KR102606636B1 - Bacillus velezensis having the effect of preventing or improving for sarcopenia and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus velezensis strain having an effect of preventing or improving sarcopenia, a microbial agent for preventing or improving sarcopenia containing the strain or its culture as an active ingredient, and the strain or its culture as an active ingredient. A probiotic probiotic active composition containing the strain or its culture as an active ingredient, a pharmaceutical composition containing the strain or its culture as an active ingredient, a health functional food composition containing the strain or its culture as an active ingredient, and the strain or its culture as an active ingredient. It relates to a quasi-drug composition containing the same, a drinking water additive containing the strain or its culture as an active ingredient, and a feed additive containing the strain or its culture as an active ingredient.

Description

Bacillus velezensis having the effect of preventing or improving sarcopenia and uses thereof}

The present invention relates to Bacillus velezensis, which has the effect of preventing or improving sarcopenia. More specifically, it relates to a Bacillus velezensis strain that has the effect of preventing or improving sarcopenia, and the strain or its culture solution as an active ingredient. A microbial agent for preventing or improving sarcopenia comprising a microbial agent for preventing or improving sarcopenia, a method for producing a microbial agent for preventing or improving sarcopenia comprising culturing the strain, and a probiotic active composition comprising the strain or its culture medium as an active ingredient. , pharmaceutical compositions, food compositions, and feed additives.

Sarcopenia is a disease in which the breakdown of muscle protein occurs more than the synthesis of protein, leading to a continuous decrease in muscle mass. Also called muscle atrophy, it can be defined as loss of size and mass of muscle cells and muscle tissue that occurs due to long-term inability to move due to aging, serious illness, nerve damage, etc. or malnutrition.

Muscles are largely divided into skeletal muscle, cardiac muscle, and smooth muscle. Among these, skeletal muscle is the tissue present in the largest amount in the human body, accounting for 40-45% of body weight. Skeletal muscles are attached to bones through tendons and play a role in producing bone movement or force. Skeletal muscle has the characteristic of being regenerated and maintained depending on the environment, but this characteristic is lost with age, and as a result, not only muscle mass decreases but also strength is lost as aging progresses.

A typical physiological change due to aging is a decrease in muscle mass and strength. It occurs due to a decrease in muscle cells due to aging and lack of activity. The human body is made up of about 600 muscles. These muscles make up half of your body weight. Muscles are formed by gathering tens of thousands of muscle fibers (muscle cells). Muscle fibers increase in size as we grow, but their number decreases with age. Functionality gradually declines. Muscle mass loss is a natural phenomenon associated with aging and can be slowed down with appropriate exercise and nutrition.

Sarcopenia is caused by low physical activity, malnutrition, environmental factors, disease, inflammation, mitochondrial abnormalities, and hormonal changes. It is reported that there are also genetic differences. Once sarcopenia occurs, it quickly worsens. When muscle loss becomes severe due to sarcopenia, the ability to store energy decreases and you feel tired easily. As your basal metabolic rate decreases, your weight changes frequently and you gain weight easily. In other words, sarcopenia not only causes a direct decrease in muscle strength as part of a metabolic disease, increasing the risk of death due to a decrease and disability in various body functions, but also increases the risk of death due to decreased metabolism and decreased immunity, as well as high blood pressure, diabetes, arthritis, obesity, etc. It can also cause an increase in the prevalence of metabolic diseases such as cancer.

In a situation where there is no definite treatment drug to completely cure sarcopenia, there is a need for the development of new materials with low side effects and high economic value for various therapeutic compositions that can prevent or treat sarcopenia.

Currently, the treatment and best prevention method is proper exercise and nutrition. Aerobic exercise and strength training should be combined. Protein intake is also important. Leucine, which is important for building muscle, cannot be produced in the body and must be consumed through food. Eggs are a representative food rich in leucine. Milk, bananas, nuts, etc. are also high in leucine. It is also helpful to consume foods rich in vitamin D such as salmon, tuna, cheese, and mushrooms.

Meanwhile, probiotics, when consumed in appropriate amounts, are effective in alleviating lactose intolerance, activating and regulating immunity, alleviating allergic reactions and inflammation, reducing blood cholesterol, suppressing colon cancer, atopic dermatitis, Crohn's disease, diarrhea, candida infection and A general term for microorganisms that have a beneficial effect on the host's health through reduction of clinical symptoms of urethral infection and competitive inhibition of pathogens (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 useful effects in the animal digestive tract and have useful effects on the host animal, such as improving the intestinal flora. Strains used as probiotics include Lactobacillus, Enterococcus, Bifidobacterium, Bacillus, Clostridium, Saccharomyces, and Aspergillus . The mechanism of action of probiotics is normalization of intestinal flora, production of antibiotics, competition for nutrients 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 production of decay products and toxins, These include digestive enzyme production, vitamin synthesis, and immune stimulation.

