KR101679045B1 - Microorganism capable of reducing body fat and use thereof - Google Patents

Abstract

The present invention relates to microorganisms selected from the group consisting of Lactobacillus paracasei (LMT1-30, accession No. KCTC13023BP), Lactobacillus plantarum (LMT1-48, accession No. KCTC13024BP), and Pediococcus acidilactici (LMT2- 46, accession No. KCTC13025BP); to a combination thereof or an extract thereof; and to uses thereof. According to the present invention, the microorganisms and the combination thereof or the extract thereof can be used to reduce the fat content in fat cells or objects.

Description

Microorganism capable of reducing body fat and use thereof TECHNICAL FIELD

Microorganisms selected from the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP) Or a combination thereof or an extract thereof, and a use thereof.

Lactobacillus is a living organism that benefits our body and is known as a microorganism that settles in the small and large intestine as part of the intestinal flora. Lactic acid bacteria are known to suppress the growth of harmful bacteria by lowering the surrounding pH through the production of various organic acids, as well as to produce antibacterial substances such as bacteriocin depending on the strain. In addition to the simple intestinal action through stimulation of the intestinal epithelial cells, lactic acid bacteria are known to play a role in alleviating irritable inflammatory reactions as they possess antibacterial and antiviral effects to inhibit harmful bacteria in the intestine, as well as the ability to regulate immune cell activity. These are used for various purposes as health functional foods, animal feed additives, etc. Recently, research on efficacy for various diseases has been actively conducted. In particular, it has been reported to have excellent effects in the improvement of atopic symptoms, which are irritable inflammatory diseases, and enteritis (IBD) and irritable bowel syndrome (IBS), which are representative diseases caused by abnormal intestinal flora. In addition, in an animal model for the treatment of rheumatoid arthritis, which is an autoimmune disease, research results for relieving arthritis symptoms by inhibiting inflammatory T cell activity as well as induction of regulatory T cell activity have been reported, and studies are being actively conducted to investigate the mechanism.

In obesity, excess calories from ingested foods are not sufficiently converted to heat, leading to fat accumulation. Eventually, the size of fat cells increases and the differentiation of fat progenitor cells into fat cells is promoted, and the number increases, resulting in excessive fat accumulation in the body. As a condition, it has emerged as a serious social problem of modern people, and numerous various methods for treating it have been proposed. Fat that has entered the body is not consumed or excreted, but is accumulated or is present in blood vessels, which can cause metabolic diseases such as hyperlipidemia, hyperglycemia, arteriosclerosis, and diabetes. In order to prevent this, the activity of lipolytic enzymes that can burn fat while generating heat by directly decomposing fat in the body or promoting the oxidation of fat is very important. It is an enzyme present in the mitochondria that plays the most important role. There is an uncoupling protein (UCP). They are mainly present in white fat, brown fat, and muscle cells. They reduce ATP production and induce heat generation. In fact, studies have reported that UCP1 expression levels are lowered in obese patients, and it is known that UCP1 expression levels are closely related to energy consumption.

Various methods have been proposed in an effort to suppress obesity, and there are methods of reducing the intake of fat itself or inhibiting the activity of triglycerides that help the body absorb fat. Various products have been introduced as a treatment for obesity, and as drugs to treat obesity, there are appetite suppressants, absorption inhibitors that block the absorption of fat in the small intestine, and metabolic stimulators that help burn calories. Phentermine and phendimetrazine, which are used as appetite suppressants, are known to suppress appetite by increasing neurotransmitters such as dopamine and norepinephrine. Side effects include insomnia, headache, hand tremor, constipation, and tolerance. Increasing, etc. are being reported. Orlistat is well known as a drug for inhibiting fat absorption, and it has a function of inhibiting the activity of lipolytic enzymes in the pancreas. Orlistat is mainly a side effect of the digestive system, such as leakage of bowel movements, oily spotting on clothes, filling of gas or an increase in the number of bowel movements. In addition, there is ephedrine as a metabolic accelerator, and this evil substance has an effect of reducing body fat due to excessive consumption of calories due to the facilitation of fever, but insomnia, tremors, and increase in blood pressure have been reported as side effects.

There are many drugs that have been canceled or banned from being sold because of the recent release of such drugs, and due to serious side effects, they are limited to use for a short period of 4 weeks or are recommended to be used as an adjuvant therapy. Therefore, in the treatment of obesity, there is an urgent need to develop a safe therapeutic agent without side effects.

On the other hand, lactic acid bacteria have been reported to have immunity enhancement, antimicrobial, antioxidant, anticancer effect, antiobesity effect, antihypertension or constipation prevention effect (Prev Nutr Food Sci. 2015 Dec; 20(4): 298-302) . However, there has not been a technology related to lactic acid bacteria that has excellent anti-obesity effect enough to be commercially successful.

Recently, it was reported that the cause of obesity was due to the presence of Enterobacter cloacae, an opportunistic pathogenic bacterium in the intestine. Fei et al., The ISME Journal (2013) 7, 880-884). These are known to be part of the intestinal flora, interfering with metabolism, inducing fat accumulation in the body, leading to obesity and overweight.

Intestinal acidity (pH) may be lowered by metabolites such as lactic acid, acetic acid, citric acid, and formic acid, which are metabolites released as lactic acid bacteria grow. Gram-negative bacteria such as Enterobacter cloacae are limited in their growth at low pH.

The inventors of the present invention isolated three strains that specifically inhibit the growth of Enterobacter cloacae by organic acids contained in the lactic acid bacteria culture, not simply by acidity (pH). The invention was completed.

One aspect is a group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a microorganism selected from, or a combination thereof or an extract thereof.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a composition for use in reducing the fat content in adipocytes, comprising a microorganism selected from or a combination thereof or an extract thereof as an active ingredient.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a composition for use in reducing the fat content in an individual comprising a microorganism selected from or a combination thereof or an extract thereof as an active ingredient.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a method for reducing the fat content in fat cells comprising; contacting the microorganism selected from, or a combination thereof, or an extract thereof with adipocytes.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a method for reducing the fat content of a subject comprising; administering to the subject a microorganism selected from, or a combination thereof, or an extract thereof.

One aspect is a group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). Microorganisms selected from or a combination thereof are provided.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides an extract of a microorganism or a combination thereof selected from. The extract may be a protein extract of a microorganism or a combination thereof. The extract may be a supernatant remaining after dissolving a microorganism or a combination thereof, removing a precipitate by centrifugation.

In the microorganism or a combination thereof or an extract thereof, the combination is Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP) may be mixed in an arbitrary ratio based on the weight. The mixing ratio may be, for example, 1:0.3 to 3.0:0.3 to 3.0.

The microorganism or a combination thereof, or an extract thereof, has antibacterial activity against E. cloacae, which causes obesity, has acid resistance, has bile acid resistance, and inhibits the differentiation of adipocytes into adipocytes. Active, inhibiting the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 among adipocytes, inhibiting the accumulation of fat in adipocytes, and body weight when administered to an individual And reducing one or more selected from the group consisting of the volume of adipose tissue in the body, and reducing one or more selected from the group consisting of triglycerol and leptin content in blood when administered to an individual. It may be to have.

