KR20220109667A - Lactobacillus plantarum having inhibitory effect on osteoclast differentiation and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum strain having an osteoclast differentiation inhibiting effect, to a microbial preparation for inhibiting osteoclast differentiation containing the strain or the culture medium thereof as an active ingredient, to a probiotic bioactive composition containing the strain or the culture medium thereof as an active ingredient, to a pharmaceutical composition containing the strain or the culture medium as an active ingredient, to a composition for foods (healthy functional food) containing the strain or the culture medium thereof as an active ingredient, to a drinking water additive containing the strain or the culture medium thereof as an active ingredient, and to a feed additive containing the strain or the culture medium thereof as an active ingredient. The Lactobacillus plantarum strain and the culture medium thereof according to the present invention can be very usefully used for healthy functional food requiring prevention or alleviation of osteoporosis, a pharmaceutical composition requiring treatment thereof, or a feed additive or animal medicine for improving bone health of livestock or companion animals.

Description

Lactobacillus plantarum having inhibitory effect on osteoclast differentiation and uses thereof

The present invention relates to Lactobacillus plantarum subsp. plantarum having an effect of inhibiting differentiation of osteoclasts. More specifically, a Lactobacillus plantarum strain having an osteoclast differentiation inhibitory effect, a microbial preparation for inhibiting osteoclast differentiation comprising the strain or a culture solution thereof as an active ingredient, osteoclast differentiation inhibition comprising the step of culturing the strain It relates to a method for producing a microbial preparation for use, a probiotic probiotic composition comprising the strain or a culture medium thereof as an active ingredient, a pharmaceutical composition, a food (health functional food) composition, a drinking water additive and a feed additive.

Bone (骨) of living things starts from birth and continues until adolescence, when the skeleton matures and growth ends. is done when Bone remodeling is a process in which old bone is removed and replaced with new bone after growth has already been completed. In the bone remodeling unit, a dynamic, constantly repeating dynamic that balances the activity of osteoblasts and osteoclasts. It has homeostasis that maintains a phosphorus equilibrium.

Physiologically, bone continuously undergoes bone remodeling to replace the bone matrix with new tissue (bone turnover), and this process is in a state of balance between bone resorption and bone formation. Progression (Jerome CP. 2004. Hormonal therapies and osteoporosis. ILAR J. 45, 170-178).

When the balance between osteoblasts and osteoclasts is disrupted, and bone resorption is enhanced more than bone formation, the calcification of the bone tissue is reduced and the compactness of the bone is thinned. Because the bones are weak, they are easy to fracture even with a small impact. A disease caused by these symptoms is osteoporosis (Burckhardt P, Burnand B. 1993. The effect of treatment with calcitonin on vertebral fracture rate in osteoporosis. Osteoporos Int. 3, 24-30.).

Osteoporosis refers to a condition in which the bone matrix is pathologically thin, resulting in decreased bone density and weakened bone strength, increasing the risk of fracture. The strength of the skeleton is primarily formed and maintained by the deposition of minerals in the bone matrix and the bone matrix, which is clinically reflected in bone density. Osteoporosis is classified into primary osteoporosis and secondary osteoporosis according to the cause (Jeffcoat MK, Chesnut CH, 1993. 3rd. Systemic osteoporosis and oral bone loss: evidence shows increased risk factors. J Am Dent Assoc. 124, 49-56.).

Among them, the main clinical problem is postmanopasual osteoporosis, which is type 1 of primary osteoporosis. The reason that menopause is the main cause of bone metabolic diseases is that when estrogen in the body decreases, the skeletal reactivity to parathyroid hormone increases, and as bone resorption increases, calcium in the bones flows into the blood, resulting in an increase in serum calcium concentration. It has been reported that bone resorption increases and vitamin D production decreases due to decreased hormone secretion, which leads to decreased calcium ion absorption in the gastrointestinal tract, increased urinary calcium excretion, and activation of osteoclasts due to increased IL-6. (Recker RR, Saville PD, Heaney RP. 1977. Effect of estrogens and calcium carbonate on bone loss in postmenopausal women. Ann Intern Med. 87, 649-55.).

Osteoclasts play a very important role in inducing bone resorption in bone diseases such as periostitis and rheumatoid arthritis, including osteoporosis. Therefore, many efforts have been made to discover alternative drugs that can treat bone diseases by inhibiting the differentiation of osteoclasts (Huang H, Ryu J, Ha J, Chang EJ, Kim HJ, Kim HM, Kitamura T, Lee ZH, Kim). HH. 2006. Osteoclast differentiation requires TAK1 and MKK6 for NFATc1 induction and NF-kappaB transactivation by RANKL. Cell death and differentiation, 13, 1879-1891.).

Until now, the effect related to the differentiation of osteoclasts is not well known. Therefore, in this study, the action of ABE as an inhibitor on osteoclast differentiation was revealed, and furthermore, the potential for development as an alternative drug to prevent or treat bone resorption was investigated.

