KR100940125B1 - Composition for Improving Bone Metabolism Comprising Milk Component Fermented by Latobacillus casei - Google Patents

Abstract

The present invention is Lactobacillus casei ( Lactobacillus) Casei ) provides a composition for improving bone metabolism or a pharmaceutical composition for the prevention or treatment of bone metabolic diseases comprising a fermented milk of the strain as an active ingredient. Milk fermented product by the Lactobacillus casei strain of the present invention promotes the proliferation and differentiation of osteoblasts, increases bone strength and bone density while inhibiting the activity of osteoclasts, thereby improving, preventing or treating bone metabolic diseases such as osteoporosis. It can be very useful.

Lactobacillus casei, milk, fermentation, milk protein, bone metabolism, osteoporosis, bone density, osteoblasts, osteoclasts

Description

Composition for Improving Bone Metabolism Comprising Milk Metabolism Compensing Milk Component Fermented by Latobacillus casei

The present invention is Lactobacillus casei ( Latobacillus) Casei ) relates to a composition for improving bone metabolism comprising a fermented product of milk by the strain as an active ingredient.

Bones support the soft tissues and weight of the body and surround internal organs to protect internal organs from external shocks. It is also one of the important parts of the human body that not only structurally supports muscles or organs, but also stores calcium and other essential minerals in the body, such as phosphorus and magnesium. Thus, the bones of grown adults do not stop, and until the day they die, the old bones are removed and the bones of the new bones are replaced with new bones. This is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu Kakusan Koso ., 50 (6 Suppl): 664-669 (2005). It is known that two types of cells are involved in aggregate formation. One of the two cells is an osteoblast that produces bone, and the other is an osteoblast that destroys bone. Osteoblasts are involved in the bone formation process of producing new bone, osteoclasts are involved in the resorption process of destroying old bone, and the process of breaking (absorbing) and producing (reforming) bones stimulates another step. , A nearly fully linked system (Christenson, Clinical Biochemistry, 30, 573-593, 1997).

Osteoblasts produce RANKL (receptor activator of nuclear factor-κB ligand) and osteoprotegerin (OPG), its decoy receptor. When RANKL binds to the receptor activator of nuclear factor (RANK), a receptor on the surface of osteoclast progenitor cells, osteoclast progenitors mature into osteoclasts and cause bone resorption. However, when OPG binds to RANKL, the binding between RANKL and RANK is blocked, which inhibits the formation of osteoclasts and prevents more bone resorption from occurring than required (Theill LE. Et al., Annu Rev Immunol . , 20: 795-823 (2002); Wagner EF. et al., Curr Opin Genet Dev . , 11: 527-532 (2001).

Absorption or destruction of old bone is caused by osteoclasts from blood cells (hematopoietic stem cells), which puncture the bone and release a small amount of calcium into the bloodstream to maintain body function (William J. et al. , Nature ., 423: 337-342 (2003).

Osteoblasts produced from bone cells, on the other hand, fill the pores with collagen and cover the calcium and phosphorus hydroxyapatite to reconstruct the skeleton to form new bones (Stains JP. Et al., Birth) . Defects Res C Embryo Today., 75 (1): 72-80 (2005)). It takes about 100 days for bones to begin to break down and rebuild into new bones (Schwarz EM. Et at., Curr Opin Orthop . , 11: 329-335 (2000). In infants, bone calcium changes 100% within a year, but in adults, about 10-30% of the skeleton is remodeled through this process every year. Bone metabolic disease occurs when osteoblast activity decreases in bone remodeling systems, resulting in decreased bone formation, increased osteoclast activity, increased bone resorption, or both factors. Such diseases are representative.

Osteoporosis is a condition in which bone mass decreases due to various causes and the risk of fracture is continuously increased due to the deterioration of the microstructure of bone tissue. Osteoporosis is a condition in which minerals (particularly calcium) and substrates that make up bone are reduced. Formation is broken, resulting in osteoclasts being increased than osteogenic (Iqbal MM., South Med J. , 93 (1): 2-18 (2000)). Normal bone inside has a dense structure like a net, but in the case of osteoporosis, the gap between the structures becomes wider and the microstructure becomes thinner and weaker, so that the bone can be easily fractured even with a small impact (Stepan JJ. Et al. ., Endocr Regul . , 37 (4): 225-238 (2003). Osteoporosis is a postmenopausal osteoporosis that causes rapid bone loss (2–3% per year) at the beginning of menopause and increases the risk of vertebral compression and carpal fractures, and diseases of any age (endocrine) Diseases, gastrointestinal disorders, malignant tumors) or secondary osteoporosis caused by drugs (adrenal cortical hormones, chemotherapy, thyroid hormones, anticonvulsants, anticoagulants, methotexate, cyclosporine, GnRH, etc.), alcohol, smoking, or accidents (Rosep CJ., N Engl J Med ., 353 (6): 595-603 (2005); Davidson M., Clinicain Reviews., 12 (4): 75-82 (2002)).

