. Bone absorption can be promoted, leading to osteoporosis. On the other hand, OPG (Osteoprotegerin) is a factor that is received by RANK instead of RANKL, and OPG can inhibit the differentiation of osteoclasts by interfering with the binding of RANK and RANKL.

The present inventors analyzed the mRNA expression levels of RANKL, OPG, and BSP, which are bone metabolism-related markers, through qRT-PCR to confirm the effect of fermented milk administration of the present invention on bone metabolism. Specifically, after homogenization of the thigh and bone cortex of the experimental animal, total RNA was extracted using TRIzol reagent (Thermofisher, USA). The purified total RNA was used for cDNA synthesis. PCR was performed using Biorad RT-PCR, and after processing at 95°C for 3 minutes, a total of 44 cycles were repeated at 94°C for 10 seconds and at 60°C for 30 seconds. Primers for qRT-PCT analysis are shown in Table 5. The degree of total transcription was normalized from the β-actin transcription level (Fig. 7 to 9).

Genetic name Forward primer (5' to 3') Reverse primer (5' to 3') RANKL CCC ATC GGG TTC CCA TAA AGT C GCC TGA AGC AAA TGT TGG CGT A OPG ACG GTT TGC AAA AGA TGT CC GTG AGC TGC AGT TGG TGT GT BSP AGA AAG AGC AGC ACG GTT GAG T GAC CCT CGT AGC CTT CAT AGC C

As shown in FIG. 7, it was confirmed that the A41 and SRK414 fermented milk decreased the differentiation of osteoclasts by significantly reducing the expression of RANKL mRNA in the A41 group and the SRK414 group compared to the Dex group.

In addition, as shown in FIG. 8, the expression level of OPG mRNA was also significantly increased in all fermented milk administration groups except 393 and KSK to confirm the inhibition of osteoclast differentiation.

Finally, as shown in FIG. 9, the ultimate osteoblast differentiation marker BSP (Bone sialoprotein) expression level also shows relatively high expression levels in the A41, SRK414 and B719 groups, thereby promoting osteoblast differentiation and promoting the therapeutic effect of osteoporosis. Was confirmed to be excellent.

Based on the above results, when the fermented milk fermented with the L. plantarum A41 strain and L. fermentum SRK414 strain of the present invention is administered to an animal model, osteoblasts in the body promote OPG production and inhibit the production of RANKL to differentiate osteoclasts It was confirmed that it exhibits a preventive effect against secondary osteoporosis by inhibiting and promoting differentiation of osteoblasts.

Korea Research Institute of Bioscience and Biotechnology KCTC13686BP 20181025 Korea Research Institute of Bioscience and Biotechnology KCTC13687BP 20181025