Early on, in Europe and Japan, strains such as L. rhumnosus GG (Valio, Finland) and L. casei Shirota (Yakult, Japan) were isolated to develop probiotics with excellent functions, and various strains were tested for their wide range of physiological activities. Research is actively underway. Likewise, in Korea, research is actively underway to isolate strains derived from fermented foods such as kimchi and sauces, animals, and humans and use them as probiotics, but securing the strains is the most important key.

Bacillus velezensis , also known as a probiotic, is a spore-forming bacillus that secretes over 20 types of enzymes to recycle nutrients. It is an excellent source of vitamins B1, B2, and K and has cholesterol-lowering activity in vivo. It is known to improve physiological activities such as stimulating IgG production and inhibiting the proliferation of Caco-2 colon cancer cells, strengthening immune function, and aiding digestion. In addition, it has the function of inhibiting the growth of pathogenic bacteria such as typhoid bacteria, paratyphus bacteria, erythrobacteria, and cholera bacteria, and has been used in the treatment of various intestinal diseases.

Accordingly, while trying to solve the problem of sarcopenia, the present inventors completed the present invention by confirming that the Bacillus belegensis strain significantly improves sarcopenia in vivo.

The present invention was developed in response to the above-described needs, and the present inventors first discovered that the Bacillus velezensis strain alleviates sarcopenia, and developed a method for preventing sarcopenia containing the strain or its culture fluid as an active ingredient. Or a microbial preparation for improvement, a method for producing a microbial preparation for preventing or improving sarcopenia comprising culturing the strain, a probiotic active composition containing the strain or its culture medium as an active ingredient, pharmaceutical composition, health The present invention was completed by confirming that it can be applied as a functional food composition, drinking water additive, and feed composition.

In order to achieve the object of the present invention, the present invention provides a Bacillus belegensis strain that has the effect of preventing or improving sarcopenia.

The Bacillus belegensis strain was isolated from soil in Gunsan, Jeollabuk-do. The isolated strain was identified through 16S rDNA sequence analysis and was confirmed to be Bacillus velezensis (KCTC13417).

The term 'sarcopenia' used in the present invention refers to a disease in which muscle protein decomposition occurs more than protein synthesis, resulting in a continuous decrease in muscle mass.

The term “prevention” used in the present invention may refer to any act of suppressing or delaying sarcopenia by administering the composition for preventing or improving sarcopenia according to the present invention to an individual.

As used herein, the term “improvement” may mean any action that reduces at least the degree of a parameter related to the condition being treated, such as a symptom.

The Bacillus belegensis strain showed high oxidative stress inhibition effect (Table 1) and antioxidant activity (Table 2), and was confirmed to have a very strong muscle loss inhibition effect (Table 3, Figure 1), showing excellent physiological activity, In particular, the potential of biological materials applicable to sarcopenia was confirmed.

In addition, the present invention provides a microbial agent for preventing or improving sarcopenia comprising the above strain or its culture medium as an active ingredient.

The microbial agent for preventing or improving sarcopenia of the present invention can be manufactured using Bacillus belegensis strain as an active ingredient. The microbial preparation for preventing or improving sarcopenia according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste, dust, scattering material or granule, but is not limited thereto.

Additionally, the present invention provides a method for producing a microbial agent for preventing or improving sarcopenia, comprising culturing the strain. The strain of the present invention can be cultured according to methods commonly used in the art.

Additionally, the present invention provides a probiotic active bacterial composition containing the above strain or its culture medium as an active ingredient. In a general characteristic test, the strain of the present invention showed a survival rate of more than 50.8% in artificial gastric fluid adjusted to pH 2.0 (Table 4), and in resistance to artificial bile acid, it showed a survival rate of more than 50.8% in a 1.0% Oxgall (artificial bile) solution. It showed a survival rate of over 52.2% (Table 5) and a high heat resistance of about 78.4% when exposed to 70°C for 10 minutes (Table 6), confirming that it has excellent characteristics as a probiotic live cell.

In addition, the present invention provides a pharmaceutical composition for prevention, improvement or treatment containing the above strain or its culture medium as an active ingredient.

The pharmaceutical composition for preventing, improving or treating sarcopenia according to the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier and provided as food, medicine, feed additive, drinking water additive, etc. there is.

As used in the present invention, the term 'pharmaceutically acceptable carrier' may mean a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound.

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

In addition, if necessary, other common additives such as antioxidants, buffers, and/or bacteriostatic agents can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. can be additionally added to form solutions such as aqueous solutions, suspensions, emulsions, etc. It can be formulated and used in dosage form, pills, capsules, granules, or tablets.