The acid resistance may be viable for 2 hours or more at pH 2.5 and 37°C in MRS medium. Viable time is, for example, 4 hours or more, 10 hours or more, 15 hours or more, 24 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 2 hours to 24 hours, 2 hours to 48 hours, 2 hours To 60 hours, or 2 to 72 hours.

The bile acid resistance may be viable for 2 hours or more at 37° C. in an MRS medium containing 0.3% bile acid. Viable time is, for example, 4 hours or more, 10 hours or more, 15 hours or more, 24 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 2 hours to 24 hours, 2 hours to 48 hours, 2 hours To 60 hours, or 2 to 72 hours.

The intestinal epithelial cells may be small intestine, large intestine, or a combination thereof.

The adipocyte progenitor cell may be 3T3-L1.

The microorganism or a combination thereof or an extract thereof may inhibit the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 among adipocytes. The inhibition is 10% or more, 15% or more, 20% of the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4 compared to the case in which the microorganism or a combination thereof or an extract thereof is not present. More than, 25% or more, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100 %, or 90% to 100% reduction.

The microorganism or a combination thereof or an extract thereof may be one that inhibits the accumulation of fat in adipocytes. The inhibition is 10% or more, 15% or more, 20% or more, 25% or more of fat accumulation based on the number of microorganisms (number) or weight (g) compared to the case in which the microorganism or a combination thereof or an extract thereof is not present, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% To 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to It could be a 100% reduction.

When the microorganism or a combination thereof or an extract thereof is administered to an individual, it may reduce one or more selected from the group consisting of body weight and the volume of adipose tissue in the body. The inhibition is 10% or more, 15% or more, 20% or more, 25% or more of fat accumulation based on the number of microorganisms (number) or weight (g) compared to the case in which the microorganism or a combination thereof or an extract thereof is not present, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% To 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to It could be a 100% reduction.

The microorganism, or a combination thereof, or an extract thereof, when administered to an individual, may reduce one or more selected from the group consisting of triglycerol and leptin content in blood. The inhibition is 10% or more, 15% or more, 20% or more, 25% or more of fat accumulation based on the number of microorganisms (number) or weight (g) compared to the case in which the microorganism or a combination thereof or an extract thereof is not present, 30% or more, 35% or more, 45% or more, 50% or more, 55% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 100% or more, 10% To 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to It could be a 100% reduction.

Another aspect is the above-described Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a composition comprising a microorganism selected from the group consisting of, a combination thereof, or an extract thereof as an active ingredient.

Microorganisms selected from the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP) Or the combination or the extract is as described above.

The composition has antibacterial activity against E. cloacae, which causes obesity, has acid resistance, has bile acid resistance, has activity of inhibiting the differentiation of adipocytes into adipocytes, adipocytes Inhibiting the expression of one or more genes selected from the group consisting of PPARν, C/EBPα, FAS, and FABP4, inhibiting the accumulation of fat in adipocytes, body weight and volume of adipose tissue in the body when administered to an individual It may have one or more properties selected from the group consisting of reducing one or more selected from the group consisting of, and reducing one or more selected from the group consisting of triglycerol and leptin content in blood when administered to an individual.

Therefore, the composition suppresses the proliferation or removal of E. cloacae , inhibiting the differentiation of adipocytes into adipocytes, among adipocytes, one or more genes selected from the group consisting of PPARν, C/EBPa, FAS, and FABP4 Inhibits the expression of, inhibits the accumulation of fat in adipocytes, when administered to an individual, reduces one or more selected from the group consisting of body weight and the volume of adipose tissue in the body, and when administered to an individual, triglycerol in blood, And leptin content. These uses may be used alone or in combination thereof.

The composition may be food or pharmaceutical. The composition may contain a food pharmaceutically or pharmaceutically acceptable carrier.

In the composition, the microorganism or a combination or extract thereof may be included in a "food effective amount" or a "therapeutically effective amount". In the above composition, "therapeutically effective amount" means an amount sufficient to exhibit a therapeutic effect when administered to an individual in need of treatment. The term “treatment” refers to treating a disease or medical condition, such as an obesity disease, in an individual, eg, a mammal, including humans, which includes: (a) preventing the occurrence of the disease or medical condition, Ie, prophylactic treatment of the patient; (b) alleviation of the disease or medical condition, ie causing the elimination or recovery of the disease or medical condition in the patient; (c) inhibition of the disease or medical condition, ie slowing or stopping the progression of the disease or medical condition in the subject; Or (d) alleviating the disease or medical condition in the subject. The "effective amount" can be appropriately selected by a person skilled in the art. The "effective amount" may be 0.01mg to 10,000mg, 0.1mg to 1000mg, 1mg to 100mg, 0.01mg to 1000mg, 0.01mg to 100mg, 0.01mg to 10mg, or 0.01mg to 1mg.

The composition can be administered orally. Accordingly, the composition may be formulated in various forms such as tablets, capsules, aqueous solutions or suspensions. In the case of tablets for oral use, excipients such as lactose and corn starch, and lubricants such as magnesium stearate may be usually added. In the case of capsules for oral administration, lactose and/or dried corn starch may be used as a diluent. If an aqueous suspension for oral use is desired, the active ingredient can be combined with emulsifying and/or suspending agents. If necessary, certain sweetening and/or flavoring agents can be added.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a method for reducing the fat content in fat cells comprising; contacting the microorganism selected from, or a combination thereof, or an extract thereof with adipocytes.

In the above method, the contacting may be cultivation of a microorganism or a combination thereof or an extract thereof in a medium of adipocytes. The method may be an in vitro or in vivo method.

Another aspect is the group consisting of Lactobacillus paracasei LMT1-30 (accession number KCTC13023BP), Lactobacillus plantarum LMT1-48 (accession number KCTC13024BP), and Pediococcus ashdilactici LMT2-46 (accession number KCTC13025BP). It provides a method for reducing the fat content of a subject comprising; administering to the subject a microorganism selected from, or a combination thereof, or an extract thereof.

In the above method, a person skilled in the art can appropriately select the route of administration during administration according to the patient's condition. The administration may be oral or topical.

In the above method, the dosage varies according to various factors such as the condition of the patient, the route of administration, and the judgment of the attending physician, as described above. Effective dosages can be estimated from dose-response curves obtained in vitro or in animal model tests. The proportion and concentration of the compound of the present invention present in the composition to be administered can be determined according to chemical properties, route of administration, therapeutic dosage, and the like. The dosage may be administered to an individual in an effective amount of about 1 μg/kg to about 1 g/kg per day, or about 0.1 mg/kg to about 500 mg/kg per day. The dosage may vary depending on the age, weight, sensitivity, or symptoms of the individual.

In the above method, the individual may be a mammal, not a human.

The microorganism according to an aspect, or a combination thereof or an extract thereof, may be used to reduce the fat content in adipocytes or to reduce the fat content in an individual.