On the other hand, probiotics are, when consumed in an appropriate amount, alleviation of lactose intolerance, immune activity and regulation, alleviation of allergic reactions and inflammation, reduction of blood cholesterol, inhibitory effect on colorectal cancer, atopic dermatitis, Crohn's disease, diarrhea, Candida infection and Microorganisms (Cho, Jung-Whan, Kim, Dong-Hyeon, Kim, Hyun-Sook, Kim, Hong-Seok, Hwang, Dae-Geun, Song, Kwang-Young, Yim, Jin-Hyuk, Choi, Dasom, Lim, Jong-Soo and Seo, Kun-Ho Seo. 2014. Effect of Probiotics on Risk Factors for Human Disease: A Review. Korean J. Dairy Sci. Technol. 32, 17-29).

Probiotics are microorganisms that have a useful action in the animal digestive tract, and have a useful action on the host animal, such as improving the intestinal flora. The strains used as probiotics include Lactobacillus, Enterococcus, Bifidobacterium, Bacillus, Clostridium, Saccharomyces, and Aspergillus genera. The mechanism of action of probiotics is normalization of the intestinal flora, production of antibiotics, nutrient competition to prevent the establishment of pathogens, production of organic acids by lowering pH, inhibition of harmful enzymes of pathogens such as β-glucuronidase, azoreductase, and nitroreductase, inhibition of the production of decay products and toxins, Digestive enzyme production, vitamin synthesis, immune stimulation, etc.

Early in Europe and Japan, strains such as L. rhumnosus GG (Valio, Finland) and L. casei Shirota (Yakult, Japan) were isolated and probiotics with excellent functions were developed. Research is actively underway. Similarly in Korea, research to isolate fermented foods such as kimchi and soy sauce, as well as animal and human-derived strains and use them as probiotics, is being actively conducted, but securing the strain is the most important key.

The present inventors have completed the present invention by discovering the effect of Lactobacillus plantarum to inhibit the differentiation of osteoclasts while studying the bioactive properties of the probiotic.

The present invention was derived from the above needs, and the present inventors first discovered that the Lactobacillus plantarum subsp. plantarum strain inhibits osteoclast differentiation, and the strain or its culture medium as an active ingredient A microbial preparation for inhibiting osteoclast comprising, a method for producing a microbial preparation for inhibiting osteoclast comprising the step of culturing the strain, a probiotic probiotic composition comprising the strain or a culture solution thereof as an active ingredient, a pharmaceutical composition, The present invention was completed by confirming that it can be applied as a food (health functional food) composition, a drinking water additive, and a composition for feed.

In order to achieve the object of the present invention, the present invention provides a Lactobacillus plantarum strain having an osteoclast differentiation inhibitory effect.

The Lactobacillus plantarum strain was isolated from commercially available fermented kimchi. As a result of identifying the isolated strain through nucleotide sequence analysis by 16S rDNA analysis, it was confirmed as Lactobacillus plantarum subsp. plantarum, KCTC3108.

As used in the present invention, the term 'osteoclast' refers to a cell that decomposes and absorbs bone.

As used herein, the term "inhibition" may refer to any action of inhibiting or delaying the differentiation of osteoclasts by administering the microbial agent for inhibiting osteoclast differentiation according to the present invention to an individual.

As used herein, the term “improvement” may refer to any action that at least reduces a parameter related to a condition to be treated, for example, the degree of symptoms.

As used herein, the term "treatment" refers to any act of administering the Lactobacillus plantarum strain and its culture medium of the present invention to a subject suspected of osteoporosis to improve or benefit the symptoms of osteoporosis due to inhibition of osteoclast differentiation. can mean

The Lactobacillus plantarum strain promotes osteoblast differentiation (Table 3), confirms that it has an effect of strongly inhibiting osteoclast differentiation (Table 4), and thus has excellent physiological activity, particularly inhibition of osteoclast differentiation (FIG. 1) ), the potential of biological materials that can be applied to bone health was confirmed.

In addition, the present invention provides a microbial preparation for inhibiting osteoclast differentiation comprising the strain or a culture solution thereof as an active ingredient.

The microbial preparation for inhibiting osteoclast differentiation of the present invention can be prepared using a Lactobacillus plantarum strain as an active ingredient. The microbial preparation for inhibiting osteoclast differentiation according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, dispersion material or granules, but is not limited thereto.

In addition, the present invention provides a method for preparing a microbial agent for inhibiting osteoclast differentiation comprising the step of culturing the strain. The method of culturing the strain of the present invention can be cultured according to a method commonly used in the art.

In addition, the present invention provides a probiotic probiotic composition comprising the strain or a culture medium thereof as an active ingredient. The strain of the present invention exhibited a survival rate of about 64% when exposed to a bile acid-resistant 1.0% bile solution (Table 6), and showed a high survival rate of about 88.1% even after heat treatment at 70° C. for 10 minutes (Table 6). 7), it was confirmed to have very excellent properties as probiotic live bacteria.

In addition, the present invention provides a pharmaceutical composition for inhibiting osteoclast differentiation comprising the strain or a culture medium thereof as an active ingredient.

The pharmaceutical composition for inhibiting differentiation of osteoclasts according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as a food, drug, feed additive, and drinking water additive.