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

The pharmaceutical composition of the present invention can be used as a single preparation, or can be prepared and used as a combination preparation by additionally containing a drug known to have a recognized sarcopenia treatment effect, and is formulated using a pharmaceutically acceptable carrier or excipient. It can be manufactured in unit dosage form or by placing it in a multi-dose container.

In another aspect, the present invention provides a method for preventing or improving sarcopenia, comprising administering the pharmaceutical composition for preventing or improving sarcopenia to a subject. As used in the present invention, the term “individual” may refer to any animal, including humans, that has developed or is likely to develop sarcopenia.

The prevention or treatment method of the present invention may specifically include administering the composition in a pharmaceutically effective amount to an individual who has developed or is at risk of developing sarcopenia.

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

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

As used in the present invention, the term "administration" means introducing the pharmaceutical composition of the present invention into a patient by any appropriate method, and the route of administration 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 can be manufactured into various dosage forms depending on the desired administration method.

The frequency of administration of the composition of the present invention is not particularly limited, but may be administered once a day or divided into multiple doses.

In addition, the present invention provides a composition for health functional food that can prevent or improve sarcopenia, comprising the strain or its culture medium as an active ingredient. Since ingestion of the strain of the present invention has an effect of improving sarcopenia, it can be provided as a health food for preventing or improving sarcopenia using it as an active ingredient. For example, these health foods for preventing or improving sarcopenia can be consumed for the prevention and treatment of muscular diseases including muscle atrophy, muscle strength loss, etc.

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

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. These include alcoholic beverages and vitamin complexes.

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

The food composition may include a foodologically acceptable carrier.

The food composition may be a health functional food. Functional food is the same term as food for special health use (FoSHU), and refers to food with high medical or medical effects that has been processed to efficiently exhibit bioregulatory functions in addition to supplying nutrients. , the food can be manufactured in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to achieve useful effects in preventing or improving sarcopenia.

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

The health functional food can be manufactured by a method commonly used in the industry, and can be manufactured by adding raw materials and components commonly added in the industry. In addition, unlike general drugs, it is made from food, so it has the advantage of not having any side effects that may occur when taking the drug for a long time, and it can be highly portable.

According to another embodiment of the present invention, the present invention provides a quasi-drug composition for preventing or improving sarcopenia containing the above strain or its culture medium. The composition of the present invention can be added to a quasi-drug composition for the purpose of preventing or treating sarcopenia.

According to another embodiment of the present invention, the present invention provides the strain or its culture medium.

Provided is a drinking water additive for preventing or improving sarcopenia, including: The drinking water additive of the present invention can be used by separately preparing a composition containing the strain or its culture medium in the form of a drinking water additive and mixing it with drinking water, or by adding it directly when preparing drinking water. The drinking water additive of the present invention may be in a liquid or dry form, and preferably in a dried powder form. The drying method for producing the drinking water additive of the present invention in dried powder form is not particularly limited, and methods commonly used in the art can be used. The drinking water additive of the present invention may additionally include other additives as needed. Non-limiting examples of the additives that can be used include binders, emulsifiers, and preservatives added to prevent the quality of drinking water from deteriorating; amino acids, vitamins, enzymes, probiotics, and flavoring agents added to increase the utility of feed or drinking water. , non-protein nitrogen compounds, silicate agents, buffers, colorants, extractants or oligosaccharides, and may additionally include feed mixtures, etc. These may be used singly or two or more types may be added together.

Since the Bacillus belezensis strain of the present invention is very effective in preventing, improving, and treating sarcopenia, the Bacillus velgensis strain and its culture medium are health functional foods requiring prevention or improvement of sarcopenia or pharmaceuticals requiring treatment. It can be very useful as a feed additive or animal medicine to improve sarcopenia in livestock or companion animals.

Figure 1 shows the effect of treatment with Bacillus belegensis culture medium to inhibit protein reduction caused by Dexamethasone (Dex).

Hereinafter, the present invention will be described in detail through examples and experimental examples. However, the following examples and experimental examples only illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[Example 1]

<Preparation of Bacillus belegensis cells and culture medium>

Bacillus bellegensis (KCTC13417) was inoculated into Brain heart infusion (BHI, Difco Laboratories, U.S.A.) medium, cultured with shaking at 30°C for 48 hours to grow the strain, then centrifuged to obtain the strain and culture supernatant, respectively. It was used as a sample in the following experimental example.