A composition for use in reducing fat content in adipocytes according to another aspect, or a composition for use in reducing fat content in a subject, a composition for use in reducing fat content in adipocytes, or reducing fat content in a subject Can be used to make.

According to the method of reducing the fat content of fat cells or the method of reducing the fat content of an individual according to another aspect, it is possible to efficiently reduce the fat content of the fat cells or the fat content of the individual.

1 shows a scanning electron microscope (Scanning Electron Microscopy) picture of the selected three types of lactic acid bacteria.
2 is a view showing the number of cells of E. cloacae KCTC 1685 cultured in a medium containing a culture medium corrected to the same acidity (pH) as the lactic acid bacteria culture medium.
FIG. 3 is a diagram showing the number of selected three microorganisms attached to intestinal epithelial cells.
FIG. 4 is a diagram showing an electron micrograph (FIGS. 4A to 4F) and the degree of fat accumulation in the cells (FIG. 4G) of adipocytes of 3T3-L1 adipocytes cultured in the presence of a culture medium of three selected microorganisms.
FIG. 5 shows the results of confirming the expression of PPARν, C/EBPα, FAS, and FABP4 genes related to obesity in 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected three microorganisms at the mRNA level.
6 is a diagram showing a change in body weight over time when three kinds of selected microorganisms are administered in obesity-induced mice.
7 is a diagram showing the body fat volume when three kinds of selected microorganisms are administered in obesity-induced mice.
Figure 8 is a case of administration of three selected microorganisms in obesity-induced mice, triglycerides in serum (Figure 8a), total cholesterol (Figure 8b), high-density lipoprotein (HDL) (Figure 8c), low-density lipoprotein (LDL) ( Fig. 8d), leptin (Fig. 8e), and adiponectin (Fig. 8f) is a view showing the results of measuring the content.
9 is a diagram showing the result of classifying the intestinal microorganisms in the feces at the class (strong) level when three types of selected microorganisms are administered in obesity-induced mice.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Recently, it has been reported that the cause of obesity is due to the presence of obesity-causing bacteria in the intestine (The ISME Journal (2013) 7, 880-84, Na Fei, etc.). Na Fei et al. proved that the obese patients were actually obesity-causing bacteria by administering E. cloacae to mice and inducing obesity after examining the intestinal flora of obese patients. E. cloacae is an opportunistic pathogenic bacterium and is a Gram-negative bacterium. These are known to be part of the intestinal flora, interfering with metabolism, inducing fat accumulation in the body, leading to obesity and overweight. Therefore, reducing the proportion of E. cloacae in the intestinal flora can be expected to reduce body fat.

In the present invention, the intestinal acidity (pH) is lowered by metabolites such as lactic acid, acetic acid, citric acid, formic acid, etc., which are metabolites released as the lactic acid bacteria grow, and at this time, Gram-negative bacteria such as obese bacteria are limited in their growth at low pH. Based on these facts, E.clocae If you select and use the lactic acid bacteria with the best growth inhibition ability, you can expect obesity treatment. In addition, there is no need to worry about serious side effects, which are the limitation of obesity treatment drugs, and synergistic effects can be expected with complex functions such as immunity enhancement and regulation along with improvement of obesity.

Indicator bacteria used in the experiment were Enterobacter cloacae (E. cloacae KCTC 1685) (hereinafter also referred to as'indicator bacterium 1'), Enterobacter cloacae subspecies Cloace ( Enterobacter). subsp. cloacae KCTC 2361) (hereinafter also referred to as ``indicator 2''), which was sold and used by the Korea Research Institute of Bioscience and Biotechnology Biological Resource Center. In addition, lactic acid bacteria were directly isolated and identified from traditional foods such as kimchi and milk gal, and these lactic acid bacteria were selected through screening for the inhibition of growth of obesity bacteria. Among them, three strains with high antibacterial activity against obesity bacteria, namely Lactobacillus paracasei. LMT1-30 (hereinafter also referred to as'LMT1-30'), Lactobacillus plantarum LMT1-48 (hereinafter referred to as'LMT1-48'), and Pediococcus acidilactici LMT2-46 (hereinafter referred to as'LMT2-46') can be secured. there was.

These three strains were deposited with the Korea Cell Line Bank (KCTC), microbial metabolism characterization (API kit), morphological analysis using electron microscope, stability test in the intestine through acid resistance and bile resistance analysis, and adipocyte differentiation of adipocytes. The effect of reducing body fat was verified through ability verification and animal experiments. It will be described in detail below.

Example 1. Isolation and identification of strains

1. Isolation and selection of strains

Kimchi, which was used to isolate lactic acid bacteria, was prepared directly at home and stored at 4℃. For the isolation of total lactic acid bacteria from kimchi, it was plated on MRS medium (Difco Co., USA) and cultured at 30°C. The pretreatment method for kimchi is to take 20g of kimchi at 4℃ aseptically, dilute with 180ml of 0.85% NaCl solution, homogenize for 5 minutes with a stomacher, and then sterilize the tube containing 9ml of 0.85% NaCl solution. Then, a sample was prepared by diluting it step by step in the prepared tube. After that, it was spread on MRS plate medium and cultured at 37°C for 2-3 days, and the colonies that appeared were separated by shape and color, and purified again. For the separated colonies, 128 colonies whose pH of the culture medium decreased to 4.5 or less were selected while culturing at 37° C. for 24 hours in an MRS liquid medium having a pH of 6.8. Subsequently, it was identified by 16S rRNA sequencing, and lactic acid bacteria that inhibit the growth of obesity-related bacteria were selected among the isolated 128 lactic acid bacteria. SEQ ID NOs: 1, 2, and 3 are the nucleotide sequences of the 16S rRNA genes of L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46.

In order to select the lactic acid bacteria that inhibit the growth of obesity-related bacteria from among the 128 lactic acid bacteria isolated above, MRS agar plate added with ^{indicator bacteria 1, Enterobacter cloase subspecies Cloase, which are obesity-related bacteria at 1×10 6 CFU/ml} 5 µl of the lactic acid bacteria was dropped and cultured at 37° C. for 24 hours to select 42 types of lactic acid bacteria having inhibitory ability as shown in Table 1.

