KR20220153141A - Probiotics mixture with prevention, improvement or treatment effect on metabolic disease and uses thereof - Google Patents

Abstract

The present invention relates to: complex probiotics comprising a Bacillus bellegensis strain, a Bacillus licheniformis strain, an Enterococcus faecium strain, and a Lactobacillus plantarum strain having prevention, improvement, or treatment effects on metabolic diseases; a microbial agent for prevention, improvement, or treatment of metabolic diseases containing the complex probiotics or a culture medium thereof as an active ingredient; a probiotic bioactive composition containing the strain or a culture medium thereof as an active ingredient; a pharmaceutical composition containing the strain or a culture medium thereof as an active ingredient; a food (health functional food) composition containing the strain or a culture medium thereof as an active ingredient; a drinking water additive composition containing the strain or a culture medium thereof as an active ingredient; and a feed additive containing composition containing the strain or a culture medium thereof as an active ingredient. The complex probiotics of the present invention can be very usefully used for health functional food required for preventing or improving metabolic diseases or pharmaceuticals required for treatment thereof and feed additives or drugs for animals for improving metabolic diseases of livestock or companion animals.

Description

Probiotics mixture with prevention, improvement or treatment effect on metabolic disease and uses thereof {Probiotics mixture with prevention, improvement or treatment effect on metabolic disease and uses thereof}

The present invention is Bacillus velezensis , Bacillus licheniformis , Enterococcus faecium , and Lactobacillus plantarum, which have metabolic disease prevention, improvement, or treatment effects. It relates to a composite probiotic using as a main probiotic, and more specifically, the composite probiotic having an effect of preventing, ameliorating, or treating metabolic diseases caused by an obese diet, preventing metabolic diseases containing the composite probiotic or its culture medium as an active ingredient, A composite probiotic preparation having an improvement or therapeutic effect, a method for producing a composite probiotic preparation having an effect of preventing, improving, or treating metabolic diseases caused by an obese diet, including culturing the strain, the composite probiotic preparation or a culture medium thereof It relates to a probiotic probiotic composition containing as an active ingredient, a pharmaceutical composition, a food (health functional food) composition, a drinking water additive and a feed additive.

Metabolic diseases are obesity, diabetes, and hypercholesterolemia caused by abnormal metabolic processes such as insulin resistance, abnormal sugar and lipid metabolism, and decreased immune function due to rapid increase in adipose tissue due to excessive accumulation of surplus nutrients and chronic inflammation. , hyperlipidemia, arteriosclerosis, osteoporosis, sarcopenia, etc. are collectively referred to (Koopman, R. J., Swofford, S. J., Beard, M. N., and Meadows, S. E. 2009. Obesity and metabolic disease. Prim Care 36, 257-270).

Among them, obesity appears as an early symptom of representative metabolic diseases. The main cause of obesity is the conversion and accumulation of surplus nutrients due to high-calorie food intake and low metabolic activity to fat. induce the occurrence of

Anti-obesity drugs known so far are largely divided into satiety boosters, fat absorption inhibitors, and antipsychotic appetite suppressants according to the mechanism of action. There are ExoliseTM (Atopharma, France), but they cause steatorrhea, intestinal gas, abdominal distension, and bowel incontinence, and are accompanied by side effects such as heart disease, respiratory disease, and nervous system disease, and their efficacy is low. has a problem.

To treat obesity, it is to reduce body weight by burning excess fat or to improve metabolic imbalance. Current treatment of obesity aims not only to lose weight, but also to improve metabolic abnormalities by removing factors that cause cardiovascular disease at an early stage. In addition, studies on suppressing obesity by controlling dietary intake and energy consumption have been actively conducted.

Obesity disrupts metabolic and immune homeostasis as well as the balance of the gut microbiome. It has been reported that altered gut microbiota in obesity has an important link between metabolism and immune dysfunction. For example, insulin resistance is induced by toxins in serum taken up in 'leaky gut' and increases systemic inflammation. Gut dysbiosis disrupts immune homeostasis and promotes the development of various inflammatory and metabolic diseases. Obesity-associated microbiome changes lead to increased intestinal permeability and circulating endotoxin levels, resulting in low-grade systemic inflammation. How members of the microbial community interact with each other and with the host immune system to prevent or protect against obesity is under extensive research. Certain bacterial classes (texa) (eg, Akkermansia muciniphila ) have been shown to modulate these symptoms by reducing endotoxemia and promoting the accumulation of regulatory T cells in adipose tissue.

Various bacteria make up the microflora in the intestine, and a high-fat diet causes abnormalities in these microbiota and increases the concentration of lipopolysaccharide (LPS), resulting in low-level chronic inflammation. This inflammatory environment causes metabolic abnormalities It is known to induce obesity and type 2 diabetes (Hersoug, L. G., Moller, P., and Loft, S. 2016. Gut microbiota-derived lipopolysaccharide uptake and trafficking to adipose tissue: implications for inflammation and obesity. Obes Rev 17 , 297-312;Moreno-Navarrete, J. M., Ortega, F., Serino, M., Luche, E., Waget, A., Pardo, G., Salvador, J., Ricart, W., Fruhbeck, G. , Burcelin, R., and Fernandez-Real, J. M. 2012. Circulating lipopolysaccharide-binding protein (LBP) as a marker of obesity-related insulin resistance. Int J Obes (Lond) 36, 1442-1449).

Probiotic bacteria, including lactic acid bacteria, which are known to contribute to the health of the host through the regulation of the immune and metabolic systems, suppress the abnormality of the intestinal microbial flora caused by a high-fat diet, thereby lowering LPS-induced inflammation, thereby reducing insulin resistance in obesity and type 2 diabetes. has been reported to improve It has been reported that probiotics can achieve beneficial effects by regulating the secretion of hormones such as adiponectin, which plays an important role in improving insulin resistance in the intestine and fat cells, and glucagon-like peptide 1 (GLP-1), which helps to feel full. In another study, when lactic acid bacteria were administered to rats on a high-fat diet, it was reported that fatty liver and hyperlipidemia were improved due to increased fatty acid oxidation (Saez-Lara, M. J., Robles-Sanchez, C., Ruiz-Ojeda, F. J., Plaza-Diaz, J., and Gil, A. 2016. Effects of Probiotics and Synbiotics on Obesity, Insulin Resistance Syndrome, Type 2 Diabetes and Non-Alcoholic Fatty Liver Disease: A Review of Human Clinical Trials. Int J Mol Sci 17, 589-595; Yeonju Lee. 2017. Inhibitory effect of lactobacillus strains on the induction of metabolic abnormalities in high-fat diet rats. Chonnam National University).

In the past, fat cells have been recognized as cells that simply store surplus energy in the body in the form of neutral fat and act as a shock absorber from the outside. However, it has recently been known that fat cells play an important role in maintaining homeostasis and regulating energy metabolism as an endocrine organ that secretes adipocytokines that regulate fasting, metabolism, and insulin sensitivity. As remodeling of fat cells, a major tissue of energy metabolism, has been confirmed to improve energy imbalance through increased energy consumption, interest has recently increased as a preventive and therapeutic strategy for metabolic diseases such as obesity, diabetes, and fatty liver. The relationship between intestinal microbes and energy metabolism regulatory signal pathways is continuously being identified, and the regulatory mechanism for the association between intestinal microbes and metabolic abnormalities is identified by discovering key regulators of the host that mediate energy metabolism control by gut microbes. Research is actively under way.

On the other hand, probiotics reduce lactose intolerance, activate and regulate immunity, alleviate allergic reactions and inflammation, reduce blood cholesterol, inhibit colorectal cancer, atopic dermatitis, Crohn's disease, diarrhea, Candida infection and It is a collective term for microorganisms that exert a beneficial effect on the host's health by reducing clinical symptoms of urinary tract infections and competitively inhibiting 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 act usefully in the digestive tract of animals and act usefully to 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, nutrient competition to prevent the establishment of pathogens, production of organic acids by lowering the pH, inhibition of harmful enzymes such as β-glucuronidase, azoreductase and nitroreductase, inhibition of decay products and toxin production, These include production of digestive enzymes, synthesis of vitamins, and stimulation of the immune system.

While studying the physiological activity characteristics of probiotics, the present inventors found that Bacillus velezensis , Bacillus licheniformis , Enterococcus faecium and Lactobacillus plantarum strains It was confirmed that they had different physiological activity effects.

Therefore, the present inventors found that when these strains were combined in a specific ratio, they exhibited antioxidant and anti-inflammatory activities, and suppressed obesity by increasing energy metabolism. The present invention was completed by discovering that a mixture of strains having an optimal mixing ratio was prepared to prevent, ameliorate, or treat metabolic diseases in laboratory animals in which obesity was induced by feeding a high-fat diet.

Publication No. 10-2019-0122083

The present invention has been derived from the above needs, and the present inventors first discovered that the complex probiotics of Bacillus bellegensis, Bacillus licheniformis, Enterococcus faecium and Lactobacillus plantarum strains improve metabolic diseases. .

Therefore, it is intended to provide a microbial preparation for the prevention, improvement, or treatment of metabolic diseases comprising the composite probiotic or its culture medium as an active ingredient.