[Experimental Example 1]

<Test of muscle loss inhibition ability of Bacillus belegensis culture medium>

<1-1> Experiment method

① Resistance test to H ₂ O ₂

Bacillus belegensis strain was inoculated and cultured in BHI broth medium ^, centrifuged ( 7,000 was suspended in Immediately mix 500 μL of 0.5 mM H ₂ O ₂ solution with an equal amount of cell suspension and react for a total of 5 hours at 60-minute intervals, then centrifuge again (20,000× g , 1 min, 4 min.) to remove remaining H ₂ O ₂ ℃) and the pellet was recovered. The pellet was resuspended in buffer, diluted step by step, plated on BHI agar (30°C, 24 hours), the number of colonies produced was measured, and the reduction rate relative to the initial number of bacteria was shown.

② Antioxidant activity test by DPPH method

Antioxidant substances form complexes by donating electrons or hydrogen to free radicals, and 1,1-diphenyl-2-picyryl hydrazyl (DPPH) is an antioxidant. Since it receives electron hydrogen from a substance to form an irreversibly stable molecule, antioxidant activity can be measured from electron donating ability (Seo, Y.-H., Kim, I.-.J., Min , H.-K. and Park, S.-U. 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 (DPPH) was measured by dissolving 6 mg of DPPH in 100 mL of ethanol and then dissolving 6 mg of DPPH in 100 mL of ethanol. 200 μL each of the Bacillus bellegensis strain culture supernatant and sample were added to 1000 μL (mL distilled water), shaken for 10 seconds, left for 10 minutes, and then the absorbance was measured at 528 nm.

③ NO production inhibition effect by LPS

Raw 264.7 macrophages were distributed at 1×10 ⁴ /well in a 96 well plate using DMEM medium containing 10% FBS and cultured for 24 hours. After 24 hours, the attached cells were treated with culture medium at concentrations of 2.5% and 5% for 1 hour, then treated with LPS (100 ng/mL) and cultured for 20 hours. After 20 hours, the supernatant was taken and the absorbance was measured at 540 nm using Geiess reagent (0.2% N-(1-naphthy) ethylene diamine solution:0.2% sulfanilamide solution = 1:1). Inhibitory activity was evaluated by comparing with the group treated only with LPS.

Separately, in order to confirm whether the NO inhibitory activity was due to the cytotoxicity of the culture medium, to measure the amount of NO production, the supernatant was taken and the remaining culture medium was added with tetrazolium bromide salt (MTT, Sigma, St.Louis, MO). , USA) the final concentration of the solution was 500 μg/mL and cultured for about 4 hours, then the MTT solution was removed and each well was treated with 100 μL of DMSO to dissolve all fomazan produced in the wells, and then analyzed at 570 nm. Absorbance was measured. At this time, the survival rate (%) was calculated by calculating the ratio of the average absorbance of the culture medium-treated group to the average absorbance of the group treated only with LPS.

④ Inducing muscle cell culture and differentiation

C2C12 myoblasts were cultured at 37°C and 5% CO ₂ using DMEM growth medium containing 10% FBS and 100 unit/mL antibiotics. To differentiate C2C12 myoblasts into myotubes, differentiation medium containing 2% horse serum was used.

⑤ Test for the inhibition effect of protein reduction induced by Dexamethasone (Dex)

When differentiation from myoblast to myotube is induced, the number of muscle fibers increases due to fusion between cells and the diameter of the muscle fiber becomes thicker. However, when muscle atrophy is induced, the number and diameter of muscle fibers decrease through the breakdown of muscle proteins, resulting in a decrease in overall muscle mass. To construct this muscle atrophy cell model, C2C12 cells that had been sufficiently differentiated were treated with Dex and an increase in the expression of E3 ligase MuRF1 was confirmed. Under the same culture conditions, Bacillus belegensis culture medium and Dex were treated together to confirm muscle fiber diameter and MuRF1 protein expression.

<1-2> Experiment results

① Resistance test to H ₂ O ₂

The results showing the resistance of Bacillus belegensis culture medium to oxidative stress induced by H ₂ O ₂ are shown in Table 1 below.

Resistance test results for H ₂ O ₂ Reaction time with H ₂ O ₂ 0 1 hours 3 hours 5 hours Inhibition rate (%) 0 98.2± ^1.5c 45.2± ^2.6a 85.6± ^1.1b

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

As a result of the oxidative stress resistance evaluation, after 1 hour of reaction, the number of bacteria increased by about 2 times compared to the initial number, showing an inhibition rate of almost 99%, and 3 hours after the reaction, the inhibition rate was about 45.2%, and 5 hours after the reaction, the inhibition rate was approximately 99%. When elapsed, the inhibition rate was as high as 85.6%. Resistance to oxidative stress of lactic acid bacteria was measured by cell survival rate according to reaction time, and showed a cell survival rate of about 85.6% at 5 hours of reaction, showing similar resistance to the previously reported lactic acid bacterium L. brevis GK55 isolated from mustard kimchi. It was confirmed that this is believed to increase resistance by detoxifying active oxygen in the culture medium or 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 to determine the antioxidant activity of the Bacillus bellegensis culture medium.