Another important disease that can occur when the balance of bone regeneration is broken is damage due to bone metastasis of cancer cells. Bone metastasis almost always occurs in patients with breast cancer, prostate cancer or multiple myeloma (Kozlow W. et al., J Mammary Gland Biol Neoplasia . , 10 (2): 169-180 (2005)), and how long these cancer patients can live is known to be affected by bone metastasis.

Currently, food materials for the prevention of osteoporosis are limited to soy protein or soy extract in the West, and its mechanism of action is explained by phytoestrogen effect (Arjmandi et al., Journal of Nutrition, Vol. 126, 161-167). , 1996). Studies have been conducted to verify the effects of preventing or treating osteoporosis of safflower seeds (safflower seed extract) or 칡 traditionally used in herbal medicine in Korean medicine (Kim, et al., 34, 710-718, 2002). . In addition, as a result of investigating the effect on the proliferation and differentiation of osteoblasts using various extracts of natural edible natural resources in Korea, it was reported that seaweed extract increased the activity of osteoblasts (Pak et al., 34, 929). -936, 2005).

In the case of animal husbandry, whey protein supplementation increased the amount of hydroxyproline, a component of the bone matrix protein collagen, which is involved in regulating bone strength, bone cell metabolism and mineralization in ovarian-abated mice. Has been reported (Takada et al., Nutrition Research, Vol. 17, 1709-1720, 1997). Whey proteins may also enhance osteoblast proliferation in cell culture experiments (Takada et al., Biochemistry Biophysics Research Communication, Vol. 223, 445-449, 1996), while reducing bone resorption of osteoclasts (Takada et al., International). Dairy Journal, Vol. 7, 821-825, 1997), continued to search for active ingredients in whey proteins.

Yamamura et al. (Biochemistry Biophysics Research Communication, Vol. 261, 113-117, 1999) estimated that the whey protein component that promotes the proliferation of osteoblasts is a high mobility group protein (HMG1) with a molecular weight of about 10 Kda. Silver was bound during the purification of cationic chromatography and was called the milk basic protein fraction (MBP). Unlike MBP, a trace protein present in milk, proteolytic enzymes introduced into milk proteins from outside or proteinases secreted from the starter during fermentation may break down proteins to produce physiologically active peptides, especially to improve bone density. It also contributes.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors tried to study the possibility of treating bone metabolic diseases by improving bone metabolism by using the fermented milk of lactic acid bacteria. As a result, milk fermented by Lactobacillus casei strain promotes osteoblast growth and differentiation to increase bone density and bone strength, while effectively inhibiting osteoclast activity, thereby improving bone metabolic disease. The present invention has been found to be very useful in the treatment, prevention and treatment.

Therefore, the object of the present invention is Lactobacillus Casei ) To provide a composition for improving bone metabolism or preventing or treating bone metabolic diseases comprising a fermented milk of the strain as an active ingredient.

In addition, another object of the present invention to provide a method for producing a milk fermented product by Lactobacillus casei strain.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the invention, Lactobacillus casei (Lactobacillus casei) Provides a composition for improving bone metabolism comprising the fermented product of milk by the strain as an active ingredient.

The present inventors tried to study whether the fermented milk of lactic acid bacteria can treat bone metabolic diseases by improving bone metabolism. As a result, milk fermented by Lactobacillus casei strain promotes osteoblast growth and differentiation, increases bone density and bone strength, while effectively inhibiting osteoclast activity, thereby improving bone metabolism and treating bone metabolic diseases. Or it was confirmed that it is very useful for prevention.

As used herein, the term “lactic acid bacteria” refers to a group of bacteria that produce lactic acid as the main product of carbohydrate metabolism. Lactobacillus strain used to ferment milk in the present invention to obtain a milk fermented product is Lactobacillus casei ( Lactobacillus casei ). "Lactobacillus casei" in the present invention belongs to the genus Lactobacillus (genus Lactobacillus ) is a transient anaerobic (transient anaerobic) microorganisms found in the human intestine and oral cavity. This strain has a wide pH and temperature range, promotes the growth of Lactobacillus acidophilus strains that produce lactic acid, which helps promote the growth of desirable bacteria and produces the carbohydrate amylase. It is known to improve and reduce milk intolerance and constipation. Strains belonging to the Lactobacillus casei of the present invention can be easily purchased from the American Type Culture Collection (ATCC, Manassas, VA 20108 USA) and preferably include, but are not limited to, strains having the following ATCC Accession Numnber: ATCC 49178, ATCC 7469a, ATCC 9595, ATCC 27773, ATCC 11981, ATCC 39595, ATCC 15008, ATCC 13075, ATCC 334, ATCC 393, ATCC 7469, ATCC 11578, ATCC 11582, ATCC 11982, ATCC 12116, ATCC 14435, ATCC 14957, ATCC 25180, ATCC 25302, ATCC 25303, ATCC 25598, ATCC 25599, ATCC 27092, ATCC 27139, ATCC 29599, ATCC 335, ATCC 39392, ATCC 4007, ATCC 4913. Most preferably, the lactobacillus strain that can be used in the present invention is lactose. Bacillus casei ATCC 393 ( Lactobacillus casei ATCC 393).