In addition, a method for preparing a composite probiotic preparation for preventing, improving, or treating metabolic diseases including culturing the composite probiotic, a probiotic bioactive composition containing the strain or its culture medium as an active ingredient, a pharmaceutical composition, It is intended to provide 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 composite probiotic of Bacillus bellegensis, Bacillus licheniformis, Enterococcus faecium and Lactobacillus plantarum strains having a preventive, ameliorative, or therapeutic effect on metabolic diseases do.

The Bacillus bellegensis strain was isolated from soil in Gunsan, Jeollabuk-do. As a result of identifying the isolated strain by performing 16S rDNA sequencing, it was identified as Bacillus velezensis (KCTC13417).

The Bacillus licheniformis strain was isolated from conventional soybean paste purchased from a public market in Gunsan, Jeollabuk-do. As a result of performing a 16S rDNA gene sequence test for strain identification, it was confirmed as Bacillus licheniformis (KCTC1918).

The Enterococcus faecium strain was used after being isolated from kimchi and identified. As a result of the 16S rDNA test, it was confirmed as Enterococcus faecium (KCTC13225).

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

As used herein, the term 'metabolic disease' refers to abnormal metabolic processes and immune function, such as insulin resistance, abnormal sugar and lipid metabolism, etc. It refers to clinical symptoms that collectively refer to diseases such as obesity, diabetes, hypercholesterolemia, hyperlipidemia, arteriosclerosis, osteoporosis, and sarcopenia, which are caused by low blood pressure.

As used herein, the term "inhibition" refers to all activities of inhibiting or delaying some or all clinical symptoms of metabolic diseases by administering the complex probiotic preparation for preventing, improving, or treating metabolic diseases according to the present invention to a subject. can mean

As used herein, the term "improvement" may refer to any activity that at least reduces a parameter associated with a condition to be treated, for example, the severity of a symptom.

As used in the present invention, the term "treatment" may refer to any action that improves or benefits the symptoms of a metabolic disease by administering the complex microbial preparation and its culture solution of the present invention to a subject suspected of having a metabolic disease.

More specifically, the present invention is a complex microbial preparation for the prevention, improvement, or treatment of metabolic diseases of the present invention using Bacillus bellegensis, Bacillus licheniformis, Enterococcus faecium and Lactobacillus plantarum strains as active ingredients can be manufactured.

At this time, the mixing ratio of the composite probiotic preparation is Bacillus bellegensis, Bacillus licheniformis, Enterococcus physeum and Lactobacillus plantarum strains at a mixing ratio of 1-3: 1-3: 1-3: 1-3 can be mixed.

More specifically, the mixing ratio of the composite probiotic preparation may be a mixture of Bacillus bellegensis, Bacillus licheniformis, Enterococcus phyceum, and Lactobacillus plantarum strains at a mixing ratio of 3:3:3:1.

The composite probiotics may be grown by individually culturing each strain, freeze-drying, and mixing at a constant ratio, or by mixing strains and then jointly culturing to proliferate. More preferably, the strains are individually cultured and finally at a constant mixing ratio Mixing can further ensure the effect.

The complex probiotics have high antioxidant activity (50%), oxidative stress resistance (82.3%), anti-inflammatory activity against inflammation caused by LPS, enhancement of innate immunity through activation of neutrophils and macrophages, and atopic dermatitis in in vitro experiments. improvement, bone loss inhibition activity through osteoblast differentiation enzyme activity (121.6%) and osteoclast differentiation cell inhibition (76.8%), muscle loss inhibition through MuRF1 gene expression inhibition, triglyceride production reduction (58.6%) and high cholesterol assimilation ability (78.2%), it is judged to have an excellent physiological activity by showing an anti-obesity effect.

In addition, the composite probiotic has an antibacterial effect against bacteria or fungi. Specific examples of the bacteria or fungi include Salmonella enterica serovar Typhi, Escherichia coli , Staphylococcus aureus , Listeria monocytogenes , Candida albicans ( Candida albicans ), Aspergillus fumigatus ( Aspergilus fumigatus ), and the like, but is not limited thereto.

In addition, the complex probiotics of the present invention suppressed rapid weight gain (from 66% to 44.2%) due to feeding of high-fat diet in metabolic disease improvement experiments using metabolic syndrome animal models, and cholesterol levels and triglycerides (307.8 mg/dl to 157.2 mg/dl), significantly lowered arteriosclerosis index and cardiovascular risk index, improved inflammatory response inhibitory factors and inflammatory markers, and significantly increased appetite suppressant factors. , it was confirmed that it has a preventive, ameliorative, or therapeutic effect on metabolic diseases.

The microbial preparation for preventing, improving, or treating metabolic diseases according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oil dispersion, paste, dust, scattering material, or granule, but is not limited thereto.

In addition, the present invention provides a method for preparing a composite probiotic for the prevention, improvement, or treatment of metabolic diseases, which includes the step of culturing the composite probiotic. The method for culturing the complex 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 bioactive composition comprising the composite probiotic or its culture medium as an active ingredient. The composite probiotics of the present invention showed relatively high acid resistance of 64.8% or more, bile acid resistance of 64.1%, and a high survival rate of 89.8% or more even after heat treatment at 70 ° C. for 10 minutes, and it was confirmed that they had very excellent characteristics as probiotic live bacteria.

In addition, the present invention provides a pharmaceutical composition for the prevention, improvement, or treatment of metabolic diseases comprising the complex probiotic or its culture medium as an active ingredient.

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

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 while not stimulating living organisms.

The type of the 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, 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 bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to form main solutions such as aqueous solutions, suspensions, and emulsions. It may be formulated into a dosage form, pill, capsule, granule or tablet for use.

The administration method of the pharmaceutical composition for preventing, improving, or treating metabolic diseases according to the present invention is not particularly limited, and may be 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 preventing, improving, or treating metabolic diseases of the present invention may be prepared in various formulations according to the desired administration method.

The pharmaceutical composition of the present invention can be used as a single formulation, or can be prepared and used as a combination formulation by further including a drug known to have a recognized therapeutic effect on metabolic diseases, and can be formulated using a pharmaceutically acceptable carrier or excipient. It can be prepared in unit dose form or introduced into a multi-dose container.

As another aspect, the present invention provides a method for preventing or treating a metabolic disease comprising administering to a subject a pharmaceutical composition for preventing, improving, or treating the metabolic disease.

As used herein, the term "subject" may refer to all animals, including humans, who have or may have a metabolic disease.

Specifically, the preventive or therapeutic method of the present invention may include administering the composition in a pharmaceutically effective amount to a subject having or at risk of developing a metabolic disease.

In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with 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 , an amount of 7 log CFU to 10 log CFU, preferably 8 log CFU / g to 9 log CFU / g, can be divided and administered once or several times a day. However, for the purpose of the present invention, the suitable total daily amount of the composite probiotic may be determined by treatment within the scope of correct medical judgment, and may be administered once or divided into several times. However, for the purposes of the present invention, a specific therapeutically effective amount for a specific patient is determined by the type and extent of the 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 condition, It is preferable to apply differently according to various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together with or concurrently used with a specific composition, and similar factors well known in the medical field.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without causing side effects in consideration of all of the above factors, 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 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 administration method 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 formulations 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 the target disease comprising the complex strain or its culture medium as an active ingredient.

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

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

When using the composition 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 conventional methods.

The food composition may include a food chemically 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 and medical effects processed to efficiently display bioregulatory functions in addition to nutrient supply. , The food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, and pills in order to obtain useful effects for preventing or improving metabolic diseases.

At this time, the content of the composite probiotic or its culture medium contained in the food is not particularly limited thereto, 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 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 can be prepared by adding raw materials and components commonly added in the art during the manufacture. In addition, unlike general drugs, there is an advantage in that there is no side effect that may occur when taking a 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 provides a drinking water additive for preventing and improving metabolic diseases, including the complex probiotic or its culture medium. The drinking water additive of the present invention may 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 directly adding it during preparation of 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 if necessary.

Non-limiting examples of the usable additives include binders, emulsifiers, and preservatives added to prevent deterioration of drinking water quality; There are amino acids, vitamins, enzymes, probiotics, flavors, non-protein nitrogen compounds, silicates, buffers, coloring agents, extractants, or oligosaccharides added to increase the effectiveness of feed or drinking water. can be further included. These may be used singly or two or more may be added together.

The Bacillus velezensis strain, Bacillus licheniformis , Enterococcus faecium and Lactobacillus plantarum complex probiotics of the present invention prevent metabolic diseases, Since the improvement or treatment effect is very excellent, the complex probiotics and their culture solutions are health functional foods requiring treatment or prevention of metabolic diseases, medicines requiring treatment, prevention, improvement, or treatment of metabolic diseases in livestock or companion animals. It can be used very usefully for feed additives for animals or veterinary medicines.

Figure 1 visually shows the effect of feeding the complex probiotic of the present invention to inhibit the increase in body weight during a 5-week feeding of experimental animals (6-week-old male C57BL/6 mice) whose obesity was induced by a high-fat diet.