Antioxidant activity (DPPH radical scavenging ability) evaluation results culture medium live bacteria dead bacteria Vitamin C DPPH radical scavenging ability (%) 44.8±2.2 45.2±3.2 41.7±2.9 100

The DPPH radical scavenging ability of Bacillus belegensis culture and live and dead cells was measured using BHI broth as a control, and was found to be 44.8, 45.2, and 41.7% in culture, live, and dead cells, respectively. Various ROS and toxic products, including free radicals, generated during the oxidation process damage cells and biologically active molecules in vivo. A high DPPH radical scavenging ability means that it has antioxidant activity due to its high ability to reduce or cancel out free radicals. Although it is lower than the positive control group Vitamin C, it is similar to the radical scavenging ability of other lactic acid bacteria reported so far. Therefore, it can be said that Bacillus belegensis culture medium and live and dead cells have antioxidant power, and based on this, antioxidant functional products can be developed.

③ NO production inhibition effect by LPS

Raw 264.7 macrophages were treated with LPS to evaluate the inhibitory effect of Bacillus belegensis culture medium on NO produced by inducing iNOS. Table 3 shows the NO production rate along with the cell survival rate indicating cytotoxicity.

Inhibition of NO production and cell survival rate by LPS NO production rate Cell survival rate (%) control group 0 ^100d LPS treatment group 29.6± ^1.4c 72.8± ^1.3a LPS+culture medium 2.5% 14.6± ^1.1b 79.2± ^1.2b LPS+culture medium 5% 10.1± ^2.5ab 82.1±1.9 ^B.C. LPS + culture medium 10% 6.7± ^2.1a 85.9± ^1.2c

Different English letters ( ^ad ) for the NO production inhibition value and cell viability value indicate statistical significance (p<0.05).

NO production increased by LPS was reduced in a concentration-dependent manner in the Bacillus belegensis culture medium, and cell survival rate was also increased, showing that the inflammatory response caused by LPS was improved without cytotoxicity.

④ Confirmation of protein reduction inhibition effect

As shown in Figure 1, when muscle atrophy was induced by treating sufficiently differentiated C2C12 cells with Dex, the expression of MuRF1 protein, an E3 ligase, increased, and the same protein decreased, resulting in a decrease in myotube diameter of about 42.6%. However, as a result of treatment with Bacillus belegensis culture medium, MuRF1 expression increased by Dex decreased in a concentration-dependent manner, and myotube diameter was also reduced to about 24.3%, confirming that muscle atrophy caused by Dex was recovered. In other words, the Bacillus bellegensis culture medium reduced the expression of MuRF1 protein, an E3 ligase that increased due to Dex treatment, thereby inhibiting muscle fiber atrophy and restoring myotube diameter.

[Experimental Example 2]

<Test to evaluate the characteristics of Bacillus belegensis as a live bacterium>

<2-1> Experiment method

① Test strain

To determine the basic characteristics of Bacillus belegensis as a probiotic, acid resistance, bile resistance, and heat resistance were measured. At this time, Bacillus subtilis (ATCC23857) was used as a comparison strain purchased from American Type Culture Collection (ATCC, USA).

The antibacterial activity of Bacillus belegensis is effective against 4 types of bacteria ( Salmonella enterica serovar Typhi (ATCC19430), Escherichia coli O157:H7 (ATCC 43895), Staphylococcus aureus (ATCC25923) , Listeria monocytogenes (ATCC 19113)), and 1 type of yeast ( Candida albicans ATCC 24433) and one type of mold ( Aspergilus fumigatus ATCC 96918) were investigated using the paper disk method. The bacteria and fungi were purchased and used from ATCC.

② Resistance (acid resistance) test to artificial stomach acid

Artificial gastric acid is prepared by inoculating 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, incubating for 2 hours, plating on plate medium, and incubating at 37°C for 24 hours. The cells were cultured and the number of viable bacteria was counted.

③ Resistance (bile resistance) test to artificial bile fluid

Artificial bile was prepared and used by adding Oxgall to BHI Broth at concentrations of 0.3, 0.5, and 1.0%, respectively. A 1% suspension of 8.0 log CFU/mL was inoculated into the artificial bile prepared above, cultured for 2 hours, smeared on a plate medium, and cultured at 37°C for 24 hours to count the number of viable bacteria.