“Milk” used as a fermentation raw material in the present invention means milk of various animal origins, for example, cow's milk, goat's milk, or horse's milk. It includes, but is not limited to. It may also be used in the form of their whole milk or skim milk or their powder.

As used herein, the term “fermented milk” refers to a mixture of all fermentations obtained after milk fermentation by the Lactobacillus casei strain of the present invention.

Milk fermentation product of the present invention can be prepared by a conventional lactic acid bacteria fermentation method. Lactic acid bacteria fermentation methods are known to those of ordinary skill in the art. For example, after sterilizing milk as a fermentation ingredient, Lactobacillus casei ( Lactobacillus) casei ) is prepared by inoculating strains and culturing them. Cultivation for fermentation can be carried out by selecting suitable conditions. For example, if the strain of lactic acid bacteria is anaerobic, the oxygen of the medium is replaced with an inert gas such as carbon dioxide gas or nitrogen gas in order to make the medium anaerobic condition before the culture, or the oxygen reaction. Oxygen is removed from the medium using an oxygen reaction agent. If the lactic acid bacteria strain is aerobic, suitable aerobic conditions are selected.

Lactobacillus using other culture conditions casei ) can be adjusted according to the type of strain. For example, the culture may be performed by adding milk, which is a fermentation raw material, to the medium at a concentration of 0.001-40% on a solids basis and at a temperature range of 20-40 ° C. for about 15-48 hours. The medium may additionally be added various carbohydrates, nutrients, plants or microorganisms. Nutrients such as carbohydrates such as glucose, sucrose, fructose-glucose syrup and glucose-fructose syrup, meat extract, yeast extract, peptides and minerals Ingredients, grains, grain seed shoots, vegetable ingredients, such as vegetables, fruits, nuts and herbs, animal ingredients and microbial culture ingredients may further be added within the range of about 0.001-99% by mass.

For details on the cultivation and fermentation of microorganisms, see Kubitschek, HE, Introduction to Research. with Continuous Cultures . Englewood Cliffs, NJ: Prentice-Hall, Inc., 1970; Mandelstam, J., et al., Biochemistry of Bacterial Growth , 3rd ed. Oxford: Blackwell, 1982; Meynell, GG, et al., Theory and Practice in Experimentalal Bacteriology , 2nd ed. Cambridge: Cambridge University Press, 1970; Gerhardt, P., ed., Manual of Methods for General Bacteriology , Washington: Am. Soc. Microbiol, 1981, which is incorporated herein by reference.

The fermented milk of the Lactobacillus casei strain of the present invention has the effect of promoting the proliferation and differentiation of osteoblasts to enhance bone density and bone strength, while inhibiting the activity of osteoclasts. Therefore, the milk fermented product of the present invention can be very useful for improving the imbalanced bone metabolism in which the balance of activity and proliferation of osteoclasts and osteoblasts is broken.

According to a preferred embodiment of the present invention, the present invention provides a composition for improving bone metabolism or a composition for the prevention or treatment of bone metabolic diseases comprising a fermented digest of milk protein by Lactobacillus casei strain as an active ingredient.

As used herein, the term “milk protein” refers to all proteins contained in milk, including, for example, casein, whey protein and other trace milk proteins.

The term “fermented digest of milk protein” in the context of the present invention means a mixture of all digests obtained after fermentation of milk protein by Lactobacillus casei strain.

Milk fermentation product of the present invention includes a fermentation product of milk protein produced by fermentation by Lactobacillus casei strain, these milk protein fermentation products promote osteoblast proliferation and differentiation, increase bone density and bone strength, osteoclasts It is expected to act to inhibit the activity of. Therefore, according to the present invention, it is possible to provide a milk fermentation product having an effect of improving bone metabolism without separately adding proteolytic enzymes or protein degradation products from the outside during fermentation.