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

[Experimental Example 1]

<Preparation of test strain>

The test strains of the present invention, Bacillus bellegensis (KCTC13417), Bacillus licheniformis (KCTC1918), Enterococcus phyceum (KCTC13225) and Lactobacillus plantarum (KCTC3108) strains were prepared and Brain heart infusion (BHI, Difco), respectively. Laboratories, U.S.A.), inoculated into a liquid medium, and cultured with shaking at 30° C. for 48 hours to propagate the strain, and then centrifuged to obtain the strain. The strain was lyophilized, prepared into powder, stored frozen at -20 ° C, and used in the following experiments.

[Examples 1 to 5] Preparation of composite probiotic mixture

Powders of test strains in cryopreservation were prepared by weighing 30% by weight of Bacillus bellegensis, 30% by weight of Bacillus licheniformis, 30% by weight of Enterococcus phyceum, and 10% by weight of Lactobacillus plantarum, and then selected excipients and powders After mixing well using a mixer, 2 g of each was dispensed into sterilized wrapping paper and sealed to prepare a composite probiotic test sample of the present invention. The excipients were configured by minimizing the types of excipients in order to grasp the physiological efficacy of the test strain mixture and to avoid as much as possible that other ingredients other than the strains affect the active effect. When the strain mixture strain is 50% by weight, the excipients are corn starch (20% by weight), anhydrous sugar (16% by weight), dextran (12% by weight), sorbitol (1% by weight), fish collagen (12% by weight) ) was selected.

[Experimental Example 2] <Physiological activity effect of complex probiotics>

<2-1> Experiment method

① Antioxidant activity test by DPPH method

Example 1 was inoculated and cultured in BHI broth, centrifuged (7,000× g , 20 minutes, 4° C.), and then strain precipitates and culture supernatants were separated. The culture supernatant was stored in a refrigerator after filtering with 0.45 μL filter. The strain precipitate was washed three times with PBS solution and suspended in the same buffer according to the cell number of about 10 ⁸ CFU/mL. The suspension was separated into two parts, one part was stored in a refrigerator and used as a live cell sample. The other half was placed in a test tube with a lid, put in a water bath previously fixed at 90 ° C, heat treated for 15 minutes to kill, and then centrifuged to obtain dead cells. Live cells and dead cells were used after diluting with the same amount of distilled water immediately before the test. The hydrogen donating ability for DPPH was measured by adding 200 μL each of the culture supernatant, live cell, and dead cell of the test strain to 1,000 μL of 100 M DPPH solution (DPPH 6 mg, EtOH 100 mL, distilled water 100 mL), shaking for 10 seconds, and then shaking for 10 seconds. After allowing to stand for a minute, the absorbance was measured at 528 nm.

② Resistance test for H ₂ O ₂

Example 1 was inoculated and cultured in BHI broth, centrifuged (7,000× g , 20 minutes, 4°C), washed three times with PBS solution, and suspended in the same buffer to adjust the cell number to about 10 ⁸ CFU/mL. . 500 μL of 0.5 mM H ₂ O ₂ solution and the same amount of cell suspension were immediately mixed _and reacted for a total of ₅ hours at 60 minute intervals, followed by centrifugation again (20,000 × g , 1 minute, 4 °C) to recover the pellet. The pellet was resuspended in buffer, then diluted step by step, and plated on BHI agar (30°C, 24 hours) to measure the number of colonies generated, and the reduction rate for the initial number of bacteria was shown.

③ Anti-inflammatory effect test

The culture supernatant of Example 1 was measured for LPS-induced NO production rate and macrophage survival rate in Raw 264.7 macrophages. That is, Raw 264.7 macrophages were divided into DMEM medium containing 10% FBS in a 96 well plate at 1×10 ⁴ /well and cultured for 24 hours. After 24 hours, the attached cells were treated with 2.5% and 5% culture medium for 1 hour, then treated with lipopolysaccharide (LPS, 100 ng/mL) and cultured for 20 hours. After 20 hours, the supernatant was taken and absorbance was measured at 540 nm using Geiess reagent (0.2% N-(1-naphthy) ethylene diamine solution: 0.2% sulfanilamide solution = 1:1). The inhibitory activity was evaluated by comparing with the group treated only with LPS. Separately, in order to confirm that the NO inhibitory activity is due to the cytotoxicity of the culture medium, the supernatant was taken to measure the amount of NO production, and a tetrazolium bromide salt (MTT, Sigma, St.Louis, MO, USA) solution was added to the remaining culture medium at the final concentration. After incubation for about 4 hours after making it 500 μg / mL, the MTT solution was removed, DMSO was treated with 100 μL of each well to dissolve all the fomazan produced in the well, and absorbance was measured at 570 nm. At this time, the survival rate (%) was calculated by obtaining the ratio of the average absorbance of the culture solution-treated group to the average absorbance of the LPS-only treated group.

④ Evaluation of immune activity

In order to confirm the immunological activity of Example 1, the immunological activity of live cells obtained using the above method and the culture supernatant was measured. The immune activity confirmation experiment was conducted in two ways. First, the oxidative burst was prepared by mixing dog whole blood with cell medium (RPMI1640) in a ratio of 1:1 to confirm the activity of neutrophils, and then 25 μL of viable cells or metabolites were added to a 1.5 mL tube. 100 μL of whole blood was added, mixed well, and allowed to stand for 30 minutes. 25 μL of 50 μM PMA (blank: PBS) and 25 μL of 30 μg/mL DHR123 solution (blank: PBS) were added and allowed to stand for 15 minutes. Remove the supernatant after centrifugation (250 × g , 5 minutes). After resuspending the pellet, add 1 mL RBC lysis buffer and let it stand in the dark for 7 minutes. After washing in FACS buffer (PBS, 1% FBS, 0.1% sodium azide), it was floated in 0.3 mL FACS buffer and the mean fluorescence intensity (MFI) value of rhodamine 123 was measured by flow cytometry. Phagocytosis activity was measured to determine the phagocytic activity of macrophages. 100 μL of whole blood and 100 μL of live bacteria or metabolites were added to a 1.5 mL tube, mixed well, and allowed to stand for 30 minutes. After centrifugation (200 × g , 5 minutes), the supernatant was discarded, the pellet was resuspended, and 100 μL of Fluorosphere beads solution (Polysciences, Inc) was added and allowed to stand for 30 minutes. After centrifugation (250 × g , 5 minutes), the supernatant was removed, and 1 mL RBC lysis buffer was added and left in the dark for 7 minutes. After washing with FACS buffer, it was floated again in 0.3 mL FACS buffer and the MFI (mean fluorescence intensity) of the beads was measured by flow cytometry.

⑤ Atopic dermatitis improvement effect test

As the composite probiotic culture medium of the present invention inhibited NO production by LPS (Table 3), the expression of Nuclear factor of activated T-cells 5 (NFAT5) and cytokines, which are inflammatory transcription factors, were examined to investigate the mechanism. saw. HaCat cells, human keratinocytes, were dispensed into a 6-well plate in DMEM medium containing 10% FBS, and when grown to about 80%, 2.5% and 5% concentrations of the culture medium were treated for 1 hour. After 1 hour, 10 ng/mL TNF-α and 500 μM H ₂ O ₂ were treated and cultured for 24 hours.

⑥ Osteopenia inhibitory effect test

In order to examine the effect of the composite probiotic culture medium of the present invention on the differentiation of osteoblasts and osteoclasts, the cell proliferation rate of each cell and the activity level of ALP, an activity indicator enzyme, were measured. MC3T3-E1 cells were cultured in α-MEM medium with 10% FBS and 1% antibiotics at 37°C and 5% CO2. To induce bone cell differentiation, 10 mM β-glycerol phosphate (Sigma Co., St. Louis, MO, USA) and 50 μg/mL of vitamin C were added as a differentiation induction medium, and the medium was changed every 3 days. did

The cultured MC3T3-E1 cells were adjusted to 1×10 ⁴ cells/well, dispensed into a 96 well plate, cultured for 24 hours, exchanged with a differentiation induction medium, cultured for 24 hours, and then treated with the culture medium. After 3 days, after washing with PBS, 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 absorbance was measured at 405 nm. ALP activity was derived by measuring p -nitrophenol (p-NP) generated from p-NPP, preparing a standard graph for p-NP, and then comparing the activity with the control group.

The tibia of a 4-5 week old ICR mouse was removed, both ends were cut, and bone marrow cells were collected by passing through α-MEM essential medium, treated with 50 ng/μL M-CSF (macrophagy-colony stimulation factor), and cultured for 24 hours. cultured for a while. After washing the non-adherent cells with α-MEM, they were dispensed to 5×10 ⁴ cells/well in 96 wells and cultured in α-MEM medium supplemented with 50 ng/μL M-CSF for 3 days. Thereafter, 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. Cells were divided into 96 wells to be 5×10 ⁴ cells/well, treated with differentiation factors and culture medium, and cultured for 4 days. After removing the medium, washing it with PBS, dispensing 100 μL of the substrate solution (1.36 mg/mL 4-nitrophenyl phosphate disodium salt, 10 mM tartate, 50 mM citrate buffer, pH 4.6) to the cells fixed with 10% formaldehyde and incubating at 37℃. reacted for 30 minutes. After the reaction, the enzyme reaction solution was transferred to a new plate, and the reaction was stopped with 0.1 N NaOH, and absorbance was measured at 405 nm. TRAP activity was expressed as a percentage of the absorbance of the sample relative to the absorbance of the control.