Thereafter, the first 42 lactic acid strains selected were placed on a disc paper in the same manner, and an antimicrobial activity comparison experiment was conducted. At this time, in order to select each of the lactic acid bacteria strains with the best antibacterial activity at each species level _{, only the culture medium (OD 600} ≒2.0) was collected, 100 µl was added dropwise, and cultured at 37°C for 24 hours, and then the second selection was performed. Finally, three types of lactic acid bacteria with the best ability to inhibit the growth of obese bacteria were selected.

number Lactobacillus (Strains) Indicator strain growth inhibition ring (mm) E. cloacae
KCTC 1685 E. subsp. Cloacae KCTC 2361 One Lactobacillus plantarum LMT1-2 15 15 2 Lactobacillus plantarum LMT1-3 13 13 3 Lactobacillus plantarum LMT1-13 12 12 4 Lactobacillus plantarum LMT1-33 12 12 5 *Lactobacillus plantarum LMT1-48 18 18 6 Lactobacillus plantarum LMT3-1 14 14 7 Lactobacillus plantarum LMT3-26 17 18 8 Lactobacillus plantarum LMT3-66 12 12 9 *Lactobacillus paracasei LMT1-30 16 16 10 *Pediococcus acidilactici LMT2-46 18 18 11 Lactobacillus casei LMT1-31 11 11 12 Lactobacillus casei LMT1-71 14 13 13 Lactobacillus sakei LMT1-21 11 11 14 Lactobacillus sakei LMT1-36 13 14 15 Lactobacillus sakei LMT1-66 15 15 16 Lactobacillus brevis LMT1-46 12 12 17 Lactobacillus brevis LMT1-73 12 12 18 Leuconostoc lactis LMT2-35 12 12 19 Lactobacillus parabuchneri LMT1-47 11 11 20 Weissella cibaria LMT2-10 14 13 21 Weissella halotolerans LMT2-48 11 11 22 Pediococcus pentosaceus LMT1-1 14 13 23 Pediococcus pentosaceus LMT1-63 14 14 24 Pediococcus pentosaceus LMT2-64 14 14 25 Pediococcus pentosaceus LMT2-68 12 12 26 Pediococcus pentosaceus LMT3-5 12 12 27 Pediococcus pentosaceus LMT3-41 13 13 28 Leuconostoc mesenteroides LMT1-8 14 13 29 Leuconostoc mesenteroides LMT1-26 13 13 30 Leuconostoc mesenteroides LMT1-50 13 13 31 Leuconostoc mesenteroides LMT2-4 11 12 32 Leuconostoc mesenteroides LMT1-75 11 11 33 Leuconostoc mesenteroides LMT1-79 12 12 34 Lactobacillus fermentum LMT3-3 12 12 35 Lactobacillus fermentum LMT3-67 12 12 36 Leuconostoc citreum LMT2-9 14 14 37 Enterococcus faecalis LMT2-19 11 11 38 Enterococcus faecium LMT2-13 12 12 39 Enterococcus faecium LMT2-14 11 12 40 Enterococcus faecium LMT3-60 11 11 41 Enterococcus faecium LMT3-61 11 11 42 Lactococcus lactis subsp. Cremoris LMT2-37 12 12

As a result of examining the antibacterial activity of the three selected lactic acid bacteria, the antibacterial activity could be measured by the diameter of a clear zone that was clearly created around the disk. The culture medium of L. paracasei LMT1-30 is 16 mm, the culture medium of L. plantarum LMT1-48 is 18 mm, and the culture medium of P. ashdilactici LMT2-46 is 18 mm. Indicator bacteria 1, Enterobacter cloase subspecies It showed antimicrobial activity against cloase, and it was confirmed that it has minimal antimicrobial activity.

Therefore, the lactic acid bacteria culture medium of the present invention has excellent antibacterial activity against obesity-related bacteria.

2. Identification of the selected strain

(One) Mycological Characteristic analysis

The selected three types of lactic acid bacteria were cultured in MRS plate medium (Difco, USA), and the colony morphology was observed. The colony morphology in the MRS plate medium of the selected lactic acid bacteria is shown in Table 2 below.

1 is a scanning electron microscope (Scanning Electron) of the selected three types of lactic acid bacteria Microscopy) photographs are shown. As shown in Figure 1, rod-shaped bacillus and spherical shape similar to typical Lactobacillus genus and Pediococcus genus.

LMT1-30 LMT1-48 LMT2-46 shape circle circle circle size 0.5-1mm 1mm 1mm color cream color cream color cream color Opacity opacity opacity opacity bump Convex Convex Convex surface lubricity lubricity lubricity Aerobic growth + + + Anaerobic growth + + +

(2) 16S rDNA analysis

For the PCR reaction of the isolated lactic acid bacteria, the Universal bacterial primer was commissioned to Macrogen Co., Ltd. using 27F (SEQ ID NO: 16) and 1492R (SEQ ID NO: 17) to amplify the 16S rRNA gene, which is the base conserving sequence of bacteria. Analysis was carried out. The results were analyzed using NCBI blast (http://www.ncbi.nlm.nih.gov/).

The inventors of the present invention are LMT1-30 lactic acid bacteria, LMT1-48 lactic acid bacteria and LMT2-46 lactic acid bacteria, respectively, " Lactobacillus paracasei (Lactobacillus paracasei) LMT1-30 (Accession No.: KCTC13023BP)", " Lactobacillus plantarum (Lactobacillus plantarum) LMT1-48 (Accession No.: KCTC13024BP)" and "Pediococcus ashdi lactissi ( Pediococcus acidilactici ) LMT2-46 (Accession No.: KCTC13025BP)" and it was deposited on May 13, 2016 with the Korean Collection for Type Cultures (KCTC) located at the Korea Research Institute of Bioscience and Biotechnology.

(3) selected Lactobacillus Sugar fermentation properties

Sugar fermentation characteristics were investigated according to the experimental method of the supplier using the API 50 CHL kit (Biomerieux, France). The sugar fermentation properties of the lactic acid bacteria L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46 are shown in Table 3 below.

LMT1-30 LMT1-48 LMT2-46 Glycerol - - - Erythritol - - - D- Arabinose - - - L- Arabinose + - + D-Ribose + + + D- Xylose - - - L- Xylose - - - D- Adonitol - - - Methyl-β-D- Xylopyranoside - - - D- Galactose + + + D-Glucose + + + D-Fructose + + + D- Mannose + + + L- Sorbose + - - L- Rhamnose + - + Dulcitol - - - Inositol - - - Mannitol + + + D- Sorbitol + + + Methyl-α-D- mannopyranoside + + + Methyl-α-D- glucopyranoside + - + N- Acetylglucosamine + + + Amygdalin + + + Arbutin + + + Esculin + + + Salicin + + + D- Cellobiose + + + D-Maltose + + + D-Lactose + + + D- Melibiose + + + D- Saccharose + + + D- Trehalose + + + Inulin - - + D- Melezitose + + + D- Raffinose + + + Amidon - - - Glycogen - - - Xylitol - - - Gentiobiose + + + D- Turanose + + + D- Lyxose - - - D- Tagatose + - - D- Fucose - - - L- Fucose - - - D- Arabitol - - + L- Arabitol - - - Potassium Gluconate + + + Potassium 2- Ketogluconate - - - Potassium 5- Ketogluconate - - -

Table 3 is a table showing the analysis results of sugar fermentation pattern of lactic acid bacteria according to the present invention.