According to another preferred embodiment of the present invention, the present invention comprises the milk fermented product by the Lactobacillus casei strain fraction fraction (Molecualr Weight Cut-Off, MWCO) 350-35000 fractions of the milk fermented product as an active ingredient Provided are compositions for improving bone metabolism or preventing or treating bone metabolic diseases.

In the present invention, the "fraction" may be performed using a method of filtration, dialysis, or osmosis, and an ultrafiltration membrane, a microfiltration membrane, a nanofiltration membrane, and a nanofiltration membrane. ) Or reverse osmosis membrane, or the like, but may not be limited thereto.

In the present invention, the fraction molecular weight (MWCO, Molecular Weight Cut-Off) of the fraction of milk fermentation is preferably in the range of 350-35000. If the fraction molecular weight is less than 350, it is disadvantageous due to the small size of impurities other than the active ingredient in the milk fermentation, which requires a long time in the process, and the activity having the effect of improving bone metabolism in the milk fermentation when the fraction molecular weight exceeds 35000. There is a disadvantage in that the components are lost. More preferably, the fractional molecular weight is 700-17500, even more preferably 1750-7000, and most preferably the fractional molecular weight is 3500.

The composition of the present invention may be provided in the form of a food, in particular a functional food composition. Functional food compositions of the present invention include ingredients that are commonly added in the manufacture of food, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, flavoring agents or natural carbohydrates may be included as additional ingredients in addition to the milk fermented product as an active ingredient. For example, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrins, cyclodextrins, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, and the like). As flavoring agents, natural flavoring agents (eg, taumartin, stevia extract, etc.) and synthetic flavoring agents (eg, saccharin, aspartame, etc.) can be used. Considering easy access to food, the functional food composition of the present invention is very useful for the prevention and treatment of bone metabolic diseases as well as for improving bone metabolism.

The composition of the present invention may be provided in the form of a pharmaceutical composition. According to a preferred embodiment of the invention, the invention is (a) Lactobacillus casei ( Lactobacillus casei ) a pharmaceutically effective amount of milk fermentation by the strain; And (b) provides a pharmaceutical composition for the prevention or treatment of bone metabolic diseases comprising a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention comprises an active ingredient and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as 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, and the like. It is not limited. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex of the patient, degree of disease symptom, food, time of administration, route of administration, rate of excretion and response to reaction. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment. On the other hand, the dosage of the pharmaceutical composition of the present invention is preferably 0.001-1000 mg / kg (body weight) per day.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and when administered parenterally, may be administered by intravenous infusion, subcutaneous infusion, intramuscular injection, intraperitoneal injection, transdermal administration, or the like. It is preferable that the route of administration is determined according to the type of the disease to which the pharmaceutical composition of the present invention is applied.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention can be very usefully used for the prevention or treatment of bone metabolic diseases. The term "bone metabolic disease" in the context of the present invention refers to a disease due to the balance of activity and proliferation of osteoclasts and osteoblasts. Thus, “bone metabolic disease” as used herein includes diseases or conditions caused by overactivity or hyper-proliferation of osteoclasts, and is associated with low activity or low growth of osteoblasts. It also includes the disease or condition that results from it. As used herein, the term "disease due to overactivation or overgrowth of osteoclasts" refers to a disease caused by excessive activation or excessive proliferation of osteoclasts.

According to a preferred embodiment of the present invention, the composition of the present invention can be used for the treatment of diseases due to excessive bone resorption by osteoclasts.

According to another preferred embodiment of the present invention, the composition of the present invention is useful for the treatment and prevention of osteoporosis and related osteoopenic diseases.

Diseases treatable and preventable with the compositions of the present invention include osteoporosis, especially hypercalcemia associated with premenopausal osteoporosis, Paget's disease, bone neoplasms, and Types of osteoporosis and related diseases include, but are not limited to: menopausal osteoporosis, type I or postmenopausal osteoporosis, type II or senile osteoporosis, juvenile osteoporosis, idiopathic Osteopathic osteoporosis, endocrine abnormality, hyperthyroidism, hypogonadism, ovarian agensis or Turner's syndrome, hyperadrenocorticism or hyperadrenocorticism Cushing's syndrome, hyperparathyroidism, bone marrow abnormalities, Multiple myeloma and related diseases, systemic mastocytosis, disseminated carcinoma, Gaucher's disease, connective tissue abnormalities, osteogenic imperfecta, homo Homocystinuria, Ehlers-Danlos syndrome, Marfan's syndrome, Menke's syndrome, immobilization or weightlessness, Sudeck's atrophy atrophy, chronic obstructive pulmonary disease, chronic heparin administration and chronic ingestion of anticonvulsant drugs.