⑦ Muscle loss suppression effect test

When differentiation is induced from myoblasts to myotubes, the number of muscle fibers increases and the diameter of muscle fibers becomes thicker due to fusion between cells. However, when muscle atrophy is induced, the number and diameter of muscle fibers decrease through the breakdown of muscle protein, resulting in a decrease in overall muscle mass. C2C12 myoblasts were cultured in DMEM growth medium containing 10% FBS and 100 unit/mL antibiotics at 37°C and 5% CO ₂ conditions. To differentiate C2C12 myoblasts into myotubes, a differentiation medium containing 2% horse serum was used.

To construct an amyotrophic cell model, sufficiently differentiated C2C12 cells were treated with Dex to confirm an increase in the expression of the E3 ligase MuRF1. Under the same culture conditions, the test strain culture medium having a concentration of 10 ⁸ CFU/mL and Dex were treated together to confirm muscle fiber diameter and MuRF1 protein expression.

⑧ Obesity inhibitory effect test

3T3-L1 preadipocytes were cultured in DMEM medium (Gibco, BRL) containing 10% FBS at 37°C and 5% CO ₂ conditions. After culturing 3T3-L1 preadipocytes in a 6-well plate for cell culture to confluent state, differentiation medium (MDI) containing 10 μg/mL insulin, 0.1 mM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthin (IBMX) and cultured for 2 days, and then exchanged with medium containing 10 μg/mL insulin every 2 days. 3T3-L1 cells were fully differentiated into adipocytes and then converted into brown adipocytes by treatment with 50 nM Triodothyronine (T ₃ ). The T ₃ treatment group was used as a positive control, and the culture medium of the test strain was treated with T3 to measure the expression of PPARα and PGC1α, which are energy metabolism promoters, through western blot analysis.

⑨ Cholesterol assimilation ability (absorption inhibition ability)

In order to confirm the cholesterol assimilation (absorption inhibition) effect of the composite probiotic of the present invention, it was measured using the method of Rudel and Morris. Specifically, the culture solution of the trial product sample of the composite probiotic of the present invention was added to MRS liquid medium supplemented with 0.5% (w/v) oxgall (Sigma-Aldrich, USA) and 0.1 g/L water-soluble cholesterol (Sigma-Aldrich). % (v/v) was inoculated, followed by anaerobic culture at 37°C for 24 hours (GasPak EZ Anaerobe Container System, Becton Dickinson and company, USA), followed by centrifugation (9,950× g , 5 minutes, 4°C). After adding 2 mL of 33% (w/v) KOH and 3 mL of 95% (v/v) ethanol aqueous solution to 1 mL of the supernatant obtained through the centrifugation, they were mixed well for 1 minute, and in a water bath at 60 ° C. After holding for 10 minutes, it was cooled with cold running water, 5 mL of hexane was added, mixed well, and 1 mL of distilled water was added to resuspend the mixture, and then left at room temperature for 10 minutes for layer separation. 3 mL of the separated upper layer of hexane was transferred to a clean test tube and concentrated with nitrogen gas. 4 mL O-phthaladehyde reagent (0.5 mg O-phthalaldehyde/glacial acetic acid 1 mL, Sigma-Aldrich) was added to the concentrated sample. After adding and mixing well, the mixture was allowed to stand at room temperature for 10 minutes, and 2 mL of concentrated sulfuric acid was slowly added and mixed well. After 10 minutes, the absorbance was measured at 550 nm.

⑩ Statistical analysis

Statistical analysis of the experimental results was performed using the 'Independent-Sample-T-Test method', a software dedicated to statistics in SPSS 13.0, and expressed as the average value ± standard difference. In addition, multiple comparison was indicated by the LSD method, and *, #: p<0.05, **, ##: p<0.01 means that the level of difference is indicated.

<2-2> Experimental results

① Antioxidant activity by DPPH method

Antioxidants donate electrons or hydrogen to free radicals to form complexes, and 1,1-diphenyl-2-picyryl hydrazyl (DPPH) is an antioxidant. Since an irreversibly stable molecule is formed by receiving electron hydrogen from a material, the antioxidant activity can be measured from the 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).

Table 1 shows the results of examining the antioxidant activity by measuring the DPPH radical scavenging ability of the culture medium, live cells and dead cells of the composite probiotic sample of the present invention. BHI broth was used as a negative control, and Vitamin C was used as a positive control, and when the value of Vitamin C was set to 100, the value of the obtained result was expressed as a percentage. The raw materials used as excipients in the development of the prototype composite probiotic of the present invention are prepared so as not to affect the antioxidant activity of the strain mixture, and the results obtained can be evaluated as the activity of the strain mixture.

Antioxidant activity (DPPH) radical scavenging ability evaluation result Complex probiotic sample type ¹⁾ culture supernatant probiotics dead cells Example 1 42.1±3.6 53.6±1.8 ^* 52.4±1.5 ^*

^{*, #} : p<0.05, ^{**, ##} : p<0.01

¹⁾ The result value represents the percentage value of the result obtained when the value of Vitamin C, a positive control, is set to 100.

Antioxidant activity of the composite probiotic sample of the present invention showed DPPH radical scavenging activity of 42.1% in the culture medium, 53.6% in viable cells, and 52.4% in dead cells. In previous studies, the DPPH radical scavenging ability of B. lichenifomis , the main raw material strain of the composite probiotic of the present invention, was very high, ranging from 54.9% (culture medium) to 64.2% (live cells) and 65.7% (dead cells). It is believed to act as a major causative agent.

② Resistance to oxidative stress induced by H ₂ O ₂

The results of testing the resistance to oxidative stress induced by H ₂ O ₂ of the composite probiotic of the present invention are shown in Table 2.

During the respiratory metabolism process for energy production in vivo, a certain amount of oxygen creates harmful substances called reactive oxygen species (ROS), that is, free radicals and hydrogen peroxide (H ₂ O ₂ ), which are highly reactive, cause oxidative stress. Oxidative stress induces harmful biochemical reactions in living organisms and is known to cause various vascular diseases including atherosclerosis, mutations, cancer, neurodegenerative diseases, immune dysfunction and aging. Meanwhile, it has been reported that some microorganisms can detoxify reactive oxygen species by producing antioxidant enzymes such as Mn-SOD and Cu-SOD, and decompose hydroxyl radicals by producing antioxidants such as NADH, NADPH, glutathione, and uric acid. Lac. plantarum, Lac. Lactic acid bacteria that produce SOD, such as lactis , have metal ion chelating activity, so they inhibit lipid peroxidation with high antioxidant power and are known to be effective in improving inflammation of the large intestine.

Results of resistance test against oxidative stress induced by H ₂ O ₂ Strain growth rate (%) ¹⁾ 1 h 3 h 5 h Example 1 38.4±1.2 62.7±1.6 ^* 82.3±1.5 ^**

^{*, #} : p<0.05, ^{**, ##} : p<0.01

^{1) The} result value is expressed as a percentage value based on the growth rate of the cells recovered after exposure to H ₂ O ₂ for a certain period of time based on the number of bacteria immediately before treatment.

After 1 hour of H ₂ O ₂ treatment, 38.4% of oxidative stress resistance was exhibited, and as time passed, growth was restored, showing 62.7% after 3 hours and 82.3% of scavenging effect after 5 hours. Differences over time were found to have statistical significance. The effect of resistance to oxidative stress of the composite probiotic of the present invention is the raw material strains B. lichenifomis , B. velezensis, Lac. It is thought to be due to the active effect of strains such as plantarum .

③ Anti-inflammatory effect test

LPS increases the production of NO by iNOS from L-arginine in immune cells. Excessive production of NO intensifies the inflammatory response and is known to be involved in the mechanism of development of various diseases including chronic inflammatory diseases, autoimmune diseases, and cancer. have. On the other hand, since RAW264.7 macrophages are sensitive to LPS stimulation and produce NO well, they are used to evaluate anti-inflammatory effects. Table 3 shows the NO production rate and Raw 264.7 macrophage survival rate when raw 264.7 macrophages were treated with LPS to produce iNOS-induced NO when 5% of the composite probiotic culture medium of the present invention was treated. Negative control refers to untreated, and positive control indicates that the sample culture medium was not treated with LPS alone. The NO production inhibition index (NO inhibition index) shows the difference between the cell viability for the amount of NO production and the untreated value of 1. The lower the value, the lower the inhibitory effect, and the higher the index value, the higher the inhibitory effect.

In Raw 264.7 macrophages, the effect of treatment with the complex probiotic of the present invention on LPS-induced NO production and cell viability NO production (%) survival rate (%) NO production inhibition index ¹⁾ voice control phrase 100 100 One positive control 127.3±3.2 ^* 71.4±2.7 ^* 0.441 ^* Example 1 109.1±1.8 ^# 82.7±0.1 ^# 0.736 ^#

^{*, #} : p<0.05, ^{**, ##} : p<0.01

^{1) The} NO production inhibition index (Inhibition Index) is the difference between the cell survival rate for the amount of NO production and the untreated value of 1.

The amount of NO production (127.3%) increased by LPS was reduced to 109.1% by treatment with the complex probiotic culture medium of the present invention, and at this time, the cell viability increased from 71.4% to 82.7%, and the NO production inhibition index also showed from 0.441 to 0.736. It was shown that the composite probiotic culture solution of the present invention improved the inflammatory response caused by LPS without cytotoxicity.