④ Heat resistance test

The comparative strain and the Bacillus belegensis strain were cultured at 36.5°C for 20 hours (using BHI broth) to produce at least 7 log CFU/mL, then treated for 10 minutes at temperature conditions of 50, 60, and 70°C (using a waterbath), and plated. After plating on the medium, the cells were cultured at 37°C for 24 hours and the number of growing bacteria was counted.

⑤ Antibacterial test

The Bacillus belegensis strain was inoculated into BHI broth and cultured at 36.5°C for about 20 hours to produce at least 7 log CFU/ml. Then, the culture medium was centrifuged, the supernatant was collected, filtered, and prepared as a stock test sample solution. The test sample solution was concentrated 20-fold using a vacuum concentrator, and then 10-fold and 5-fold concentrates were prepared using BHI, respectively, and then used for measuring antibacterial activity below.

Four types of tested bacteria Sal. enterica serovar Typhi, E. coli O157:H7, Sta. aureus, Lis. monocytogenes was plated on BHI (Difco Laboratories) plate medium at an amount of 7 log CFU/ml or more, and the paper was added to the Bacillus bellegensis strain culture test sample solution prepared at the four concentrations (stock solution, 5-fold, 10-fold, and 20-fold concentrate) prepared above. After sufficiently soaking the disk and attaching it to the center, it was incubated at 37°C for 20 hours and the diameter of the clear zone created around the paper disk was measured to determine the presence and extent of antibacterial activity. The test yeast , Can. albicans was plated on sabouraud dextrose (Difco Laboratories) plate and tested against the fungus Asp. fumigatus ^was smeared on PDA plate medium at 1 The diameter was measured to determine the presence and extent of antibacterial properties.

<2-2> Experiment results

① Resistance to artificial stomach acid (acid resistance) test results

The results of testing the survival rate of Bacillus belegensis strains under artificial gastric acid conditions are shown in Table 4 below.

Comparison of artificial gastric acid resistance of Bacillus belegensis strains Strain name Number of viable bacteria (Log CFU/mL) pH 6.0 pH 4.0 pH 2.0 Bacillus velezensis 7.91 ± 0.04 ^c 4.89 ± 0.03 ^{b, Y} 3.56 ± 0.13 ^a,Y Bacillus subtilis 7.94 ± 0.03 ^c 3.67 ± 0.25 ^b,X 1.46 ± 0.75 ^a,X

Among the resistance values for artificial gastric acid, the different English letters ( ^ac ) in the results for each strain indicate statistical significance (p<0.05).

Among the resistance values to artificial gastric acid, different English letters ( ^XY ) in the results between strains indicate statistical significance (p<0.05).

As a result of the test, the growth of the test strain, Bacillus belegensis, increased to 7.91 log CFU/mL under artificial gastric acid conditions of pH 6, and 4.89 log CFU/mL under artificial gastric acid conditions of pH 4, showing a survival rate of about 69.8%. , under conditions of pH 2, the survival rate was approximately 50.8% at a level of 3.56 log CFU/mL. Meanwhile, the comparison strain, Bacillus subtilis (ATCC23857), showed a survival rate of about 20.8% at a level of 1.46 log CFU/mL under artificial gastric acid culture conditions of pH 2, showing that the test strain, Bacillus belegensis, has higher artificial gastric acid resistance. appear.

② Resistance (bile resistance) test to artificial bile fluid

When Bacillus belegensis strains were prepared and treated with artificial bile fluid (Oxgall) at different concentrations, the results of testing the survival rate under artificial bile fluid conditions are shown in Table 5 below.

Resistance (bile resistance) test results of Bacillus belegensis strains to artificial bile Strain name Number of viable bacteria (Log CFU/mL) oxgall 0.3% oxgall 0.5% oxgall 1.0% Bacillus velezensis 7.82 ± 0.04 ^c 4.54 ± 0.07 ^b 4.18 ± 0.06 ^a,Y Bacillus subtilis 7.32 ± 0.04 ^c 4.38 ± 0.08 ^b 3.41 ± 0.08 ^a,X

Among the resistance values to artificial bile acids, the different English letters ( ^ac ) in the results for each strain indicate statistical significance (p<0.05).

Among the resistance values to artificial bile acids, different English letters ( ^XY ) in the results between strains indicate statistical significance (p<0.05).

As a result of the test, the Bacillus belegensis strain was 7.82 log CFU/mL, about 97.75%, under oxgall 0.3% artificial bile solution conditions, and 4.54 log CFU/mL, about 56.7%, 1.0% under 0.5% artificial bile solution conditions. showed a survival rate of about 52.2% at a level of 4.18 log CFU/mL. The comparison strain, Bacillus subtilis, showed a survival rate of about 42.6% at a level of 3.41 log CFU/mL under 1.0% artificial bile acid conditions, and the test strain, Bacillus belegensis, had a higher survival rate than Bacillus subtilis (ATCC23857), a representative Bacillus genus strain. It showed bile acid resistance.