In addition, the composition of the present invention is rheumatoid arthritis, periodontal disease (peridontal disease), periprosthetic osteolysis, other autoimmune diseases, neoplastic destruction of bone and bone resorption disease associated with cancer Can be used for the treatment or prevention of, but is not limited thereto.

The compositions of the present invention are also not explicitly described herein but can be used for the treatment of diseases caused by overactivation or overproliferation of osteoclasts and secondary diseases thereof.

According to the most preferred embodiment of the present invention, diseases caused by hyperactivation or hyperproliferation of the osteoclasts are osteoporosis, Paget's disease, hypercalcemia, rheumatoid arthritis, metastatic Metastatic bone destruction, cancer and immune diseases.

The term "prevention" in the specification of the present invention means not inhibiting the occurrence of a disease or disorder in an animal that has not been diagnosed as having a disease or disorder but is prone to such disease or disorder. As used herein, the term “treatment” means (i) inhibiting the development of a disease or condition; (Ii) alleviation of the disease or condition; And (iii) elimination of the disease or condition.

According to another aspect of the present invention, the present invention provides a method for preparing a milk fermentation fraction for bone metabolism improvement comprising the following steps: (a) Lactobacillus casei strain comprising milk Inoculating the medium to be treated; (b) culturing the medium of step (a); And (c) obtaining the fraction by using the milk fermented product produced in step (b) with a molecular weight of cut (Molecular Weight Cut-Off, MWCO) of 350-35000.

Lactobacillus strains that can be used in the method of the present invention are Lactobacillus casei strains, which can be easily purchased from the American Type Culture Collection (ATCC, Manassas, VA 20108 USA).

As used herein, "milk" refers to milk of various animal origins.

Production of the milk fermented product according to the steps (a) and (b) of the present invention can be carried out through a conventional lactic acid bacteria fermentation method. Lactic acid bacteria fermentation method is known to those skilled in the art, it can be carried out by selecting a suitable culture conditions for fermentation.

Details of lactic acid bacteria Lactobacillus casei, fermentation raw materials, fermentation methods and culture conditions at the time of fermentation that can be used in the method of the present invention are described in the section about “composition comprising milk fermented product as an active ingredient” of the present specification. Since it is the same as, the description is omitted to avoid excessive complexity of the specification.

According to a preferred embodiment of the present invention, the present invention provides a method for producing a milk fermented product, characterized in that the medium of step (a) is a medium to which whey protein is additionally purified. The addition of the separated and purified whey protein to the medium can increase the amount of fermentative degradation products of milk proteins, especially whey proteins, in the final milk fermentation.

By performing the fractionation step as in step (c), milk protein fermentation products that promote osteoblast proliferation and differentiation, increase bone density and bone strength, and inhibit osteoclast activity can be separated from milk fermentation products.

In the present invention, the "fraction" may be performed using a method of filtration, dialysis, or osmosis, and an ultrafiltration membrane, a microfiltration membrane, a nanofiltration membrane, and a nanofiltration membrane. ) Or reverse osmosis membrane, or the like, but may not be limited thereto.

In the present invention, the fraction molecular weight (MWCO, Molecular Weight Cut-Off) at the time of fractionation of milk fermentation is preferably in the range of 350-35000. If the fractional molecular weight is less than 350, it is disadvantageous due to the small size of impurities other than the active ingredient in the milk fermentation, which requires high pressure and long time in the process, and the effect of improving the metabolism in the milk fermentation when the fractional molecular weight exceeds 35000. There is a disadvantage that the active ingredient is lost. More preferably, the fractional molecular weight is 700-17500, even more preferably 1750-7000, and most preferably the fractional molecular weight is 3500.

The present invention provides a composition for improving bone metabolism or preventing or treating bone metabolic diseases comprising milk fermented product by Lactobacillus casei strain as an active ingredient. Milk fermented product as an active ingredient of the composition of the present invention promotes proliferation and differentiation of osteoblasts, increases bone density and bone strength, and inhibits osteoclast activity, thereby improving, preventing or treating bone metabolic diseases such as osteoporosis. It can be very useful.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example  One: Lactobacillus Kasei ( Lactobacillus casei Milk by strain Fermented product  Produce

Lactobacillus casei ATCC 393 ( Lactobacillus casei ATCC 393) was inoculated in skim milk powder (12% SNF, Solids Not Fat), incubated at 37 ° C. for 18 hours, and centrifuged to obtain a supernatant. The pH of the recovered supernatant was adjusted to 6.8, dialyzed for 48 hours using a membrane having a molecular weight of 3,500 (Molcular Weight Cut Off, MWCO), and then lyophilized to prepare milk fermented product 1.