④ Evaluation of immune activity

Table 4 shows the results of testing the immunological activity of the live cells of the composite probiotic of the present invention and the culture supernatant.

Evaluation of immune activity of the prototype composite probiotic of the present invention Sample Neutrophil activity (%) Macrophage activity (%) probiotics culture supernatant probiotics culture supernatant voice control phrase 11.26±1.26 12.05±1.87 9.56±2.35 10.72±1.65 Positive control 1 678.24±18.74 ^* 681.06±23.32 ^* Positive control 2 52.3±3.69 ^* 54.36±2.74 ^* Example 1 445.14±11.57 ^# 98.73±12.36 ^# 490.71±21.31 ^# 289.94±26.11 ^#

^{*, #} : p<0.05, ^{**, ##} : p<0.01

When compared with the values of negative control (DHR) and positive control 1 (DHR+PMA) in the activity test for oxidative damage, the probiotics of the composite probiotic of the present invention were neutrophils more than 678.24% of the positive control and 681.06% of the culture supernatant. It did not increase the activity of 445.14% and 98.73%, respectively, confirming that the activity of the culture supernatant was lower than that of viable cells. In macrophage activity, the activity was highly increased in both viable cells and culture medium when compared to positive control 2 (beads). Live bacteria showed 490.71% activity, and metabolites showed 289.94% activity. It was observed that treatment with live cells activated neutrophils and macrophages more than treatment with metabolites in the culture medium.

⑤ Atopic dermatitis improvement effect test

<Reduction of inflammatory transcription factors NFAT5 and cytokines by LPS in Raw 264.7 macrophages>

Nuclear factor of activated T-cells 5 (NFAT5) has been identified as an important regulator of NFκB and is known as a major target for anti-inflammatory research as it is activated in inflammatory diseases such as rheumatoid arthritis, sclerosis, diabetes, and nephropathy.

Effect of treatment with the composite probiotic culture medium of the present invention on reducing the inflammatory transcription factor NFAT5 and cytokines by LPS in Raw 264.7 macrophages Test item ¹⁾ NFAT5 cytokines TNF-α IL-6 IL-1β voice control phrase - - - - Example 1 +++ +++ +++ +++

¹⁾ Indication of the activity of the test item - : no effect, +: weak (30% or less), ++: normal (31 - 70%), +++: strong (71-100%)

As the complex probiotic culture medium of the present invention inhibited NO production by LPS, the expression of NFAT5, an inflammatory transcription factor, and cytokines were observed to investigate the mechanism, and the results are shown in Table 5. As a result of the test, all prototype samples in Raw 264.7 macrophages inhibited LPS-induced NO production and suppressed the expression of inflammatory transcription factor NFAT5 and cytokines TNF-α, IL-6 and IL-1β.

<Reduction of skin inflammatory transcription factors and cytokines by TNF-α>

The anti-inflammatory activity of the test strains was identified in HaCat cells, which are human-derived skin keratinocytes, and a skin inflammatory model by TNF-α in skin cells similar to atopic dermatitis was established. The results of testing the anti-inflammatory effect on are shown in Table 6.

Effect of the composite probiotic culture solution and probiotics of the present invention on the reduction of skin inflammatory transcription factors and cytokines induced by TNF-α in HaCat cells Test item ¹⁾ NFAT5 cytokines IL-6 IL-1β culture supernatant probiotics culture supernatant probiotics culture supernatant probiotics voice control phrase - - - - - - Example 1 ++ +++ ++ +++ ++ +++

¹⁾ Indication of the activity of the test item - : no effect, +: weak (30% or less), ++: normal (31 - 70%), +++: strong (71-100%)

The complex probiotic of the present invention for TNF-α-induced dermatitis reduced the protein expression of NFAT5, an inflammatory transcription factor, and decreased the expression of IL-6 and IL-1β, which were inflammatory cytokines.

The protein expression of NFAT5 and p65, which was increased by TNF-α, is reduced, which results in a decrease in inflammatory cytokines such as IL-6 and IL-1β. That is, as with the anti-inflammatory effect of inhibiting NO production in Raw 264.7 macrophages when treated with the complex probiotic of the present invention (Table 3), the protein expression of NFAT5 and p65, which are inflammatory regulators in skin cells, was reduced. It is also thought to reduce gene expression of inflammatory cytokines.

⑥ Osteopenia inhibitory effect test

Osteoblasts have basic phosphatase (ALP) in their cell membranes. This enzyme is found in high concentrations in the cell outer membrane and calcified tissue, transports inorganic phosphate during the calcification process, regulates cell division or differentiation, and regulates osteoblasts. It is known as a marker of cell activity. Osteoclasts are differentiated from mononuclear macrophages and are generally regulated by M-CSF and TNF-related activation induced cytokine (TRANCE, RANKL). induce differentiation. As osteoclasts differentiate, they form multinucleated mature osteoclasts according to cell fusion, which attach to the bone surface and act to resorb bone. In addition, osteoclasts have TRAP and calcitonin receptors, and are active in acid production. Because of its characteristics, TRAP is used as a marker for osteoclasts.

The results of testing the effect of the culture supernatant of the composite probiotic of the present invention on the differentiation of osteoblasts and osteoclasts and the activity of ALP and TRAP enzymes, which are activity indicators for each cell, are shown in Table 7.

Effects of treatment of the culture solution of the prototype composite probiotic of the present invention on the proliferation of osteoblasts and osteoclasts and the activity of enzymes related to bone loss, ALP and TRAP Osteoblasts (%) Osteoclasts (%) growth rate ALP activity growth rate TRAP activity voice control phrase 100 100 100 100 Example 1 105.4±1.3 ^* 121.6±1.5 ^** 81.2±0.8 ^* 76.8±0.8 ^**

^{*, #} : p<0.05, ^{**, ##} : p<0.01

According to the treatment of the culture supernatant of the composite probiotic of the present invention, the osteoblast proliferation rate was 102.3% and the ALP enzyme activity was 116.3%, which was higher than that of the negative control, but the osteoclast proliferation rate was 84.6% and the TRAP enzyme activity was 82.1%. Contrary to the osteoblast differentiation growth rate, it was inhibited in all samples. The bone loss inhibitory effect of the composite probiotic of the present invention is found in Lac. It is thought that platarum inhibits the growth of osteoclasts by inhibiting the activity of TRAP enzyme, which induces osteoclast differentiation, and exhibits the effect of suppressing bone loss such as osteoporosis.

⑦ Muscle loss suppression effect test

In C2C12 myoblast cells, in order to measure the effect of the culture medium of the test strains to inhibit protein reduction in myoblasts, sufficiently differentiated C2C12 cells were treated with Dexamethasone (Dex) to confirm the increase in the expression of the E3 ligase MuRF1, and then under the same culture conditions , The muscle fiber diameter and MuRF1 protein expression were confirmed by treating the test strain culture medium having a concentration of 10 ⁸ CFU/mL and Dex together (Table 8).

Effect of treatment of the culture supernatant of the composite probiotic of the present invention on inhibition of protein reduction induced by Dexamethasone (Dex) Strain Items tested ¹⁾ MuRF1 Myotube diameter (%) control +++ 43.1±0.5 Example 1 - 28.7±0.6 ^**

^{*, #} : p<0.05, ^{**, ##} : p<0.01

¹⁾ Indicates the activity level of the test item - : None, + : Weak (less than 30%), ++ : Normal (31 - 70%), +++ : Strong (71-100%).

As a result of inducing muscular atrophy by treating fully differentiated C2C12 cells with Dex, MuRF1 protein expression increased and the protein decreased in the same way, resulting in a myotube diameter of about 42.6%. However, as a result of treating the culture supernatant of the complex probiotic of the present invention, the expression of MuRF1, which was increased by Dex, was completely reduced, and the myotube diameter was about 24.3%, which inhibited muscle fiber atrophy and restored the myotube diameter.

⑧ Obesity inhibitory effect test

Adipocytes can be divided into white adipocytes that store energy and brown adipocytes that, on the contrary, release energy sources as heat. Brown adipocytes are being studied as new targets as they have been reported to decompose body fat with heat. Recently, it was revealed that Beige cells similar to brown adipocytes exist in white adipose tissue (Kershaw & Flier, 2004). The differentiation process of brown adipocytes is carried out by multiple factors through several steps. In particular, PGC-1/PPARα induces the differentiation conversion from white adipocytes to brown adipocytes, and UCP-1, which consumes energy similarly to brown adipocytes, As it is expressed, it promotes energy metabolism from mitochondria. When 3T3-L1 preadipocytes differentiate into adipocytes, PPARγ, a key transcription factor regulating adipogenesis, is highly expressed in the late stage of differentiation, resulting in intracellular fat accumulation. It was tested whether the treatment of the culture supernatant of the complex probiotic of the present invention inhibits the formation of lipid droplets during the differentiation of 3T3-L1 preadipocytes into adipocytes. When 3T3-L1 preadipocytes are differentiated into adipocytes, the results of testing the effect of the treatment of the stock strain culture on the expression of obesity-related factors and the accumulation of fat globules (increase in TG content) are shown in Table 9.