③ Heat resistance test

The heat resistance test results of Bacillus belegensis strains are shown in Table 6 below.

Comparison of heat resistance of Bacillus belegensis strains Strain name Number of viable bacteria (Log CFU/mL) 50℃ 60℃ 70℃ Bacillus velezensis 8.82 ± 0.04 ^c,Y 7.93 ± 0.02 ^b,Y 5.49 ± 0.06 ^a,Y Bacillus subtilis 7.91 ± 0.03 ^c,X 6.63 ± 0.07 ^b,X 4.78 ± 0.04 ^a,X

Among the results for the heat resistance test, the different English letters ( ^ac ) of the results for each strain indicate statistical significance (p<0.05).

Among the results for the heat resistance test, the different English letters ( ^XY ) in the results between strains indicate statistical significance (p<0.05).

As a result of the test, the growth of both the test strain, Bacillus belegensis, and the comparison strain, Bacillus subtilis, increased at 50°C, and when heated at 60°C for 10 minutes, the test strain had 7.93 log CFU/mL and the comparison strain had 6.63 log. CFU/mL is indicated. When the test strain was exposed to 70°C for 10 minutes, it showed a survival rate of about 78.4% at 5.49 log CFU/mL, and the comparison strain showed a survival rate of about 68.2% at 4.78 log CFU/mL at 70°C. Bacillus velegensis was found to have somewhat high heat resistance and was confirmed to have excellent properties as a probiotic bacteria.

④ Antibacterial test

The antibacterial test results for six types of pathogens of Bacillus belegensis strains are shown in Table 7 below.

Antibacterial test results test strain antibacterial activity Culture stock solution 5x concentrate 10x concentrate 20x concentrate Salmonella enterica serovar Typhi (ATCC 19430) - - + ++ Escherichia coli O157:H7
ATCC 43895 - - + ++ Staphylococcus aureus
(ATCC 25923) - + ++ +++ Listeria monocytogenes
(ATCC 19113) + ++ +++ +++ Candida albicans
(ATCC 24433) - - - + Aspergilus fumigatus
(ATCC 96918) - + + ++

'-' or '+' in Table 1 above is determined based on the diameter size of the live bacterial inhibition ring, where '-' means no antibacterial or antifungal activity, and '+' means that the diameter size of the inhibition ring is less than 14.0 mm. '++' means that the low-resolution ring diameter size is in the range of 14.0 - 17.0 mm, and '+++' means that the low-resolution ring diameter size exceeds 17.0 mm.

When the culture supernatant of the Bacillus belegensis strain was concentrated 5-, 10-, and 20-fold, the culture stock solution showed strong growth inhibitory activity against L. monocytogenes , and S. aureus was shown at the 5-fold concentrate. Sal. Enterica serover Typhi and E. coli O157:H7 showed a growth inhibition effect in 10-fold concentrate. Can. albicans strain showed the lowest inhibitory effect, and Asp. fumigatus , inhibitory activity was found starting from 10-fold concentration. There are many reports on strains of the genus Bacillus that exhibit antibacterial activity, which are mainly due to the production of antibacterial substances such as bacteriocins. Although there are differences in activity, Bacillus velegensis is thought to have antibacterial and antifungal activities.

[Statistical analysis]

Statistical analysis of the experimental results was performed using SPSS 13.0 statistical software, 'Independent-Sample-T-Test', and was expressed as the average value ± standard difference. In addition, multiple comparisons were expressed using the LSD method, P>0.05: the difference is not clear, P<0.05: the difference is clear.

<Preparation Example 1> Preparation of microbial preparation

Preparations for preventing or improving sarcopenia containing Bacillus velgensis are manufactured by drying Bacillus velgensis using a conventional freeze-drying method and then mixing it with excipients or encapsulating it.

<Preparation Example 2> Preparation of fermented milk

Fermented milk is produced using Bacillus belegensis of the present invention according to a method commonly performed in the art. Prepare a culture solution ( ¹

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

<3-1> Manufacturing of beverages

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

<3-2> Preparation of powder

Prepare a powder by mixing 20 mg of Bacillus belegensis freeze-dried powder (1×10 ⁹ CFU/g), 100 mg of lactose, and 10 mg of talc and filling it in an airtight envelope.

<3-3> Preparation of tablets

Tablets are prepared by mixing 10 mg of Bacillus belegensis freeze-dried powder (1×10 ⁹ CFU/g), 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate and compressing them according to a normal tablet manufacturing method. .