As used herein, the term 'pharmaceutically acceptable carrier' may refer to a carrier or diluent that does not inhibit the biological activity and properties of the administered compound without irritating the organism.

The type of carrier usable in the present invention is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in combination of two or more.

In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostats may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. It can be used by formulating it into a dosage form, pill, capsule, granule, or tablet.

The administration method of the pharmaceutical composition for inhibiting osteoclast differentiation according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration.

The pharmaceutical composition for inhibiting osteoclast differentiation of the present invention can be prepared in various formulations according to the desired administration method.

The pharmaceutical composition of the present invention may be used as a single formulation, may be prepared and used as a combination formulation by additionally including a drug known to have an approved osteoporosis treatment effect, and may be formulated using a pharmaceutically acceptable carrier or excipient to form a unit. It may be prepared in dosage form or by introduction into a multi-dose container.

As another aspect, the present invention provides a method for preventing or improving osteoporosis, comprising administering the pharmaceutical composition for inhibiting osteoclast differentiation to an individual. As used herein, the term "individual" may refer to any animal, including humans, that has or is likely to develop osteoporosis.

Specifically, the method for preventing or improving the present invention may include administering the composition in a pharmaceutically effective amount to an individual who has or is at risk of developing osteoporosis.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and generally the number of bacteria of the present invention per 1 g (or mL) When converted to, the amount of 7 log CFU to 10 log CFU, preferably 8 log CFU / g to 9 log CFU / g can be administered once a day divided into several times. However, for the purpose of the present invention, a suitable total daily amount of the composition containing the extract may be determined by the treating physician within the scope of correct medical judgment, and may be administered once or divided into several doses. However, for the purposes of the present invention, a specific therapeutically effective amount for a specific patient depends on the type and extent of the response to be achieved, the specific composition, including whether other agents are used if necessary, the specific composition, the patient's age, weight, general health, It is preferable to apply differently depending on various factors including sex and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently with a specific composition, and similar factors well known in the pharmaceutical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without causing side effects, and can be easily determined by those skilled in the art.

As used herein, the term “administration” means introducing the pharmaceutical composition of the present invention to a patient by any suitable method, and the administration route of the composition of the present invention is oral or parenteral as long as it can reach the target tissue. It can be administered through various routes.

The method of administration of the pharmaceutical composition according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. The pharmaceutical composition according to the present invention may be prepared in various dosage forms depending on the desired administration method.

The frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose.

In addition, the present invention provides a composition for health functional food that can prevent or improve osteoporosis comprising the strain or a culture solution thereof as an active ingredient. Since the intake of the strain of the present invention suppresses bone loss, it can be provided as a health food for preventing and improving osteoporosis using it as an active ingredient.

When the strain or its culture solution obtained in the step of culturing the strain of the present invention is used as a food additive, the strain or its culture solution can be added as it is or used with other foods or food ingredients, and can be used appropriately according to a conventional method. can The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (treatment for the purpose of prevention or health improvement).

There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include dairy products including chocolate, candy, gum, and ice cream, beverages, drinks, alcoholic beverages, and vitamin complexes.

When the composition is used as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food composition may include a food pharmaceutically acceptable carrier.

The food composition may be a health functional food. Functional food is the same term as food for special health use, FoSHU. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. in order to obtain a useful effect in inhibiting osteoclast differentiation.

At this time, the content of the strain or its culture medium contained in the food is not particularly limited thereto, but may be included in an amount of 0.01 to 100% by weight, more preferably 1 to 80% by weight, based on the total weight of the food composition. When the food is a beverage, it may be included in a ratio of 1 to 30 g, preferably 3 to 20 g, based on 100 mL.

The health functional food can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, there is an advantage in that there are no side effects that may occur when taking the drug for a long time by using food as a raw material, and it can be excellent in portability.

According to another embodiment of the present invention, the present invention is the strain or its culture

It provides a drinking water additive for inhibiting osteoclast differentiation comprising. The drinking water additive of the present invention may be used by separately preparing a composition containing the strain or its culture solution in the form of a drinking water additive and mixing it with drinking water, or by directly adding the composition to drinking water. The drinking water additive of the present invention may be in a liquid or dry state, preferably in a dried powder form. A drying method for preparing the drinking water additive of the present invention in a dried powder form is not particularly limited, and a method commonly used in the art may be used. The drinking water additive of the present invention may further include other additives as needed. Non-limiting examples of the additives that can be used include amino acids, vitamins, enzymes, probiotics, flavoring agents added to increase the utility of feed or drinking water, such as binders, emulsifiers, and preservatives, added to prevent deterioration of the quality of drinking water. , a non-protein nitrogen compound, a silicate agent, a buffer, a colorant, an extractant, or an oligosaccharide, and may further include a feed mixture, etc. in addition. These may be used alone or two or more may be added together.

Since the Lactobacillus plantarum strain of the present invention has a very good osteoclast differentiation inhibitory effect, the Lactobacillus plantarum strain and its culture medium are health functional foods or pharmaceutical compositions requiring osteoporosis prevention or improvement, livestock or companion animals It can be very usefully used as a feed additive or veterinary medicine for improving bone health.