In addition, Lactobacillus casei ATCC 393 ( Lactobacillus casei ATCC 393) was inoculated in skim milk powder (12% SNF, Solids Not Fat) containing 1% whey protein concentrate (WPC, Alacen 878), incubated at 37 ° C. for 18 hours, and centrifuged to obtain a supernatant. The recovered supernatant was prepared in the same manner as in the preparation of the milk ferment 1, milk ferment 2 was prepared. In the following experiment to confirm bone metabolism improving activity of milk fermentation, each of the milk fermenters 1 and 2 was used as milk fermentation product.

Example  2: Lactobacillus Kasei ( Lactobacillus casei Milk by strain Fermented product  Osteoblast growth promoting effect

The effect of the milk fermentation (milk fermentation 1 and 2) on the proliferation of osteoblasts was measured using osteoblasts (MC3T3-E1 cell line ATCC, Manassas, VA). Cell line at 37 ° C. in α-minimum essential media (α-MEM, Gibco BRL, USA) containing 10% fetal bovine serum (FBS, Gibco BRL, USA), 50 unit / ml penicillin, 50 μg / ml streptomycin , 5% CO ₂ was continuously supplied at 100% humidity condition, and the culture solution was exchanged at intervals of 2-3 days until sufficient proliferation of the cells was observed.

Osteoblast proliferation effect was measured using the MTT method. Osteoblasts were dispensed in a 96 well plate (Nalgene, USA) at a concentration of 2.0 × 10 ³ cells / well and cultured for 24 hours to confirm cell adhesion, and then mixed with various concentrations of milk fermented products in serum free media. 200 μl each well was incubated in 37 ° C., 5% CO ₂ air mixed incubator for 72 hours. As a control, skim milk without fermentation by lactic acid bacteria was added to the culture medium. 50 μl of MTT working solution was added to the cultured cell medium, and cultured again under the same conditions for 4 hours. After removing the MTT-treated medium, 150 μl of dimethyl sulfoxide (DMSO) was added to each well. After dispensing, the absorbance was measured at 550 nm using an ELISA plate reader (VERSA max, Molecular devices, USA). Each experiment was repeated four times to measure cell activity. The MTT analysis results are shown in FIG. 1.

According to the results shown in Figure 1, Lactobacillus casei ( Lactobacillus casei The fermentation of ATCC 393) showed an effect of significantly improving the proliferation of osteoblasts compared to the control. In addition, the osteoblast proliferation effect was shown to depend on the concentration of fermentation added, it was confirmed that the milk fermentation of Lactobacillus casei ATCC 393 promotes osteoblast activity in a concentration-dependent manner.

Example  3: Lactobacillus Kasei ( Lactobacillus casei ) milk Fermented products  Other Lactobacillus Share milk Fermented product  Comparison of osteoblast growth promoting effect

Lactobacillus acidophilus 4356 and Lactococcus sp. KU107, which are known as strains with high milk protein degradation capacity, were selected, and fermented milk by these strains was the same as described in Example 1. After the preparation by the method, the effect of these milk fermentation on osteoblast proliferation was evaluated in comparison with the milk fermentation of Lactobacillus casei strain of the present invention.

The results of the analysis are shown in FIG. In Figure 2 fermented products of Lactobacillus ashophilus ( Lactobacillus acidophilus 4356), Lactococcus sp. KU107, the osteoblast proliferation effect is 90-110% of the control (non-lactic acid bacteria fermented skim milk) control and On the other hand, the osteoblast proliferation promoting effect of the fermentation of the Lactobacillus casei ATCC 393 of the present invention was found to be significantly higher as 160-180% of the control group. Therefore, the milk fermented product by Lactobacillus casei ( Lactobacillus casei ) was confirmed that the effect of promoting the activity of osteoblasts is superior to the milk fermented product by other strains with excellent protein degradation ability. These results demonstrate that the effect of promoting the osteoblast activity of the milk fermentation of the present invention is a specific effect by the milk fermentation of Lactobacillus casei, not other lactic acid bacteria strains.

Example  4: Lactobacillus Kasei ( Lactobacillus casei ) milk Fermented products  Osteocalcin ( osteocalcin ) mRNA  Effect on expression

Osteocalcin mRNA by Huang et al. ( Bone , Vol. 34, 799-808, 2004) using reverse transcriptase polymerase chain reaction (RT-PCR) as a direct biochemical indicator to verify osteoblast differentiation. Expression level was measured. Osteocalcin (OCN), which is a factor that affects the proliferation and differentiation of osteoblasts, is a non-relative protein present in bone and plays a key role in osteoblast activity and bone formation. Therefore, OCN mRNA is a biochemical indicator of osteoblast differentiation. (Wang et al., Journal of Bone and Mineral Research, Vol. 14, 893-905, 1999).