3. Functionality

(1) Measurement of the minimum antibacterial activity of lactic acid bacteria culture medium

In order to measure the minimum antimicrobial activity of the selected lactic acid bacteria culture medium having excellent antimicrobial activity, it was measured using an agar well diffusion assay. That is, the _{culture supernatant (OD 600} ≒2.0) was dropped on the MRS agar plate to which the ^{indicator strain was added at 1×10 6} CFU/ml, and cultured at 37° C. for 24 hours, and then the formation of an inhibitory ring was observed. The activity titer was expressed as AU (activity unit) per ml of the culture medium. That is, the reciprocal of the maximum dilution factor for forming an inhibitory ring was taken by sequentially diluting the culture medium twice, and this value was multiplied by the conversion factor converted to 1 ml, and expressed as AU/ml as shown in Table 4 below.

Indicated strain Antibacterial activity (AU/ml) LMT1-30 LMT1-48 LMT2-46 E. cloacae KCTC 1685 800 1600 1600 E. subsp. cloacae KCTC 2361 800 1600 1600

As a result of measuring the minimum antimicrobial activity of the lactic acid bacteria culture medium of the present invention, the culture medium of L. paracasei LMT1-30 showed minimal antibacterial activity at 800 AU/ml, and the culture medium of L. plantarum LMT1-48, and P. ae The culture medium of Sidilactici LMT2-46 showed minimal antimicrobial activity at 1600 AU/ml.

(2) Confirmation of dependent antibacterial activity by lactic acid bacteria culture medium components

The final pH of the selected lactic acid bacteria culture medium having excellent antibacterial activity (OD ₆₀₀ ≒2.0) was measured and shown in Table 5, and the MRS liquid medium was adjusted to be the same as the pH of each lactic acid bacteria culture medium with HCl. The lactic acid bacteria culture medium and the pH-adjusted MRS liquid medium were mixed so that the ratio of the lactic acid bacteria culture medium was 0%, 5%, 10%, 15%, and 20%, respectively, and cultured in MRS liquid medium at 37°C for 24 hours. Bacillus 1 was inoculated so that the initial number of bacteria was 1×10 ⁶ CFU/ml, and cultured at 37° C. for 24 hours. Samples after cultivation were recovered from each lactic acid bacteria culture medium with different %, diluted in MRS liquid medium, plated on MRS plate medium, incubated at 37°C for 24 hours, and then the number of colonies on the plate medium was counted. Shown in. 2 is a diagram showing the number of cells ^{of KCTC 3108 T} cultured in a medium containing a lactic acid bacteria culture medium of varying amounts.

E. cloacae (CFU) Culture medium
Addition ratio
(%) LMT1-30
PH 3.99 after incubation LMT 1-48
PH 3.96 after incubation LMT2-46
PH 3.94 after incubation HCl calibration medium
(pH 3.99) Lactobacillus
Culture filtrate
(pH 3.99) HCl calibration medium
(pH 3.96) Lactobacillus
Culture filtrate
(pH 3.96) HCl calibration medium
(pH 3.94) Lactobacillus
Culture filtrate
(pH 3.94) 100:0
(0) 2.4x10 ¹⁰ 2.4x10 ¹⁰ 2.4x10 ¹⁰ 2.4x10 ¹⁰ 2.4x10 ¹⁰ 2.4x10 ¹⁰ 95:5
(5) 2.0x10 ¹⁰ 5.7x10 ⁹ 2.0x10 ¹⁰ 1.4x10 ⁹ 2.0x10 ¹⁰ 1.8x10 ⁹ 90:10
(10) 1.0x10 ¹⁰ 9.6x10 ⁷ 9.9x10 ⁹ 3.0x10 ⁷ 9.8x10 ⁹ 3.7x10 ⁷ 85:15
(15) 1.5x10 ⁸ 4.6x10 ⁶ 1.5x10 ⁸ 1.4x10 ⁶ 1.4x10 ⁸ 1.8x10 ⁶ 80:20
(20) 3.8x10 ⁶ 9.6x10 ⁵ 3.6x10 ⁶ 5.5x10 ⁵ 3.4x10 ⁶ 6.3x10 ⁵

As a result, in a liquid medium containing 5% of each of the lactic acid bacteria cultures of L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46, respectively, 72% and 93% , And 91% of the indicator bacteria 1 was inhibited, and the growth inhibition of indicator bacteria 1 was relatively decreased in the medium to which 5% of the MRS liquid medium, which was pH-corrected with HCl solution, was added to the same as the pH of each final culture medium.

Therefore, it was confirmed that the growth of indicator bacteria 1 was inhibited by pH, and particularly important, it was confirmed that the growth of indicator bacteria 1 was specifically inhibited by organic acids contained in the lactic acid bacteria culture medium, not by simple acidity (pH). (Fig. 2 and Table 5).

4. Stability

(1) of lactic acid bacteria Acid resistance Research

In order for lactic acid bacteria to exert its efficacy as a probiotic in the intestine, it must pass through a low pH stomach after ingestion. In order to investigate the acid resistance of lactic acid bacteria, inoculate the sterilized MRS liquid medium and incubate for 16 hours at 37°C, and then adjust the pH to 2.5 with HCl and inoculate 1% of the lactic acid bacteria into the sterilized MRS liquid medium for 2 hours at 37°C. During incubation. Samples immediately after inoculation of lactic acid bacteria and after 2 hours of culture were collected, diluted in MRS liquid medium, plated on MRS plate medium, incubated at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of lactic acid bacteria. The number of lactic acid bacteria in the experimental group, which was performed in the same manner in the neutral MRS liquid medium in which the pH was not adjusted, and the number of lactic acid bacteria after 2 hours incubation in the pH 2.5 MRS liquid medium, were compared and shown in Table 6 below.

Lactobacillus (CFU/ml) LMT1-30 LMT1-48 LMT2-46 Control 2.5x10 ⁹ 5.1x10 ⁹ 1.3x10 ⁹ pH 2.5 MRS 1.3x10 ⁹ 3.5x10 ⁸ 3.0x10 ⁹

As a result, the lactic acid bacteria selected in the present invention were very excellent in resistance to acid at pH 2.5. The characteristic of these lactic acid bacteria is that the proper number of lactic acid bacteria is maintained at a pH lower than pH 3, which is close to the physiological pH of the stomach, so it can be expected that the intestinal reach rate will be very high when ingested because the number of surviving lactic acid bacteria is high even at low pH due to gastric acid secretion.

(2) of lactic acid bacteria Bile resistance Research

In order to investigate the internal bile of lactic acid bacteria, an experiment was conducted by the following method. Lactobacillus was inoculated in sterilized MRS liquid medium and incubated for 24 hours at 37°C. Considering that the concentration of bile salts in the small intestine was around 0.1%, the lactic acid bacteria 1 were added to the MRS liquid medium containing 0.3% bile salts. % Was inoculated and incubated for 2 hours at 37°C. Samples immediately after inoculation of lactic acid bacteria and after 2 hours of culture were collected, diluted in MRS liquid medium, plated on MRS plate medium, incubated at 37°C for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of lactic acid bacteria. The number of lactic acid bacteria in the experimental group, which was performed in the same manner in the MRS liquid medium without 0.3% bile hydrochloric acid, and the number of lactic acid bacteria after 2 hours incubation in the MRS liquid medium containing 0.3% bile hydrochloric acid, were compared and shown in Table 7 below.