1 shows the effect on the cell viability of osteoblasts and osteoclasts according to the treatment of Lactobacillus plantarum.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

[Example 1]

<Preparation of cells and culture medium of Lactobacillus plantarum>

Brain heart infusion (BHI, Difco Laboratories, U.S.A.) medium was inoculated with Lactobacillus platarum, grown by shaking at 30 ° C. for 48 hours to proliferate the strain, and then centrifuged to obtain the strain and the culture supernatant, respectively. It was used as a sample.

[Experimental Example 1]

<Test of osteoclast differentiation inhibitory activity of Lactobacillus plantarum>

<1-1> Experimental method

① Resistance test to H ₂ O ₂

After inoculating and culturing Lactobacillus plantarum in BHI broth medium, centrifugation (7,000 × g , 20 minutes, 4 ℃), and then washed 3 times with PBS solution to adjust the cell number to about 10 ⁸ CFU/mL and the same buffer was suspended in 500 μL of 0.5 mM H ₂ O ₂ solution and the same amount of cell suspension were immediately mixed and reacted at 60-minute intervals for a total of 5 hours, followed by centrifugation again to remove residual H ₂ O ₂ (20,000 × g , 1 min, 4 ℃) to recover the pellet. After resuspending the pellets in buffer, diluting them step by step, and measuring the number of colonies produced by plate culture on BHI agar (30℃, 24 hours), the reduction rate for the initial number of bacteria was shown.

② Antioxidant activity test by DPPH method

Antioxidant substances donate electrons or hydrogen to free radicals to form a complex, and 1,1-diphenyl-2-picyryl hydrazyl (DPPH) is an antioxidant. Since it receives electron hydrogen from a sexual substance and forms an irreversibly stable molecule, the antioxidant activity is measured from electron donating ability (Seo, YH, Kim, IJ, Min, HK and Park, SU. 1999. Fatty acid composition) and antioxidative activity in wax corn ( Zea mays L.) F1s. Kor. J. Food Sic. Technol. 31, 1415-1420).

The hydrogen donating ability for 1,1-diphenyl-2-picyryl hydrazyl (1,1-diphenyl-2-picyryl hydrazyl; DPPH) was 100 M DPPH solution (6 mg of DPPH was completely dissolved in 100 mL of ethanol) After adding 200 µL of each of the Lactobacillus platarum culture supernatant and the sample to 1000 µL of distilled water (mL of distilled water), shaking for 10 seconds, leaving it for 10 minutes, and then measuring the absorbance at 528 nm.

③ Cell culture and osteoblast differentiation

MC3T3-E1 cells were cultured at 37° C. and 5% CO ₂ condition by adding 10% FBS and 1% antibiotics to α-MEM medium. To induce osteoblast differentiation, 10 mmM β-glycerol phosphate (Sigma Co., St. Louis, MO, USA) and 50 μg/mL of vitamin C were added and used as a differentiation induction medium, and the medium was changed every 3 days. did.

④ ALP (alkaline phosphate) activity of osteoblasts

The cultured MC3T3-E1 cells were adjusted to 1×104 cells/well, aliquoted in a 96 well plate, cultured for 24 hours, exchanged with a differentiation induction medium, and cultured for 24 hours, followed by treatment with the culture medium. After 3 days, it was washed with PBS, and then 20 μL of 0.1% Triton X-100 was added and dissolved at 37°C for 30 minutes. 5 μL of the supernatant of the lysed cells was used for protein quantification, and 20 μL of 0.1 N glycine and 10 μL of 100 mM p -notrophenylphosphate (p-NPP) were added to the remaining supernatant, followed by reaction at 37° C. for 30 minutes. After the reaction, the reaction was stopped with 200 μL of 0.1N NaOH, and the absorbance was measured at 405 nm. ALP activity was derived by measuring p -nitrophenol (p-NP) generated from p-NPP, creating a standard graph for p-NP, and comparing the activity with the control group.

⑤ Isolation of bone marrow cells and differentiation of osteoclasts

The tibias of 4-5 weeks old ICR mice were removed, both ends were cut, and bone marrow cells were collected by passing through α-MEM essential medium, and treated with 50 ng/μL M-CSF (macrophagy-colony stimulation factor) for 24 hours. incubated during After washing the non-adherent cells with α-MEM, the cells were dispensed to 96 wells at 5×10 ⁴ cells/well, and cultured in α-MEM medium supplemented with 50 ng/μL M-CSF for 3 days. Then, 50 ng/μL M-CSF and 100 ng/μL RANKL (Receptor Activator for Nuclear Factor κB Ligand) were treated together, and the culture medium was treated by concentration and cultured for 4 days.