Effect of treatment with the composite probiotic culture solution of the present invention on the triglyceride content and expression of obesity-related factors (PPARγ, PPARα, PGC1α, UCP-1) in 3T3-L1 cells Sample Test item ¹⁾ Triglycerides (%) transcription factor PPARγ PPARα PGC1α UCP-1 voice control phrase 17.6±0.08 - - - - positive control 100 +++ - - - Example 1 58.6±0.08 ^* - +++ +++ +++

^{*, #} : p<0.05, ^{**, ##} : p<0.01

¹⁾ Indication of activity of test items - : No effect, + : Weak (30% or less), ++ : Normal (31 - 70%), +++ : Strong (71-100%).

As a result of the test, the treatment of the culture supernatant of the composite probiotic of the present invention showed an anti-obesity activity. When the positive control (MDI) was 100%, the negative control (untreated) showed a TG production rate of 17.6%, and the composite probiotic of the present invention showed a triglyceride production rate of 58.6%. Since the treatment of the composite probiotic of the present invention reduced PPARγ expression similar to the result of triglyceride accumulation, the treatment of the composite probiotic of the present invention suppresses the expression of PPARγ, an adipogenic transcription factor, in 3T3-L1 preadipocytes, thereby reducing lipid droplet production It is thought that the differentiation of captives was suppressed.

As a result of the energy metabolism promoter PPARα/PGC1α and UCP-1 expression test, the fat storage factor PPARγ, energy metabolism promoter PPARα, PGC1α and UCP-1 were not all expressed in the non-treated group, but in the composite probiotic of the present invention, they were induced by T3. The expression of PPARα and PGC1α was increased according to sample treatment, which was confirmed to promote energy metabolism by promoting UCP-1 expression.

In the process of brown adipocyte differentiation, PGC-1/PPARα induces differentiation conversion from white adipocytes to brown adipocytes and promotes energy metabolism from mitochondria according to the expression of UCP-1.

⑨ Cholesterol assimilation ability (absorption inhibition ability)

The results of confirming the cholesterol assimilation effect of the prototype composite probiotics of the present invention are shown in Table 10.

Cholesterol assimilation ability (inhibiting ability) change by treatment of the composite probiotic culture solution of the present invention Sample Cholesterol assimilation (%) 0.5% Oxgall 0.5% combined bile salts voice control phrase 17.6±0.08 10.3±0.02 Example 1 78.2±0.15 ^** 53.7±0.09 ^**

^{*, #} : p<0.05, ^{**, ##} : p<0.01

Cholesterol assimilation (absorption inhibition) was 78.2% when cultured in BHI liquid medium supplemented with 0.5% Oxgall and 0.1 g/L water-soluble cholesterol, which is an artificial bile acid condition, and 0.5% (w /v) In the case of the condition where TDCA and 0.1 g/L water-soluble cholesterol were added, it was 53.7%. The cholesterol assimilation ability was different depending on the type of bile salt, and it was confirmed that cholesterol assimilation ability was higher in the cholesterol medium supplemented with oxgall than in the cholesterol medium supplemented with sodium taurocholic acid, a form of conjugated bile salt.

[Experimental Example 3]

<Examination of Metabolic Disease Improvement Effect of Complex Probiotics Using Metabolic Syndrome Animal Model>

<3-1> Experiment method

① Experimental animals and specifications

Experimental animals were 6-week-old male C57BL/6 mice purchased from Orient Bio Co., Ltd. (Sungnam, Korea), acclimatized for 1 week, divided into 3 groups, and then fed with a diet prepared as shown in Table 11 for 5 weeks.

Regular and high-fat diet plan normal diet high fat diet carbohydrate Corn starch 44.875 34.375 Sucrose 15 15 Cellulose 5 5 Fat Lard - 10 Corn oil 10 10 Cholesterol - 0.5 protein Casein 20 20 vitamin mix One One mineral mix 3.5 3.5 DL-methionine 0.3 0.3 Choline chloride 0.2 0.2 Taurocholic acid 0.125 0.125 Sum 100 100

Experimental animals were a normal control group fed with normal feed (negative control; n = 7), a high-fat diet group fed with 65% fat-containing feed (positive control; n = 7), and a high-fat-containing feed and a group fed with the complex probiotic of the present invention. (Example 1, n=7) and the like were divided into 3 groups. The composite probiotic of the present invention was administered orally at a dose of 50 mg/mouse once a day at a fixed time (pm 5:00). Body weight was measured during the feeding period and body weight changes were recorded.

② Blood collection and tissue extraction

Blood was collected from the abdominal vena cava after anesthesia using ether after the end of the experiment, and at the same time, a certain area of the right lobe of the liver was extracted. Blood was centrifuged at 3,000 rpm for 10 minutes to separate serum, and after cryopreservation at -80 ° C, it was used in subsequent experiments.

③ Effect on blood lipid metabolism

Cholesterol and triglyceride contents in serum were measured by an Enzymatic assay, high density lipoprotein cholesterol (HDL-C) contents by selective inhibition, and low density lipoprotein cholesterol (LDL-C) contents by an automatic biochemical analyzer using the principles of the Enzymetric method.

④ Effect on arterial stiffness index (AI) and cardiovascular risk index (CRF)

For the atherogenic index (AI), Fiordaliso's calculation method AI=([Total-C] -[HDL-C])/[HDL-C] was used (Fiordaliso et al., 1995). In addition, CRF (Cardiac Risk Factor), a cardiovascular risk index, was calculated by dividing the amount of total cholesterol by the amount of HDL according to Friedewald's calculation method (Friedewald et al., 1972).

⑤ Effects on liver and kidney function

Liver function was measured through alanine transaminase (ALT) and aspartate transaminase (AST) activities in serum, and renal function was measured through creatinine and blood urea nitrate (BUN) activities in serum.

⑥ Measurement of serum adiponectin and leptin and inflammatory markers

Serum concentrations of adiponectin and leptin were analyzed by ELISA, respectively. C-response protein (CRP), an inflammation-related indicator in serum, was measured for absorbance at 450 nm using a CRP ELISA kit.

⑦ Statistical analysis

Statistical analysis of the experimental results was performed using the 'Independent-Sample-T-Test method', a software dedicated to statistics in SPSS 13.0, and expressed as the average value ± standard difference. In addition, multiple comparison was indicated by the LSD method, and *, #: p<0.05, **, ##: p<0.01 means that the level of difference is indicated.

<3-2> Experimental results

① weight change

Table 12 shows the results of measuring the effect of feeding the complex probiotic of the present invention on weight gain in model animals fed a high-fat diet.

Effect of high-fat diet and feeding of the complex probiotic of the present invention on weight gain of laboratory animals during feeding weight (g) Weight gain (%) dietary intake
(g/day) before experiment after experiment voice control phrase 25.2±0.25 35.2±0.36 38.4 23.5±2.4 positive control 26.1±0.31 43.5±0.45 ^** 66.5 24.3±2.1 Example 1 25.7±0.2 37.2±0.31 ^## 44.2 24.1±1.9

*, #: p<0.05, **, ##: p<0.01

The negative control group (general feed group) showed a weight gain of 38.4% from 25.2 g at the beginning of the experiment to 35.2 g after the end of the experiment, and the high-fat diet group (positive control group) increased from 26.1 g at the beginning of the experiment to 43.5 g after the end of the experiment. A weight gain of 66.5% was recorded. The high-fat diet and the complex probiotic feeding group of the present invention (Example 1) showed a weight gain of 44.2% from 25.7 g to 39.2 g at the start of the experiment. Compared to the control group, the weight gain was 22.3% less. As shown in FIG. 1, it was confirmed visually that the feeding of the complex probiotic of the present invention inhibits the increase in body weight in laboratory animals induced to become obese with a high-fat diet. There was no significant difference in food intake in all three experimental groups.

② Effects of complex probiotic supplementation on blood lipid metabolism in high-fat diet experimental animals

The results of measuring the effect of feeding the complex probiotic of the present invention on blood lipid metabolism in model animals fed a high-fat diet are shown in Table 13.

Effects of high-fat diet and feeding of the complex probiotic of the present invention on changes in serum cholesterol and triglycerides of experimental animals during breeding Total Cholesterol (mg/dl) HDL-
Cholesterol (mg/dl) LDL-
Cholesterol (mg/dl) Triglycerides (mg/dl) voice control phrase 97.14±12.5 20.7±4.8 121.7±11.2 137.28±21.8 positive control 199.2±18.2 ^* 18.7±2.7 177.8±18.3 ^* 307.8±18.2 ^** Example 1 131.2±12.7 ^# 33.2±2.8 ^# 132.1±9.8 ^# 157.2±12.9 ^##

*, #: p<0.05, **, ##: p<0.01

When obesity is induced by a high-fat diet, insulin resistance and hyperinsulinemia are accompanied, and fatty acids flowing into the liver are increased, resulting in an increase in blood triglyceride concentration and cholesterol concentration. At this time, LDL-cholesterol accumulates on the walls of blood vessels and causes atherosclerosis that induces cardiovascular and cerebrovascular diseases, and HDH-cholesterol quickly transports LDL-cholesterol accumulated on the walls of blood vessels to the liver and decomposes it to lower blood levels. Can prevent arteriosclerosis.