Lactobacillus (CFU/ml) LMT1-30 LMT1-48 LMT2-46 Control 7.2x10 ⁹ 5.1x10 ⁹ 1.3x10 ⁹ 0.3% bile salt 5.2x10 ⁷ 4.8x10 ⁸ 3.8x10 ⁹

As a result, since the lactic acid bacteria selected in the present invention maintained an appropriate number of lactic acid bacteria even at 0.3% higher than 0.1% similar to the actual concentration in the intestine, it can be confirmed that the three types of lactic acid bacteria can sufficiently survive in the intestine of humans or animals. It can be a basis.

(3) Investigation of intestinal fixation of lactic acid bacteria

The Caco-2 cell line was used in human epithelial colorectal adenocarcinoma cells purchased from the Korea Cell Line Bank to confirm the degree of fixation in the intestinal cells. Caco-2 cells were Dulbecco's modified Eagle's medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and _{were cultured at 5% CO 2} and 37°C. L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46 cultured in MRS medium were washed with PBS (Phosphate Buffered Saline), followed by DMEM without antibiotics. Suspended in a medium, and lactic acid bacteria were added to 1 ^{×10 7} CFU to Caco-2 cells forming a single layer on a 96-well cell culture plate (Corning, USA), and cultured for 2 hours at _{5% CO 2 and 37°C.} I did. To remove lactic acid bacteria that could not adhere to Caco-2 cells, wash 5 times with PBS, remove the lactic acid bacteria attached with 100 µl of 0.1% Triton X-100, and spread them on MRS solid medium for 24 hours at 37°C. After culturing during, the number of colonies on the plate medium was counted to investigate the intestinal fixation of lactic acid bacteria.

The results are shown in FIG. 3. FIG. 3 is a diagram showing the number of selected three microorganisms attached to intestinal epithelial cells. As shown in Figure 3, the novel lactic acid bacteria of the present invention L. paracasei LMT1-30 is 56.8%, L. plantarum LMT1-48 is 106.3%, and P. ashdilactici LMT2-46 is 98.9%. It was confirmed that the intestinal epithelial cells, Caco-2, had a fixative power.

Example 2. Effect of strain administration on obesity

1. Securing cell extracts of lactic acid bacteria

(1.1) L. Paracasei LMT1 -30 cell extract preparation

L. paracasei LMT1-30 was cultured in MRS medium at 37° C. for 24 hours. After centrifuging the cells, they were rinsed with PBS to remove the remaining medium components. After resuspending the lactic acid bacteria from which the medium was removed in a volume ratio of lactic acid bacteria: PBS: beads = 1: 2: 1, a protein degradation inhibitor 100 mM phenylmethylsulfonyl fluoride (PMSF) was added, and a bead beater was used. The cells were lysed using. After centrifugation at 10000 rpm for 5 minutes, the supernatant was filtered through a 0.2 µm filter to quantify protein, and 200 µg/ml of L. paracasei LMT1-30 cell extract was used in the experiment.

(1.2) L. Planta Room LMT1 -48 cell extract preparation

L. Plantarum LMT1-48 was incubated in MRS medium at 37° C. for 24 hours. After centrifuging the cells, they were rinsed with PBS to remove the remaining medium components. After resuspending the lactic acid bacteria from which the medium was removed in a volume ratio of lactic acid bacteria: PBS: beads = 1: 2: 1, a protein degradation inhibitor 100 mM phenylmethylsulfonyl fluoride (PMSF) was added, and a bead beater was used. The cells were lysed using. After centrifugation at 10000 rpm for 5 minutes, the supernatant was filtered through a 0.2 μm filter to quantify protein, and 200 μg/ml of L. plantarum LMT1-48 cell extract was used in the experiment.

(1.3) P. Ashdi Lactic LMT2 -46 cell extract preparation

P. ashdilactici LMT2-46 was incubated in MRS medium at 37° C. for 24 hours. After centrifuging the cells, they were rinsed with PBS to remove the remaining medium components. After resuspending the lactic acid bacteria from which the medium was removed in a volume ratio of lactic acid bacteria: PBS: beads = 1: 2: 1, a protein degradation inhibitor 100 mM phenylmethylsulfonyl fluoride (PMSF) was added, and a bead beater was used. The cells were lysed using. After centrifugation at 10000 rpm for 5 minutes, the supernatant was filtered through a 0.2 µm filter to quantify protein, and 200 µg/ml of P. ashdilactici LMT2-46 cell extract was used in the experiment.

(1.4) Preparation of mixture of lactic acid bacteria cell extract

Each of the lactic acid bacteria cell extracts of (1.1), (1.2) and (1.3) were mixed at the same weight ratio of 1:1:1 and used in the test as a cell extract of the complex lactic acid bacteria of the present invention.

2. 3 by selected lactic acid bacteria T3 - L1 Adipocyte differentiation inhibition ability measurement

(1) 3 T3 - L1 Induction of adipocyte differentiation

DMEM (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) was used for 3T3-L1 cells, mouse embryonic fibroblasts purchased from the Korea Cell Line Bank (KCLB). % CO ₂ and cultured at 37°C.

The procedure for differentiating 3T3-L1 cells into adipocytes is as follows. 2 days after 3T3-L1 cells grew confluently (day 0), the culture medium was added to 10% FBS, 5 µg/ml insulin (Sigma, USA), 0.5 mM isobutylmethylxanthine (IBMX) (Sigma , USA) and 1 μM dexamethanesone (Sigma, USA) was exchanged with a DMEM medium, that is, a differentiation medium, and cultured. After 2 days (day 2), the culture was performed after exchange with DMEM medium containing 10% FBS and 5 μg/ml insulin. After 2 days (day 4), the culture was performed by changing to a medium containing only 10% FBS. After 4 days (day 8), more than 90% of 3T3-L1 cells were completely differentiated into adipocytes. Until 3T3-L1 cells completely differentiate into adipocytes (day 0 to day 8), they are termed maturing 3T3-L1 pre-adipocytes, and after fully differentiated into adipocytes ( After day 8), they are called mature 3T3-L1 adipocytes.

(2) 3 T3 - L1 In fat cells Measurement of ability to inhibit fat accumulation (Oil-red-O staining assay)

Each lactic acid bacterium cell extract was added together at 200 µg/ml at the time point when the differentiation medium was added to the cultured 3T3-L1 adipocytes (day 0). After 6 days (day 6), the resulting maturing 3T3-L1 pre-adipocyte culture solution was washed 3 times with PBS to remove the culture solution, and 10% formalin (Merck millipore, Germany) The cells were fixed by adding for 1 hour. Thereafter, the oil red O (Sigma, USA) solution was reacted with the cells for 1 hour, washed with distilled water, stained with fat globules, and observed under a microscope (Figs. 4A to 4F), and isopropanol (Duksan, South Korea) was added. The fat bulb sample stained with oil red O solution was dissolved, and absorbance was measured at 520 nm using a spectrophotometer (SPECTROstar Nano, Germany) (FIG. 4g).