⑥ TRAP (tartate resistant and phosphatase) activity of osteoclasts

Cells were aliquoted in 96 wells so as to become 5×10 ⁴ cells/well, treated with differentiation factors and culture medium, and cultured for 4 days. After removing the medium, washing with PBS, and dispensing 100 μL of substrate solution (1.36 mg/mL 4-nitrophenyl phosphate disodium salt, 10 mM tartate, 50 mM citrate buffer, pH 4.6) into cells fixed with 10% formaldehyde at 37°C. was reacted for 30 min. After the reaction, the enzyme reaction solution was transferred to a new plate, the reaction was stopped with 0.1 N NaOH, and the absorbance was measured at 405 nm. TRAP activity was expressed as the absorbance of the sample as a percentage of the absorbance of the control.

<1-2> Experiment result

① Resistance test to H ₂ O ₂

Results showing the resistance to oxidative stress induced by H ₂ O ₂ of the Lactobacillus plantarum culture are shown in Table 1 below.

Resistance test results to H ₂ O ₂ Survival rate (%) according to reaction time with H ₂ O ₂ 0 1 hours 3 hours 5 hours Inhibition rate (%) 0 23.4±1.2 ^a 56.6±1.1 ^b 84.2±1.1 ^c

Different English shoulder letters ( ^ac ) of inhibition rate values for oxidative stress indicate statistical significance (p<0.05).

As a result of oxidative stress resistance evaluation, it showed a high inhibition rate of about 24.4% after 1 hour of reaction, 56.6% after 3 hours after reaction, and 84.2% after 5 hours. As a result of measuring the resistance of lactic acid bacteria to oxidative stress by cell viability according to the reaction time, it showed about 85.6 % cell viability after 5 hours of reaction. It was confirmed, and this is considered to increase resistance by detoxifying reactive oxygen species in the culture medium or by secreting antioxidant enzymes such as SOD and Catalase.

② Antioxidant activity test by DPPH method

Table 2 shows the results of measuring the electron donating ability by DPPH as the antioxidant power of the Lactobacillus plantarum strain culture medium.

Antioxidant activity (DPPH radical scavenging ability) evaluation result culture medium live bacteria dead cells vitamin C DPPH radical scavenging ability (%) 42.9±2.2 ^a 36.2±2.1 ^ab 39.7±1.9 ^b 100 ^c

Different English shoulder letters ( ^ac ) of DPPH radical scavenging activity values indicate statistical significance (p<0.05).

The DPPH radical scavenging ability of the Lactobacillus plantarum culture medium and live and dead cells was 42.9, 36.2, and 39.7% in the culture, live and dead cells, respectively, as a result of measuring the BHI broth as a control. Various ROS and toxic products, including free radicals generated during oxidation, damage cells and molecules exhibiting biological activity in vivo. When the DPPH radical scavenging ability is high, it means that it has antioxidant activity due to its high ability to reduce or offset free radicals. Therefore, it can be said that the Lactobacillus plantarum culture medium, live cells, and dead cells have antioxidant power, and based on this, antioxidant functional products can be developed.

③ Osteoblast proliferation and ALP activity promotion effect

In order to examine the effect of the Lactobacillus plantarum culture medium on the activity of osteoblasts, ALP, known as an indicator enzyme of osteoblast activity, was measured (Table 3).

Effect of promoting osteoblast proliferation and ALP activity Strain culture (%) Osteoblast proliferation rate (%) ALP activity (%) 0 100±1.2 ^a 100±0.4 ^a 2.5 102.5±2.4 ^ab 104.3±6.9 ^ab 5.0 107.7±3.2 ^b 119.8±5.2 ^b 10.0 112.1±2.6 ^b 132.9±7.4 ^c

Different English shoulder letters ( ^ac ) of the osteoblast proliferation value and the ALP activity promoting effect value of osteoblasts indicate that there is a statistical significance (p<0.05).

From the results, it was shown that as the concentration of the Lactobacillus plantarum culture solution increased, not only the cell proliferation but also the ALP activity increased by more than 30%, thereby promoting the activity of osteoblasts. Osteoblasts have basic phosphatase (ALP) in the cell membrane, which is found in high concentrations in the outer membrane and calcified tissue of cells, transports inorganic phosphate during calcification, regulates cell division or differentiation, and plays a role in osteoblast activity. known as markers. Lactobacillus plantarum is thought to help promote the differentiation of osteoblasts by activating the ALP enzyme of osteoblasts.

④ Inhibitory effect on osteoclast differentiation and TRAP activity

In order to examine the effect of the Lactobacillus plantarum culture medium on the differentiation of osteoclasts, the cell proliferation rate of osteoclasts and the degree of activity of TRAP, an index enzyme of osteoclast activity, were measured (Table 4).

Inhibitory effect on osteoclast differentiation and activity Strain culture (%) Osteoclast proliferation rate (%) TRAP activity (%) 0 100±1.2 ^b 100±1.2 ^d 2.5 102.5±4.4 ^b 85.5±2.9 ^c 5 98.7±3.2 ^b 75.8±3.2 ^b 10 78.3±3.2 ^a 64.3±2.4 ^a

Different English shoulder letters ( ^ad ) of the osteoclast proliferation value of the Lactobacillus plantarum culture medium and the TRAP activity inhibitory effect value of osteoclasts indicate that there is statistical significance (p<0.05).