<110> CKD Bio Corp <120> Composition for Preventing or Treating Secondary Osteoporosis Comprising Probiotics <130> PN180369 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 1487 <212> RNA <213> Artificial Sequence <220> <223> 16s rRNA of Lactobacillus plantarum A41 (KCTC13686BP) <400> 1 ggctcaggac gaacgctggc ggcgtgccta atacatgcaa gtcgaacgaa ctctggtatt 60 gattggtgct tgcatcatga tttacatttg agtgagtggc gaactggtga gtaacacgtg 120 ggaaacctgc ccagaagcgg gggataacac ctggaaacag atgctaatac cgcataacaa 180 cttggaccgc atggtccgag tttgaaagat ggcttcggct atcacttttg gatggtcccg 240 cggcgtatta gctagatggt ggggtaacgg ctcaccatgg caatgatacg tagccgacct 300 gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg ggaggcagca 360 gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag 420 ggtttcggct cgtaaaactc tgttgttaaa gaagaacata tctgagagta actgttcagg 480 tattgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 540 gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggttttttaa 600 gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag 660 tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa 720 caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagtatgggt 780 agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc taagtgttgg 840 agggtttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg gggagtacgg 900 ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt 960 ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca aatctaagag 1020 attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt cagctcgtgt 1080 cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt tgccagcatt 1140 aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg gatgacgtca 1200 aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg tacaacgagt 1260 tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga ttgtaggctg 1320 caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat 1380 acgttcccgg gccttgtaca caccgcccgt cacaccatga gagtttgtaa cacccaaagt 1440 cggtggggta accttttagg aaccagccgc ctaaagtggg acagatg 1487 <210> 2 <211> 1503 <212> RNA <213> Artificial Sequence <220> <223> 16s rRNA of Lactobacillus fermentum SRK414 (KCTC13687BP) <400> 2 gctcaggatg aacgccggcg gtgtgcctaa tacatgcaag tcgaacgcgt tggcccaatt 60 gattgatggt gcttgcacct gattgatttt ggtcgccaac gagtggcgga cgggtgagta 120 acacgtaggt aacctgccca gaagcggggg acaacatttg gaaacagatg ctaataccgc 180 ataacaacgt tgttcgcatg aacaacgctt aaaagatggc ttctcgctat cacttctgga 240 tggacctgcg gtgcattagc ttgttggtgg ggtaacggcc taccaaggcg atgatgcata 300 gccgagttga gagactgatc ggccacaatg ggactgagac acggcccata ctcctacggg 360 aggcagcagt agggaatctt ccacaatggg cgcaagcctg atggagcaac accgcgtgag 420 tgaagaaggg tttcggctcg taaagctctg ttgttaaaga agaacacgta tgagagtaac 480 tgttcatacg ttgacggtat ttaaccagaa agtcacggct aactacgtgc cagcagccgc 540 ggtaatacgt aggtggcaag cgttatccgg atttattggg cgtaaagaga gtgcaggcgg 600 ttttctaagt ctgatgtgaa agccttcggc ttaaccggag aagtgcatcg gaaactggat 660 aacttgagtg cagaagaggg tagtggaact ccatgtgtag cggtggaatg cgtagatata 720 tggaagaaca ccagtggcga aggcggctac ctggtctgca actgacgctg agactcgaaa 780 gcatgggtag cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta 840 ggtgttggag ggtttccgcc cttcagtgcc ggagctaacg cattaagcac tccgcctggg 900 gagtacgacc gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag 960 catgtggttt aattcgaagc tacgcgaaga accttaccag gtcttgacat cttgcgccaa 1020 ccctagagat agggcgtttc cttcgggaac gcaatgacag gtggtgcatg gtcgtcgtca 1080 gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg ttactagttg 1140 ccagcattaa gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga 1200 cgacgtcaga tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta 1260 caacgagtcg cgaactcgcg agggcaagca aatctcttaa aaccgttctc agttcggact 1320 gcaggctgca actcgcctgc acgaagtcgg aatcgctagt aatcgcggat cagcatgccg 1380 cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca 1440 cccaaagtcg gtggggtaac cttttagagc cagccgccta agtgggacag atgattaggg 1500 tga 1503 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of TRPV6 <400> 3 gatggcacga ccctttgg 18 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of TRPV6 <400> 4 cttcgggagg tacttcgaga ca 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of Calbindin-D9k <400> 5 cgctaagaaa tctcccgaag 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of Calbindin-D9k <400> 6 ctccatcacc gttcttatcc a 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of PMCA1 <400> 7 actctggggc cagcttattt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of PMCA1 <400> 8 gttcggcatg gtcaatctct 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of RANKL <400> 9 cccatcgggt tcccataaag tc 22 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of RANKL <400> 10 gcctgaagca aatgttggcg ta 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of OPG <400> 11 acggtttgca aaagatgtcc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of OPG <400> 12 gtgagctgca gttggtgtgt 20 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer of BSP <400> 13 agaaagagca gcacggttga gt 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer of BSP <400> 14 gaccctcgta gccttcatag cc 22

Claims (8)

Accession No. KCTC13687BP Lactobacillus fermentum ( Lactobacillus fermentum ) SRK414 strain, or a pharmaceutical composition for the prevention or treatment of secondary osteoporosis comprising a culture thereof as an active ingredient.
delete Food composition for preventing or improving secondary osteoporosis, including Lactobacillus fermentum SRK414 strain having accession number KCTC13687BP, or a culture thereof as an active ingredient.
The composition of claim 3, wherein the food composition is fermented milk.
delete delete delete delete

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