<3-4> Manufacturing of capsules

Mix 10 mg of Bacillus belegensis 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 according to a typical capsule manufacturing method and fill it into a gelatin capsule. Manufactures capsules.

<3-5> Preparation of mixed powder

1 g of Bacillus belegensis freeze-dried powder (1×10 ⁹ CFU/g), appropriate amount of vitamin mixture (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, appropriate amount of mineral mixture (ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, 15 mg of potassium phosphate monobasic, 55 mg of calcium phosphate dibasic, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were mixed according to a typical health functional food manufacturing method, and then granules were prepared using the usual method. It can be used to manufacture health functional food compositions. The composition ratio of the above vitamin and mineral mixture is a mixture of ingredients relatively suitable for health functional foods in a preferred production example, but the mixing ratio may be modified arbitrarily.

<Preparation Example 4> Preparation of feed composition

The feed composition is made by mixing 45 g of corn, 30 g of soybean meal, 15 g of wheat, 4 g of beef tallow, 3 g of molasses, 2 g of calcium phosphate, 0.4 g of limestone, 0.2 g of salt, and 5 g of Bacillus bellegensis supplied in the form of apo. It is manufactured.

<Production Example 5> Production of sports lotion

Sports lotion contains 0.50% by weight of Bacillus belegensis culture medium, 3.00% by weight of glycerin, 0.10% by weight of carbomer, 0.05% by weight of xanthan gum, 3.00% by weight of 1,3-butylene glycol, according to a method commonly performed in the art. Polyglyceryl-3 methylglucose distearate 1.50% by weight, glyceryl stearate 0.50% by weight, cetylaryl alcohol 0.30% by weight, jojoba oil 3.00% by weight, liquid paraffin 1.60% by weight, squalane 3.00% by weight, dimethicone 0.40% by weight %, 0.20% by weight of tocopheryl acetate, 0.10% by weight of triethanolamine, 0.1% by weight of menthol, preservatives, fragrance, trace amounts of pigment, and remaining weight% of purified water.

As described above, the Bacillus velezensis strain and its culture medium, which have the effect of preventing or improving sarcopenia, have high antioxidant properties, oxidative stress resistance, and anti-inflammatory effects, and are effective against Dex, which causes muscle loss. It reduces the increased expression of MuRF1 in a concentration-dependent manner and regenerates myotubes to recover muscle atrophy, making it a health functional food that requires prevention or improvement of sarcopenia, medicines that require treatment, quasi-drugs such as sports lotions, and those for livestock or companion animals. It can be widely used industrially, such as as a feed additive to improve bone health or in veterinary medicine.

Claims (9)

delete A microbial preparation for preventing or improving sarcopenia containing Bacillus velezensis KCTC 13417 strain or its culture fluid as an active ingredient, which has the effect of preventing or improving sarcopenia,
A microbial agent for preventing or improving sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. A method for producing a microbial agent for preventing or improving sarcopenia, comprising culturing Bacillus velezensis KCTC 13417 strain, which has the effect of preventing or improving sarcopenia, comprising:
The method of producing a microbial agent for preventing or improving sarcopenia is due to the strain or its culture reducing MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. A probiotic active composition containing Bacillus velezensis KCTC 13417 strain or its culture fluid as an active ingredient, which has the effect of preventing or improving sarcopenia. A pharmaceutical composition for preventing, improving or treating sarcopenia, comprising as an active ingredient Bacillus velezensis KCTC 13417 strain or its culture, which has the effect of preventing or improving sarcopenia,
A pharmaceutical composition for preventing, improving or treating sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. . A health functional food composition for preventing or improving sarcopenia containing Bacillus velezensis KCTC 13417 strain or its culture fluid as an active ingredient, which has the effect of preventing or improving sarcopenia,
A health functional food composition for preventing or improving sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. A quasi-drug composition for preventing or improving sarcopenia containing Bacillus velezensis KCTC 13417 strain or its culture fluid as an active ingredient, which has the effect of preventing or improving sarcopenia,
A quasi-drug composition for preventing or improving sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. A drinking water additive for preventing or improving sarcopenia containing Bacillus velezensis KCTC 13417 strain or its culture fluid as an active ingredient, which has the effect of preventing or improving sarcopenia,
A beverage additive for preventing or improving sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers. A feed additive for preventing or improving sarcopenia containing Bacillus velezensis KCTC 13417 strain or its culture solution as an active ingredient, which has the effect of preventing or improving sarcopenia,
A feed additive for preventing or improving sarcopenia, wherein the strain or its culture medium reduces MuRF1 protein expression, thereby inhibiting atrophy of muscle fibers and restoring the diameter of muscle fibers.