As a result of observing the effect of Lactobacillus plantarum culture medium on the proliferation of osteoclasts using mouse bone marrow cells, the survival rate of osteoclasts decreased slightly as the treatment concentration increased. In particular, at 10% concentration, 78.3 ± 3.2 % survival rate. And, as a result of measuring the TRAP enzyme activity, which is a cell membrane-specific enzyme of osteoclasts, it was found that when Lactobacillus plantarum cultures were treated at 2.5% and 5% concentrations, TRAP activity was inhibited by about 35.7% without cytotoxicity.

Since osteoclasts are differentiated from mononuclear macrophages and are generally regulated by M-CSF and TNFF-related activation-induced cytokine (TRANCE, RANKL), mouse bone marrow cells are isolated and osteoclast differentiation is induced using M-CSF and RANKL. do. As osteoclasts progress in differentiation, they form multinuclear mature osteoclasts according to cell fusion, which attach to the bone surface and absorb bone. Because of its characteristics, TRAP is used as a marker for osteoclasts.

As shown in FIG. 1 , it is considered that the Lactobacillus plantarum culture medium inhibits the growth of osteoclasts by inhibiting the activity of the ALP enzyme that induces osteoclast differentiation, thereby exhibiting the effect of inhibiting bone loss such as osteoporosis.

[Experimental Example 2]

<Characteristic evaluation test of Lactobacillus plantarum as a live cell>

<2-1> Experimental method

① Test strain

In order to know the basic properties of Lactobacillus plantarum as a probiotic, acid resistance, bile resistance and heat resistance were measured. At this time, as a comparative strain, Lactobacillus paracasei (KCTC13169) was provided and used by the Korea Research Institute of Bioscience and Biotechnology (KCTC).

The antibacterial activity of Lactobacillus plantarum was determined by four bacterial species ( Salmonella enterica serovar Typhi (ATCC19430), Escherichia coli O157:H7 (ATCC 43895), Staphylococcus aureus (ATCC25923) , Listeria monocytogenes (ATCC 19113)), and one yeast species ( Candida ). albicans ATCC 24433) and one type of mold ( Aspergilus fumigatus ATCC 96918) were investigated by the paper disk method. The bacteria and fungi were purchased from ATCC and used.

② Resistance to artificial gastric acid (acid resistance) test

Artificial gastric acid was inoculated with 1% of a strain suspension of at least 7.0 log CFU/mL in PPS (0.2% Photassium phosphate solution) adjusted to pH 2, 4, and 6, respectively, and cultured for 2 hours. The number of viable cells was counted by culturing.

③ Resistance to artificial bile fluid (biliary tolerance) test

For artificial bile, each artificial bile was prepared by adding Oxgall to BHI Broth at concentrations of 0.3, 0.5, and 1.0%, respectively. After 1% inoculation of a suspension of 8.0 log CFU/mL in the prepared artificial bile, the cells were cultured for 2 hours, spread on a plate medium, and cultured at 37° C. for 24 hours to count the number of viable cells.

④ Heat resistance test

The comparative strain and the Lactobacillus plantarum strain were cultured at 36.5°C for 20 hours (using BHI broth), incubated for at least 7 log CFU/mL, and then treated at 50, 60, 70°C temperature conditions (using waterbath) for 10 minutes. After plated on a plate medium, the number of viable cells was counted by incubation at 37°C for 24 hours.

⑤ Antibacterial test

After the Lactobacillus plantarum strain was inoculated in BHI broth, it was cultured at 36.5° C. for about 20 hours, culturing at least 7 log CFU/mL or more. After the test sample solution was concentrated 20 times using a reduced pressure concentrator, a 10-fold and 5-fold concentrate were prepared using BHI, respectively, and then used for the following antibacterial activity measurement.

4 test bacteria Sal. enterica serovar Typhi, E. coli O157:H7, Sta. aureus, Lis. monocytogenes is smeared on BHI (Difco Laboratories) plate medium at least 7 log CFU/mL, and the Lactobacillus plantarum strain culture test sample solution prepared at the 4 concentrations (stock solution, 5-fold, 10-fold, and 20-fold concentrate) prepared above. After immersing the paper disk sufficiently and attaching it to the center, after culturing at 37°C for 20 hours, the diameter of the clear zone created around the paper disk was measured to determine whether and the degree of antibacterial activity. The test yeast Can. albicans on sabouraud dextrose (Difco Laboratories) plate medium, the test fungus Asp. fumigatus is smeared on PDA plate medium at 1 × 10 ⁶ CFU/mL, respectively, and after immersing the paper disk in the test sample solution, attach it to the center, and incubate at 25℃ for 72 hours. By measuring the diameter, the presence and degree of antibacterial activity were measured.

<2-2> Experiment result

① Resistance to artificial gastric acid (acid resistance) test result

The results of testing the survival rate of the Lactobacillus plantarum strain in artificial gastric acid conditions are shown in Table 5 below.