As a result of measuring blood lipid metabolism according to the feeding of the complex probiotic of the present invention, the concentration of total cholesterol, LDL-cholesterol, and triglyceride in the blood, which can cause hyperlipidemia, was measured to be high in the positive control group. The lipid concentration of Example 1) was reduced similarly to that of the control group fed a normal diet, and was significantly lower than that of the positive control group. The total cholesterol concentration of the negative control group was measured as 97.1 mg/dL, whereas the positive control group significantly increased to 199.2 mg/dL. Example 1 (combined probiotic supplement of the present invention) was detected at 131.2 mg/dL, which was significantly lower than that of the positive control. This result was the same in LDH-cholesterol and triglyceride concentrations. However, HDL-cholesterol was 18.7 mg/dL in the positive control, lower than that of the negative control (20.7 mg/dL), but significantly higher in Example 1, 33.2 mg/dL.

② Effects of complex probiotic supplementation on blood arteriosclerosis index and cardiovascular risk index of high-fat diet experimental animals

Table 14 shows the results of measuring the effect of feeding the complex probiotic of the present invention on the blood arteriosclerosis index and cardiovascular risk index in model animals fed a high-fat diet.

Effects of high-fat diet and feeding of the complex probiotic of the present invention on changes in blood arteriosclerosis index and cardiovascular risk index of experimental animals during feeding arteriosclerosis index Cardiovascular Risk Index voice control phrase 3.69±0.02 4.7±0.1 positive control 9.65±0.08 ^* 10.6±0.12 ^* Example 1 3.2±0.07 ^# 4.2±0.1 ^#

*, #: p<0.05, **, ##: p<0.01

The arteriosclerosis index and cardiovascular risk index increased 2.6-fold and 2.2-fold, respectively, in the positive control group compared to the negative control group, whereas in Example 1 (combined probiotic supplement of the present invention) were measured lower than the control group, respectively, significantly 3-fold and 2.5-fold, respectively. decreased twice. This result appeared similar to the results of a study confirming the cholesterol-reducing effect in rats using Enterococcus phyceum and Lactobacillus johnsonii as test strains in in vitro and in vivo experiments in previous reports. It is thought to be due to the activity of Enterococcus phyceum and the like. Therefore, the blood lipid concentration of experimental animals fed with the complex probiotic of the present invention was remarkably reduced similarly to that of the negative control group. It is thought to have improved the arteriosclerosis index or cardiovascular risk index by suppressing

③ Effects of complex probiotic feeding on liver and kidney function of laboratory animals fed high-fat diet

In model animals fed a high-fat diet, the complex probiotic feeding of the present invention was found to affect liver function indicators, alanine transaminase (ALT) and aspartate transaminase (AST) activities, and renal function indicators, serum creatinine and blood urea nitrate (BUN) activities. The results of measuring the effect are shown in Table 15.

Effects of high-fat diet and feeding of the complex probiotic of the present invention on changes in blood arteriosclerosis index and cardiovascular risk index of experimental animals during feeding Liver function indicators kidney function indicators AST
(IU/L) ALT
(IU/L) Creatinine (mg/dl) BUN
(mg/dl) voice control phrase 141.63±11.7 85.2±7.2 0.89±0.04 23.2±2.1 positive control 216.8±12.9 87.8±8.9 1.32±0.03 20.1±1.9 Example 1 189.7±17.2 87.2±7.5 1.12±0.03 22.1±1.8

Damage to the liver tissue can be confirmed by measuring the outflow of enzymes present inside the cells into the blood. The elevation of AST and ALT in the serum indicates that transaminase is released into the blood as the destruction of the liver cells progresses due to liver damage. Therefore, it can be an indicator of destruction and degeneration of liver cells. As a result of measuring AST and ALT to examine the effect of the supplementary probiotic supplement of the present invention on liver damage caused by a high-fat diet, the AST concentration was detected as 216.8 IU/L in the positive control group, but 141.63 IU/L in the negative control group. , and in Example 1, it was detected as 189.7 IU / L, indicating that HFD was the highest, but all of them were in the normal range and were not significant. ALT concentration was detected as 85.2 IU/L in the negative control, 87.8 IU/L in HFD, and 87.2 IU/L in HFD+D-F, and there was no significant difference. Therefore, it was found that liver function was not significantly affected during the experiment in all three test groups.

In order to examine the effect of the complex probiotic supplement of the present invention on renal function according to the high-fat diet, blood creatinine and BUN were measured. In the composite probiotic supplement of the present invention (Example 1), it was detected at 1.12 mg/dL, and BUN was detected at 23.2 mg/dL in the negative control group, 20.1 mg/dL in the positive control group, and 22.1 mg/dL in Example 1. . In both tests, the negative control group was the lowest and the positive control group was the highest, but like the results of liver function index, all three experimental groups showed values within the physiologically normal range of blood, so there was no statistically significant difference.

④ Effects of complex probiotic feeding on changes in serum adiponectin and leptin and inflammatory markers in high-fat diet experimental animals

In model animals fed a high-fat diet, the complex probiotic supplement of the present invention was found to be effective in serum adiponectin (an inflammatory response inhibitor), leptin (appetite suppressant factor), and inflammation-related index C-reactive protein (C-response protein, CRP). The results of measuring the effect are shown in Table 16.

Effects of high-fat diet and feeding of the complex probiotics of the present invention on changes in adiponectin, leptin and CRP in the serum of laboratory animals during breeding Adiponectin (ng/ml) Leptin (pg/ml) CRP (ng/ml) voice control phrase 215.7±24.5 0.52±0.3 71.7±5.5 positive control 68.3±19.2 ^** 0.68±0.1 98.3 ± 9.2 ^* Example 1 189.5±27.5 ^## 1.56±0.3 ^# 73.5±4.5 ^#

*, #: p<0.05, **, ##: p<0.01

Adiponectin acts on muscle and liver receptors to inhibit inflammatory responses or enhance insulin action, promote fatty acid oxidation in muscle or liver, and improve insulin sensitivity by reducing accumulation of triglycerides. Adiponectin, which is known as a defense factor against arteriosclerosis or cardiovascular disease, was found to have a reduced concentration in obese people or type 2 diabetic patients. As a result of examining the effect of the supplementary probiotic supplement of the present invention on the concentration of adiponectin in serum, it was found to be 68.3 ng/dL in the positive control group compared to 215.7 ng/dL in the negative control group. However, in Example 1, it was found to be 189.5 ng / dL, which increased by about 2.7 times compared to the positive treatment.

Leptin regulates appetite and weight by recognizing satiety in the brain. Reports that severe obesity occurs in patients who fail to produce leptin or have receptor defects suggest that leptin plays an important role in maintaining performance by promoting energy consumption. The leptin concentration of the complex probiotic supplement of the present invention (Example 1) was detected as 1.56 pg/dL, showing a significant increase compared to the negative control (0.52 pg/dL) and positive control (0.68 pg/dL). It is judged that the adiponectin and leptin concentrations increased by the inventive complex probiotic supplementation affected the effect of suppressing weight gain and improving lipid metabolism.

Excessive accumulation of fat in body adipose tissue secretes inflammatory-inducing endocrine substances that are specifically expressed in fat cells and induce metabolic diseases due to inflammatory reactions. C-reactive protein (CRP), an inflammatory response indicator, is expressed in fat intake or fat cells, and its concentration increases as body fat mass increases. As a result of the experiment, the CRP concentration significantly increased to 98.3 ng/mL in the positive control compared to 71.7 ng/mL in the negative control. However, in Example 1, it appeared similar to the negative control at 73.5 ng / mL, indicating a significantly lower concentration than the positive control. CRP is a non-specific acute-phase protein produced in the liver that induces various inflammatory reactions and is used as one of the indicators that can predict the onset of cardiovascular disease and diabetes due to abnormal lipid metabolism. In other words, it is believed that various metabolic diseases caused by obesity can be prevented by controlling abnormal lipid metabolism including body fat mass by inhibiting the inflammatory response caused by obesity even through the provision of the complex probiotic of the present invention.

[Experimental Example 4] Test of probiotics properties of composite probiotics

<4-1> Experiment method

Acid resistance, bile resistance and heat resistance were measured in order to find out the characteristics of the composite probiotic of the present invention of Example 1 as a probiotics. Antibacterial activity against four bacteria ( Salmonella enterica serovar Typhi (ATCC19430), Escherichia coli O157:H7 (ATCC 43895), Staphylococcus aureus (ATCC25923) , Listeria monocytogenes (ATCC 19113)), one yeast ( Candida albicans ATCC 24433) and mold One species ( Aspergilus fumigatus ATCC 96918) was investigated by Paper disk method. The bacteria and fungi were purchased from ATCC and used.

The acid resistance test was performed by inoculating 1% of the strain suspension with the total number of bacteria adjusted to about 7.0 log CFU/mL in PPS (0.2% Photassium phosphate solution) adjusted to pH 2, incubated for 2 hours, plated on a plate medium, and then cultured for 24 hours at 37℃. After culturing, the total number of viable bacteria was counted.

Artificial bile was used by preparing artificial bile by adding Oxgall to BHI Broth at a concentration of 1.0%. The artificial bile prepared above was inoculated with 8.0 log CFU/mL of 1% culture medium, cultured for 2 hours, plated on a plate medium, and cultured at 37° C. for 24 hours to count the total number of viable bacteria.