Lactobacillus casei ATCC 393 milk extract of the present invention as a fermented milk or a control group by adding skim milk to the medium to extract RNA from osteoblasts cultured for a certain time, and added reverse transcription buffer and random primer (1.5) at 42 ℃ The reaction was carried out for a time. 5 μl of the reacted reverse-transfer solution was added, and 13 μl of distilled water and 1 μl of each prepared primer were added to make 20 μl of the total volume. During the PCR process, the denaturation was amplified by 30 cycles of 1 min at 94 ° C, annealing at 1 ° C at 45 ° C, and 1 min at 72 ° C. The primers used for amplification were shown in the following table.

Forward primer Reverse primer Osteocalcin 5'-AGGGAGGATCAAGTC CCG-3 ' 5'-GAACAGACCTCGGCGCTA-3 β-actin 5'-CCCTGTTGCTGTAGCCGTA-3 ' 5'-CCGGTGCTGAGTATGTCG-3 '

The amplified product was electrophoresed on 1.5% agarose gel by addition of the same amount of gel loading solution (0.02% bromophenol blue, 0.02% xylene cyanol) and a 1D image analyzer (KODAK Co., USA) The density was determined as osteocalcin mRNA / β-actin mRNA (%). The results of this analysis are shown in FIG. 3. As shown in FIG. 3, the expression of osteocalcin mRNA was started after 5 days of culture, and after 10 days of culture, the mRNA amount of the hydrolyzate expressed was increased by 43% compared to β-actin.

Example  5: Lactobacillus Kasei  ( Lactobacillus casei ) milk Fermented products  Effect of Feeding on Bone Mineral Density in Ovariectomized Rats

A 10-week-old female Sprague-Dawley rat (Samtaco Bio Korea Co., Ltd.) was purchased and used. The rats were sutured after excision of the ovaries after 4 days of environmental adaptation. (Ovary extraction). After surgery, the room temperature was maintained at 25 ° C. to speed up recovery.

The ovarian non-extracted group exposed the ovaries to the outside of the abdominal cavity after laparotomy in order to give the same stress as the ovarian extraction group, and then sutured again after a time required for ovarian extraction (apparent surgery group). After 4 days of recovery, the ovarian and non-ovarian groups (15 heads each) were assigned to each treatment group.

The experimental feed was obtained by using NIH-31M rodent diet from Samtaco Bio Korea Co., Ltd. and Lactobacillus casei ) The level of fermentation was 2% of the feed. The total test period was 46 days, consisting of 4 days postoperatively and 6 weeks experimental period. After the end of the experimental period, the cervical spine was separated, and the rats were sacrificed. The femur was harvested and dried in a 110 ° C. drying oven for 3 days. The femur weight and length were measured using a balance and vernier calipers. BMD (bone mineral density) and BMC (bone mineral content) were measured using dual energy X-ray absorptionmetry (DEXA, Norland, USA). It was.

The analysis results are shown in FIG. 4. As shown in FIG. 4, the ovarian extraction group showed significantly lower bone density than the apparent surgery group, indicating that osteoporosis was induced. Lactobacillus after ovarian extraction casei ATCC 393) When the feed containing the fermented product was administered for 6 weeks, the bone density was maintained at a level similar to that of the apparent surgical group, and it was confirmed that the administration of Lactobacillus casei fermentation product helped to increase the bone density.

Example  6: Lactobacillus Kasei ( Lactobacillus casei ) milk Fermented products  Salary of Ovariectomy Rats On bone strength  Effect

After conducting animal experiments according to the method described in Example 4, the fracture strength of the femur was measured by a texture analyzer (Stable microsystem, TA-XT2, USA). Bone strength was measured using the three point bending test method. The fracture strength and fracture displacement of the fractured areas were analyzed by applying force to the bone. When fractured the femur, the length between the points was 1 cm, and pressure was applied to the anterior bone and the bone center at half the length of the femur. The pressure applied was 0.1 mm / sec and the load applied was 20 Kg. The strength of the femur is expressed as the maximum force (N) when the femur is cut.

The results of the analysis are shown in FIG. 5. As shown in FIG. 5, the ovarian extraction group showed significantly lower fracture strength than the apparent surgical group, indicating a tendency of bone weakening. This tendency reflects a phenomenon in which bone fracture is more likely to occur due to a decrease in bone density when inducing osteoporosis. Lactobacillus casei ATCC 393 of the present invention after ovarian extraction When the feed containing fermented products was administered for 6 weeks, bone strength was maintained at the level similar to that of the apparent surgical group, which showed a synergistic effect of bone strength due to the increase of bone density.