The results are shown in FIG. 4. FIG. 4 is a diagram showing an electron micrograph (FIGS. 4A to 4F) and the degree of fat accumulation in the cells (FIG. 4G) of adipocytes of 3T3-L1 adipocytes cultured in the presence of a culture medium of three selected microorganisms. As shown in FIG. 4, compared to the control group, the group treated with L. paracasei LMT1-30 cell extract was 44.8%, the group treated with L. plantarum LMT1-48 cell extract was 49.7%, and P. axidi In the group treated with the LMT2-46 cell extract, the fat accumulation was reduced by 53.4%, and in the group treated with the complex lactic acid bacteria cell extract, 53.6%.

(3) 3 T3 - L1 Related to intracellular obesity Biomarker Gene expression Significant Measure the difference

Representative genes involved in the process of differentiating 3T3-L1 cells into adipocytes include PPARν, C/EBPα, FAS, and FABP4. The effect of inhibiting 3T3-L1 adipocyte differentiation using the cell extracts of L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46 showed the mRNA expression of the gene. Real-time PCR was performed to confirm whether it was indicated by inhibition.

At the time point when the cultured 3T3-L1 adipocytes were treated with the differentiation medium (day 0), the lactic acid bacteria cell extract was treated with 200 µg/ml. After 6 days (day 6), the resulting mature 3T3-L1 adipocytes were washed three times with PBS to remove the lactic acid bacteria cell extract, and RNA was extracted using Trizol (Thermo scientific, USA). Then, after obtaining DNA complementary to RNA using RocketScript Cycle RT Premix (Bioneer, South Korea), expression of adipose cell differentiation-related genes was confirmed using SYBR green (Takara, Japan). The results are shown in FIG. 5. FIG. 5 shows the results of confirming the expression of PPARν, C/EBPα, FAS, and FABP4 genes related to obesity in 3T3-L1 adipocytes cultured in the presence of a culture medium of the selected three microorganisms at the mRNA level.

As shown in Figure 5, compared to the control group treated with lactic acid bacteria extract, the mRNA expression levels of the PPARν, C/EBPα, FAS, and FABP4 genes were 69.1% and 66.8%, respectively, in the group treated with L. paracasei LMT1-30 cells. , 55.5%, and 73.9%, respectively, and the group treated with L. plantarum LMT1-48 cells treated 78.6%, 78.8%, 68.8%, and 85.4%, respectively, P. ashdilactici LMT2-46 cells. One group was observed to be inhibited by 72%, 73.1%, 62.4%, and 84.5%, respectively, and 75.5%, 73%, 63.1%, and 74.4% respectively in the group treated with the complex lactic acid bacteria cell extract.

In addition, compared to the control group in the lactic acid bacteria culture solution treatment group, the mRNA expression levels of the PPARν, C/EBPα, FAS, and FABP4 genes were 21.6%, 37.9%, 11%, and 29% respectively in the L. paracasei LMT1-30 culture solution treatment group. The L. plantarum LMT1-48 culture solution treatment group decreased by 21.9%, 50.1%, 38.2%, and 47%, respectively, and the P. ashdilactici LMT2-46 culture solution treatment group increased by 9.2% and 27.5%, respectively. , 32.9%, and 21.9% reduction, and the complex lactic acid bacteria culture solution treatment groups were found to inhibit 2.8%, 27.5%, 17.2%, and 22.7%, respectively.

Table 8 shows the primers used in real-time PCR, and the mRNA expression of the GAPDH gene is an internal control.

number gene primer Sequence number One
PPARν
Forward 4 Reverse 5 2
C/EBPα
Forward 6 Reverse 7 3
FAS
Forward 8 Reverse 9 4
FABP4
Forward 10 Reverse 11 5
GAPDH
Forward 12 Reverse 13

3. Evaluation of In Vivo Efficacy of Selected Lactobacillus in C57BL /6J Mouse Model Induced by Obesity Bacteria E. cloacae

(One) C57BL Obesity induction and lactobacillus treatment in /6J mice

The animal used in the experiment was a 5-week-old C57BL/6J mouse (male, 20-22g) inducing obesity with a high fat diet purchased from Jackson Laboratory (Bar Harbor, USA) and fed for 1 week (Teklad global diet 2014, Envigo). , USA) to adapt to the environment. Afterwards, lactobacilli were administered directly into the stomach for 2 weeks using a sonde for oral administration once a day for the group to which lactic acid bacteria were administered, and after feeding the lactic acid bacteria, a high fat diet (Teklad custom diet TD. Induction of obesity was induced by administration of E. cloase in the same manner for 8 weeks.

Para L. casei LMT1-30 is ^{1.0 x 10 9 CFU / day,} L. Planta LMT1-48 room is ^{1.0 x 10 9 CFU / day,} P. Ke Sidi rakti when LMT2-46 is 1.0 x 10 ⁹ CFU / day, complex lactic acid bacteria (a 1:1:1 mixture by weight of L. paracasei LMT1-30, L. plantarum LMT1-48, and P. ashdilactici LMT2-46) is 1.0x10 ⁹ CFU/day , And E. cloase was administered orally at ^{1.0 x 10 8 CFU/day.} The group (n=8) consisted of a total of 7 groups as shown in Table 9 below.

P1 Normal dietary supplement group P2 High fat diet group P3 High Fat Diet + Enterobacter Cloase Administration group P4 High Fat Diet + Enterobacter Cloase + L. Paracasei LMT1 -30 administration group P5 High Fat Diet + Enterobacter Cloase + L. Planta Room LMT1 -48 administration group P6 High Fat Diet + Enterobacter Cloase + P. Ashdi Lactic LMT2 -46 administration group P7 High Fat Diet + Enterobacter Cloase + Complex lactic acid bacteria administration group

Body weight and diet were measured once a week, and after the end of the experiment period, animals were fasted for 12 hours and sacrificed by excessive anesthesia with zoletil and ethyl ether. The experimental animals were opened, whole blood was collected from the portal vein, and plasma was separated. Plasma and tissue samples were stored at -80°C until use.

(2) Measurement of weight change according to diet control

The weight of the experimental animals was measured at a constant time every day for the entire experimental period, and the results are shown in FIG. 6. 6 is a diagram showing the change in body weight over time when three kinds of selected microorganisms are administered in obesity-induced mice. In FIG. 6, the horizontal axis represents time (weeks), and the vertical axis represents the percentage weight gain, that is, (((experiment end point-high fat diet start date)/mouse weight at high fat diet start date) x 100.

As shown in FIG. 6, the weight gain in the group fed the obesity-induced high fat diet was increased, and the weight gain rate was further increased in the group fed E. chlorase along with the high fat diet. In the case of the high-fat diet and the group receiving oral administration of E. chlorase and lactobacilli, there was no significant difference in dietary intake compared to the group inducing obesity with the high-fat diet and high-fat diet and E. chlorase, but the weight was significantly greater. Decreased. Therefore, in the case of the L. paracasei LMT1-30, L. plantarum LMT1-48 administration group, P. ashdilactici LMT2-46 administration group, and the three lactic acid bacteria combination administration group, the weight gain rate by E. cloase, obese bacteria It has been found to inhibit the facilitating function.