Comparison of gastric acid resistance of Lactobacillus plantarum strains strain name Number of viable bacteria (Log CFU/mL) pH 6.0 pH 4.0 pH 2.0 Lactobacillus plantarum 7.92 ± 0.02 ^c 3.16 ± 0.03 ^b,X 1.17 ± 0.08 ^a,X Lactobacillus paracasei 7.89 ± 0.06 ^c 4.69 ± 0.12 ^b,Y 2.85 ± 0.05 ^a,Y

Among the resistance values to artificial gastric acid, different English shoulder letters ( ^ac ) of the results for each strain indicate that there is a statistical significance (p<0.05).

Among the resistance values to artificial gastric acid, different English shoulder letters ( ^XY ) of the results between strains indicate that there is a statistical significance (p<0.05).

As a result of the test, the Lactobacillus plantarum strain, the test strain, increased growth to 7.92 log CFU/mL in the artificial gastric acid condition of pH 6, and 3.16 log CFU/mL in the artificial gastric acid condition of pH 4, showing a survival rate of about 30.4%. and showed a survival rate of about 16.7% at a level of 1.17 log CFU/mL under the condition of pH 2. On the other hand, the comparative strain, Lactobacillus paracasei (KCTC13169), showed a survival rate of about 40.7% at a level of 2.85 log CFU/mL under the artificial gastric acid culture condition of pH 2. appeared to be low.

② Resistance to artificial bile fluid (biliary tolerance) test

When the Lactobacillus plantarum strain was prepared and treated by concentration of artificial bile solution (Oxgall), the results of testing the viability in the artificial bile solution condition are shown in Table 6 below.

Test result of resistance (biliary tolerance) of Lactobacillus plantarum strain to artificial bile strain name Number of viable bacteria (Log CFU/mL) oxgall 0.3% oxgall 0.5% oxgall 1.0% Lactobacillus plantarum 7.91 ± 0.02 ^c 5.85 ± 0.06 ^b 5.12 ± 0.05 ^a,Y Lactobacillus paracasei 7.92 ± 0.04 ^c 5.73 ± 0.07 ^b 4.98 ± 0.06 ^a,X

Among the resistance values to artificial bile, different English shoulder letters ( ^ac ) of the results for each strain indicate that there is a statistical significance (p<0.05).

Among the resistance values to artificial bile, different English shoulder letters ( ^XY ) of the results between strains indicate that there is a statistical significance (p<0.05).

As a result of the test, the Lactobacillus plantarum strain was about 98.7% at 7.82 log CFU/mL under the condition of 0.3% oxgall artificial bile, and 5.85 log CFU/mL at the condition of 0.5% artificial bile solution, about 73.1% and 1.0%. showed a survival rate of about 64.0% at a level of 5.12 log CFU/mL. The comparative strain, Lactobacillus paracasei, showed a survival rate of about 62.2% at a level of 4.98 log CFU/mL in the condition of 1.0% artificial bile acid, and the test strain, Lactobacillus plantarum, was a representative Lactobacillus sp. strain, Lactobacillus paracasei. It is thought to have a bile acid resistance similar to that of

③ Heat resistance test

The heat resistance test results of the Lactobacillus plantarum strain are shown in Table 7 below.

Comparison of heat resistance of Lactobacillus plantarum strains strain name Number of viable bacteria (Log CFU/mL) 50℃ 60℃ 70℃ Lactobacillus plantarum 8.91 ± 0.04 ^c 7.23 ± 0.03 ^b,Y 6.17 ± 0.08 ^a,Y Lactobacillus paracasei 8.82 ± 0.03 ^c 6.32 ± 0.07 ^b,X 5.08 ± 0.06 ^a,X

Among the result values for the heat resistance test, different English shoulder letters ( ^ac ) of the results for each strain indicate that there is a statistical significance (p<0.05).

Among the result values for the heat resistance test, different English shoulder letters ( ^XY ) between the strains indicate that there is a statistical significance (p<0.05).

As a result of the test, both the test strain, Lactobacillus plantarum and the comparative strain, Lactobacillus paracasei, increased growth at 50°C, and when heated at 60°C for 10 minutes, the test strain was 7.23 log CFU/mL and the control strain was 6.32. Log CFU/mL is shown. When the test strain was exposed to 70 ° C for 10 minutes, it showed a survival rate of about 88.1% at 6.17 log CFU / mL, and the comparative strain Lactobacillus paracasei at 70 ° C. at 5.08 log CFU / mL level of about 72.5%. The survival rate was shown, and the test strain, Lactobacillus plantarum, appeared to have a rather high heat resistance, and thus it was confirmed that it had excellent properties as a live probiotic.

④ Antibacterial test

The results of the antibacterial test for 6 pathogens of the Lactobacillus plantarum strain are shown in Table 8 below.

Antibacterial test result of Lactobacillus plantarum strain test strain antibacterial activity undiluted culture solution 5x concentrate 10-fold concentrate 20 times concentrate Salmonella enterica serovar Typhi + + ++ +++ Escherichia coli O157:H7 + + ++ +++ Staphylococcus aureus - - - + Listeria monocytogenes - - - + Candida albicans - - - + Aspergilus fumigatus - - - +

'-' or '+' in Table 8 is determined based on the diameter size of the live cell inhibition ring, '-' means no antibacterial or antifungal activity, and '+' indicates that the diameter size of the inhibition ring is less than 14.0 mm Meaning, '++' means that the diameter of the ring of inhibition is in the range of 14.0 - 17.0 mm, and '+++' means that the diameter of the ring of inhibition exceeds 17.0 mm.