The heat resistance test was cultured at 36.5 ° C for 20 hours (using BHI broth) and cultured at least 7 log CFU / mL, treated at 70 ° C for 10 minutes (using waterbath), plated on a plate medium, and then cultured at 37 ° C for 24 hours. After culturing, the total number of viable bacteria was counted.

After inoculating the composite probiotic sample of the present invention (Example 1) into BHI broth, culturing at 36.5 ° C for about 20 hours to obtain at least 7 log CFU / ml, and then centrifuging the suspension, taking the supernatant and filtering to obtain a test solution. After preparation, it was used for the following antibacterial activity measurement. 4 types of test bacteria Sal. enterica serovar Typhi, E. coli O157:H7, Sta. aureus, Lis. After smearing monocytogenes on BHI plate medium at 7 log CFU/ml or more, immersing the paper disk sufficiently in the test sample solution prepared above, attaching it to the center, and incubating it at 37 ° C for 20 hours. The diameter of the clear zone created around the paper disk was measured to determine the presence and degree of antibacterial activity. The test yeast, Can. albicans on sabouraud dextrose (Difco Laboratories) plate medium, the test fungus Asp. fumigatus was smeared on a PDA plate medium at 6 log CFU/mL, then immersed the paper disk sufficiently in the test sample solution, attached to the center, and incubated at 25℃ for 72 hours. The diameter of the clear zone created around the paper disk was measured. The presence and degree of antibacterial activity were measured by measuring.

<4-2> Experimental results

The results of measuring acid resistance, bile resistance and heat resistance of the composite probiotic of the present invention are shown in Table 17.

General characteristic test results of the composite probiotic of the present invention general characteristics Acid resistance (pH 2) Bile-resistant (1.0% bile) Heat resistance (70℃, 10 minutes) Example 1 4.54 ± 0.04 5.13 ± 0.03 6.29 ± 0.12

The complex probiotics of the present invention showed growth of 4.54 log CFU/mL in artificial gastric acid conditions of pH 2, and growth of 3.36 log CFU/mL and 5.13 log CFU/mL in artificial damjeum acid conditions with 1.0% oxgall. , and the heat resistance at 70 ° C. showed a level of 6.29 log CFU / mL.

Table 18 shows the antibacterial activity test results for the culture supernatant of the composite probiotic of the present invention.

Antibacterial test results of the composite probiotic of the present invention Antibacterial activity (culture medium stock solution) Salmonella enterica
(ATCC19430) Escherichia
coli O157:H7
(ATCC43895) Staphylococcus
aureus
(ATCC25923) Listeria monocytogenes
(ATCC19113) Candida
albicans
(ATCC24433) Aspergillus
fumigatus
(ATCC96918) Example 1 ++ ++ + ++ - -

'-' or '+' in the table above is determined based on the diameter of the ring of inhibition, '-' means that there is no antibacterial or antifungal activity, and '+' means that the diameter of the circle of inhibition is less than 14.0 mm. '++' 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.

As a result of the antibacterial activity test on the six test strains, the antibacterial activity of the raw strain was found to be active even in the strain mixture, but no new activity was found. The composite probiotic culture solution of the present invention is Sal. It showed growth inhibitory activity against enterica server Typhi, E. coli O157:H7, and L. monocytogenes (ATCC19113), but showed weak activity against S. aureus strain and Can. albicans and Asp. fumigatus did not show inhibitory activity. This result shows that among the composite probiotics of the present invention, the Bacillus bellegensis strain is in the range of 30% by weight, the Bacillus licheniformis strain is in the range of 30% by weight, the Enterococcus phyceum strain is in the range of 30% by weight and Lactobacillus plantarum (KCTC3108 ) shows an increase in antibacterial activity against the four pathogenic bacteria when the strains were mixed at 10% by weight. There are many reports on probiotics showing antibacterial activity, which are mainly due to the production of antibacterial substances such as bacteriocin, and although there is a difference in activity, it is considered that the composite probiotic of the present invention has antibacterial activity but no antifungal activity. When analyzing the general characteristics and antibacterial activity results, the composite probiotics of the present invention are considered to have basic characteristics as probiotic live bacteria.

<Preparation Example 1> Preparation of a composite probiotic preparation

A preparation for the prevention, improvement, or treatment of metabolic diseases containing the composite probiotic obtained in Example 1 of the present invention is prepared by drying the composite probiotic by a conventional lyophilization method, and then mixing with an excipient or encapsulating the mixture.

<Production Example 2> Production of fermented milk

Fermented milk was prepared using the complex probiotic obtained in Example 1 of the present invention according to a method commonly performed in the art. Raw milk (74.41%) and skim milk powder (6.45%) are mixed and cultured by adding the culture seed and the composite probiotic of the present invention to prepare a culture medium (1X10 ⁹ CFU/mL).

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

<3-1> Manufacture of beverages

The beverage is prepared by mixing the ingredients of the formulations presented below according to a method commonly practiced in the art. Honey 522 mg, thioctic acid amide 5 mg, nicotinic acid amide 10 mg, riboflavin sodium hydrochloride 3 mg, pyridoxine hydrochloride 2 mg, inositol 30 mg, ortic acid 50 mg, the composite probiotic culture supernatant obtained in Example 1 of the present invention, 200 mL of water

<3-2> Preparation of powder

A powder preparation was prepared by mixing 20 mg of the lyophilized powder (1×10 ⁹ CFU/g) of the composite probiotic obtained in Example 1 of the present invention, 100 mg of lactose, and 10 mg of talc, and filling in an airtight bag.

<3-3> Manufacture of tablets

After mixing 10 mg of the lyophilized powder (1×10 ⁹ CFU/g) of the composite probiotic obtained in Example 1 of the present invention, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, a conventional tablet manufacturing method Thus, tablets are prepared by tableting.

<3-4> Preparation of capsules

10 mg of the complex probiotic lyophilized powder (1×10 ⁹ CFU/g) obtained in Example 1 of the present invention, 3 mg of crystalline cellulose, 14.8 mg of lactose, and 0.2 mg of magnesium stearate were prepared according to a conventional capsule preparation method. Capsules are prepared by mixing and filling into gelatin capsules.

<3-5> Preparation of mixed powder formulation

1 g of the freeze-dried powder of the composite probiotic obtained in Example 1 of the present invention (1×10 ⁹ CFU/g), appropriate amounts of the 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), nicotinamide 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, monobasic potassium phosphate 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 mg, calcium carbonate 100 mg, magnesium chloride 24.8 mg) were mixed according to a conventional health functional food manufacturing method, and then granulated It can be prepared and used for preparing a health functional food composition according to a conventional method. The composition ratio of the above vitamin and mineral mixture was prepared by mixing ingredients suitable for a relatively health functional food in a preferred manufacturing example, but the mixing ratio may be arbitrarily modified.

<Preparation Example 4> Preparation of feed composition

The feed composition was composed of 42 g of corn, 28 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 10 g of the composite probiotic obtained in Example 1 of the present invention. It is prepared by mixing.

As described above, the complex probiotic of the present invention and its culture medium having the effects of preventing, improving, and treating metabolic diseases of the present invention have high antioxidant, oxidative stress resistance, and anti-inflammatory effects, and are effective in preventing obesity and blood clots that cause metabolic diseases. It can be widely used industrially, such as health functional foods that require prevention or improvement of metabolic diseases by improving physiological biochemical indicators, pharmaceuticals that require treatment, feed additives or veterinary medicines that improve metabolic diseases of livestock or companion animals. .

Claims (9)

As a composite probiotic having a preventive, ameliorative, or therapeutic effect on metabolic diseases,
The complex probiotics include any one or more strains of Bacillus bellegensis, Bacillus licheniformis, Enterococcus faecium or Lactobacillus plantarum strains,
The metabolic disease is obesity, diabetes, hypercholesterolemia, hyperlipidemia, arteriosclerosis, osteoporosis, and sarcopenia caused by abnormal metabolic processes such as insulin resistance, abnormal sugar and lipid metabolism, and reduced immune function. Complex probiotics . The composite probiotic of claim 1 contains 30% by weight of Bacillus bellegensis, 30% by weight of Bacillus licheniformis, 30% by weight of Enterococcus phyceum, and 10% by weight of Lactobacillus plantarum. A complex probiotic having a. A microbial agent for the prevention, improvement or treatment of metabolic diseases comprising the probiotic of claim 1 or its culture medium as an active ingredient. A method for producing a composite probiotic product comprising the composite probiotic of claim 1 or its culture medium as an active ingredient. A probiotic bioactive composition comprising the complex probiotic of claim 1 or its culture medium as an active ingredient. A pharmaceutical composition for the prevention, improvement or treatment of metabolic diseases comprising the probiotic of claim 1 or its culture medium as an active ingredient. A health functional food composition for the prevention or improvement of metabolic diseases, comprising the complex probiotic of claim 1 or its culture medium as an active ingredient. A drinking water additive for the prevention or improvement of metabolic diseases comprising the complex probiotic of claim 1 or its culture medium as an active ingredient. A feed additive for the prevention or improvement of metabolic diseases comprising the complex probiotic of claim 1 or its culture medium as an active ingredient.

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