Example  7: Lactobacillus Kasei ( Lactobacillus casei ) milk Fermented products  Effect of Feeding on Osteoclast Activity in Ovariectomized Rats

Tartrate resistant acid phosphatase activity, a bone resorption expression factor of osteoclasts, was measured by the following method. The femur and femoral heads were cut with a saw, and then liquid nitrogen was added to the mortar and ground. About 200 mg of the ground femoral and femoral heads were homogenized after addition of 1 ml of 0.15 M NaCl solution containing 3 mM NaHCO ₃ . The homogenized femoral head and femoral head were centrifuged at 1000 x g for 20 minutes at 4 ° C to obtain supernatant. 50 μl of the supernatant was added to a 96 well plate, and then 150 μl of a substrate solution containing 7.6 mM 4-nitrophenyl phosphate disodium salt dexahydrate, 100 mM sodium acetate, and 50 mM sodium tartrate was added and reacted at 37 ° C. for 1 hour. Then 50 μl of 3 M NaOH solution was added to terminate the reaction. Absorbance at 405 nm was measured using a Veras Max microplate reader manufactured by Molecular Devices. Standard curves were prepared using p-nitrophenol. The amount of free phosphate released for the unit time was expressed by dividing the protein amount.

The results of the analysis are shown in FIG. 6. As shown in FIG. 6, the ovarian extraction group exhibited significantly higher tartrate resistant acidic phosphatase activity than the apparent surgical group to actively promote bone resorption when osteoporosis is induced due to ovarian extraction. Appeared. Lactobacillus casei after ovarian extraction ATCC 393) For six weeks of feeding fermented feed, the activity of tartrate-resistant acidic phosphatase remained similar to that of the apparent surgical group. The above result was Lactobacillus casei milk Taking fermented products not only improved osteoblast activity in osteoporosis-induced osteoporosis-induced animal model (Example 1-6), but also effectively inhibited osteoclast activity, and showed an effect of improving bone density.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is Lactobacillus casei of the present invention ( Lactobacillus) Casei ATCC 393) This is a graph showing the results of measuring the effect of milk fermentation by the strain on osteoblasts (MC3T3-E1) proliferation by MTT analysis method.

Figure 2 is a graph showing the result of comparing the effect of milk fermentation of osteoclast proliferation of milk fermentation by the Lactobacillus casei ATCC 393 strain of the present invention with the effect of milk fermentation of other lactic acid strains excellent in protein degradation ability.

3 is a photograph and graph showing the results of measuring the osteocalcin mRNA expression promoting effect of osteoblasts of milk fermented product by Lactobacillus casei ATCC 393 strain of the present invention using RT-PCR.

Figure 4 is a graph showing the results of measuring the mineral density of the femur of rats ( Lactobacillus casei ATCC 393) of the rats in which the milk fermented product by the strain of the present invention by DEXA.

5 is a graph showing the results of measuring the fracture strength of the femur of rats taking the milk fermented product by the Lactobacillus casei ATCC 393 strain of the present invention using a texture analyzer.

Figure 6 Lactobacillus casei of the present invention ( Lactobacillus This is a graph showing the results of tartrate resistant acid phosphatase activity, a bone resorption index at the end of femoral growth plate of rats fed milk fermented by casei ATCC 393) strain.

Claims (9)

Lactobacillus casei ( Lactobacillus casei ) containing milk fermentation product by strain ATCC (American Type Culture Collection) 393 as an active ingredient, the milk fermentation product has a molecular weight cut (Molecular Weight Cut-Off, MWCO) 350 A composition for improving osteoporosis, which is a fraction of milk ferment fractionated as -35000, which promotes proliferation, differentiation and activity of osteoblasts and inhibits osteoclast activity. The composition of claim 1, wherein the composition is a functional food composition for improving osteoporosis. The composition of claim 1, wherein the composition comprises (a) a pharmaceutically effective amount of milk fermentation by Lactobacillus casei ATCC 393 strain; And (b) a pharmaceutically acceptable carrier, wherein the milk fermentation product is a fraction of milk fermentation fractionated by fractional molecular weight (Molecular Weight Cut-Off, MWCO) of 350-35000 in milk fermentation product. A composition, characterized in that the prophylactic or therapeutic pharmaceutical composition. 4. The composition of any one of claims 1 to 3, wherein the milk fermentation product is a fermentation digest of milk protein. delete delete delete Method for producing a milk fermentation fraction for improving osteoporosis, comprising the following steps: (a) inoculating a medium containing milk with Lactobacillus casei ATCC 393 strain, (b) culturing the medium of step (a), and (c) fractionating the milk fermented product produced in step (b) with a fractional molecular weight (Molecular Weight Cut-Off, MWCO) of 350-35000 to obtain a fraction. 9. The method of claim 8, wherein the medium of step (a) is a medium to which whey protein is additionally purified.

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