Lactobacillus plantarum strain is a Gram-negative bacteria Sal. Enterica serover Typhi and E. coli O157:H7 showed strong growth inhibitory activity in two strains, but in the other four strains, antibacterial activity was not shown in the 10-fold concentrated solution. showed activity.

[Statistical Analysis]

Statistical analysis of the experimental results was analyzed using the 'Independent-Sample-T-Test method', a SPSS 13.0 statistical software, and expressed as the mean value ± standard difference. In addition, multiple comparisons were indicated by the LSD method, P>0.05: no significant difference, P<0.05: significant difference.

<Preparation Example 1> Preparation of microbial preparations

The formulation for inhibiting osteoclast differentiation containing Lactobacillus plantarum is prepared by drying Lactobacillus plantarum by a conventional freeze-drying method, and then mixing it with an excipient or encapsulating it.

<Production Example 2> Preparation of fermented milk

Fermented milk is prepared using the Lactobacillus plantarum of the present invention according to a method commonly performed in the art. A culture solution (1X10 ⁹ CFU/mL) is prepared by mixing raw milk (74.41%) and powdered skim milk (6.45%), and culturing by adding a culture seed and Lactobacillus plantarum.

<Preparation Example 3> Preparation of food and beverage (health functional food) composition

<3-1> Preparation of beverage

The beverage is prepared by mixing the ingredients of the formulations set forth below according to methods commonly practiced in the art. Honey 522 mg, thioctoacid amide 5 mg, nicotinic acid amide 10 mg, riboflavin hydrochloride 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, ortic acid 50 mg, Lactobacillus plantarum culture supernatant, water 200 mL

<3-2> Preparation of powder

20 mg of Lactobacillus plantarum freeze-dried powder (1×10 ⁹ CFU/g), 100 mg of lactose, and 10 mg of talc are mixed and filled in an airtight bag to prepare a powder.

<3-3> Preparation of tablets

After mixing 10 mg of Lactobacillus plantarum freeze-dried powder (1×10 ⁹ CFU/g), 100 mg of cornstarch, 100 mg of lactose, and 2 mg of magnesium stearate, tableting was performed according to a conventional tablet manufacturing method to prepare tablets. do.

<3-4> Preparation of capsules

10 mg of Lactobacillus plantarum freeze-dried powder (1×10 ⁹ CFU/g), 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate are mixed according to a conventional capsule preparation method and filled in a gelatin capsule. to prepare capsules.

<3-5> Preparation of mixed powder

Lactobacillus plantarum freeze-dried powder (1×10 ⁹ CFU/g) 1 g, vitamin mixture appropriate amount (vitamin A acetate 70 μg, vitamin E 1.0 mg, vitamin B1 0.13 mg, vitamin B2 0.15 mg, vitamin B6 0.5 mg, Vitamin B12 0.2 μg, vitamin C 10 mg, biotin 10 μg), nicotinic acid amide 1.7 mg, folic acid 50 μg, calcium pantothenate 0.5 mg, mineral mixture (ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, 15 mg of potassium monophosphate, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, 24.8 mg of magnesium chloride) were mixed according to a conventional health functional food manufacturing method, and then granules were prepared, According to the method, it can be used to prepare a health functional food composition. The composition ratio of the above vitamin and mineral mixture is relatively suitable for health functional food, but the mixing composition is a preferred example, but the mixing ratio may be arbitrarily modified.

<Preparation Example 4> Preparation of feed composition

The composition for feed is prepared by mixing 45 g of corn, 30 g of soybean meal, 15 g of wheat, 4 g of tallow, 3 g of molasses, 2 g of calcium phosphate, 0.4 g of limestone, 0.2 g of salt, and 5 g of freeze-dried Lactobacillus plantarum powder. manufactured.

From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the manufacturing examples described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

As described above, the Lactobacillus plantarum subsp. plantarum strain and its culture medium having the osteoclast differentiation inhibitory effect of the present invention have high antioxidant properties and resistance to oxidative stress. It can be widely used industrially, such as health functional foods requiring prevention or improvement of osteoporosis by inhibiting growth, pharmaceuticals requiring treatment, feed additives for improving the bone health of livestock or companion animals, or veterinary drugs.

Claims (8)

Lactobacillus plantarum subsp. plantarum strain having an osteoclast differentiation inhibitory effect. A microbial preparation for inhibiting osteoclast differentiation comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A method for producing a microbial agent for inhibiting osteoclast differentiation comprising the step of culturing the strain of claim 1. A probiotic bioactive composition comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A pharmaceutical composition for inhibiting osteoclast differentiation comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A food or health functional food composition for preventing or improving osteoporosis comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A drinking water additive for preventing, improving or treating osteoporosis comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A feed additive for preventing, improving or treating osteoporosis comprising the strain of claim 1 or a culture solution thereof as an active ingredient.

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