(3) Measurement of the volume ratio of adipose tissue

After the end of the 10-week experiment period, the change in body fat in the total body volume was measured for mice in the 7 groups of experimental animals through PET-CT imaging. In general, between lumbar vertebrae 1 and 5 is regarded as an abdominal region (L1 to L5) and measured, the results are shown in FIG. 7. 7 is a diagram showing a decrease in body fat volume when three kinds of selected microorganisms are administered in obesity-induced mice. 7A is a view showing the total volume of body fat measured from lumbar spine No. 1 (L1) to lumbar spine No. 5 (L5). 7B is a diagram illustrating an example of a body fat scan area. As a scan area, L1 denotes the upper lumbar region of 1 (Lumbar 1 proximal end), and L5 denotes the lower lumbar region of lumbar 5 (Lumbar vertebrate distal end). Actually photographed experimental animals were photographed from L1 to L5 regions, and abdominal fat was shown in both visceral fat and subcutaneous fat in brown color. 7C shows an animal showing an average weight loss in each experimental group among the scan photos of the lumbar spine 2 (L2) area among the regions between L1 and L5, and compares the scan photos of the animals at the same location (L2). As shown in FIG. 7, in the group (P3) orally administered E. chloroacet together with a high-fat diet after obesity induction, the body fat ratio of the total volume was the highest, and the high-fat diet and E. chlorase and lactic acid bacteria were fed the highest. In the case of the orally administered group (P4~P7), the body fat ratio was low.

As shown in Fig. 7c, the visceral fat and subcutaneous fat of the group (P3) administered orally with E. chlorase together with the high-fat diet group and the high-fat diet (P2) showed an increase in both visceral fat and subcutaneous fat. LMT1-30 (P4), L. plantarum LMT1-48 administration group (P5), P. ashdilactici LMT2-46 administration group (P6), and three types of lactobacillus combination administration group (P7) inhibit body fat accumulation in the abdominal region. It can be seen that it represents.

(4) Serum analysis

After the end of the 10-week experiment period, blood samples collected from each mouse were collected in a tube, and the serum was separated by centrifugation. Total cholesterol, triglycerol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), leptin, and adiponectin contents were measured in the separated serum, and the results are shown in FIG. 8. Figure 8 is a case of administration of three selected microorganisms in obesity-induced mice, triglycerides in serum (Fig. 8a), total cholesterol (Fig. 8b), high-density lipoprotein (HDL) (Fig. Fig. 8d), leptin (Fig. 8e), and adiponectin (Fig. 8f) is a view showing the results of measuring the content.

As shown in FIG. 8, the levels of cholesterol, high-density lipoprotein, leptin, and adiponectin were increased in the group fed the obesity-inducing high-fat diet, and cholesterol, triglycerol, high-density lipoprotein, and the group fed E. chlorase with a high-fat diet were increased. The levels of leptin and adiponectin increased more. On the other hand, it can be seen that triglycerol and leptin significantly decreased in the group administered orally with lactic acid bacteria. It was found to be excellent in inhibiting the production of body fat, but also inhibiting the secretion of blood lipids and hormones secreted from fat. In particular, leptin was found to significantly ( p <0.05) lower blood levels than the high-fat diet group, suggesting that vital signs related to energy metabolism secreted from accumulated adipocytes were reduced. In the case of low-density lipoprotein, the effect of E. chlorase was not observed, and the effect of lactic acid bacteria was not observed.

(5) Obesity bacterial intestinal microbial analysis of changes in the Enterobacter chloride acetate-induced obese C57BL / 6J mouse model by

Before the end of the experiment in Example 2-3, the last feces were collected, and as for fecal samples, genomic DNA (gDNA) of microorganisms in feces was extracted using the UltraClean Fecal DNA Isolation Kit. The 16s rRNA gene was amplified by PCR using the extracted gDNA. The primers used at this time were barcoded primers 519F: SEQ ID NO: 14 and 806R: SEQ ID NO: 15, and the DNA product has a nucleotide sequence of about 450bp. Pyrosequencing was performed with the DNA product to analyze the intestinal flora distribution. Pyrosequencing was performed using Macrogen's MiSeq™ platform (Illumina, San Diego, USA).

The results of the analysis in which the intestinal microbial distribution is classified by class (strong) level are shown in Table 10 below.

P1 P2 P3 P4 P5 P6 P7 Bacteria 0.08% 0.06% 0.07% 0.04% 0.13% 0.02% 0.09% Actinobacteria 0.95% 0.72% 0.40% 0.69% 0.35% 0.17% 0.43% Bacteroidetes 0.43% 1.87% 2.13% 0.00% 0.00% 2.95% 1.77% Bacteroidia 73.30% 64.36% 61.80% 55.51% 53.22% 46.78% 64.39% Flavobacteriia 0.01% 0.05% 0.03% 0.13% 0.03% 0.03% 0.02% Deferribacteres 0.05% 0.00% 0.00% 0.59% 0.19% 1.80% 0.40% Alphaproteobacteria 0.75% 0.04% 0.06% 0.04% 0.01% 0.03% 0.12% Deltaproteobacteria 0.00% 0.00% 0.00% 0.00% 0.00% 0.00% 0.75% Gammaproteobacteria 0.12% 0.11% 0.23% 0.17% 2.73% 0.14% 0.17% Mollicutes 0.11% 2.91% 1.87% 3.62% 3.28% 0.67% 0.39% Verrucomicrobiae 0.00% 0.00% 0.00% 0.20% 1.62% 16.22% 5.57% Candidatus Saccharibacteria 1.87% 1.67% 1.27% 0.13% 0.51% 0.41% 0.18% Chloroplast 0.03% 0.02% 0.00% 0.00% 0.00% 0.00% 0.00% Firmicutes 0.33% 0.43% 0.36% 0.75% 0.70% 2.48% 0.61% Bacilli 7.80% 2.64% 1.87% 1.46% 2.53% 1.63% 2.26% Clostridia 13.69% 24.89% 29.76% 36.69% 34.53% 26.51% 22.69% Erysipelotrichia 0.49% 0.23% 0.14% 0.01% 0.17% 0.19% 0.16%

9 is a diagram showing the result of classifying intestinal microorganisms in feces at the class (strong) level when three types of selected microorganisms are administered in obesity-induced mice. As shown in FIG. 9, it was found that Bacteroidia , Erysipelotrichi , and Clostridia are dominant bacteria. As a result of classifying the intestinal microbes changed due to inhibition of the growth of obesity bacteria after ingestion of lactic acid bacteria, it was confirmed that the diversity of the intestinal microbes increased in the experimental groups P4 to P7 compared to the control groups P2 and P3 that consumed the high fat diet. In addition, 3 types of lactobacilli administered to the experimental group ( L. paracasei LMT 1-30, L. plantarum LMT 1-48, P. acidilactici LMT 2-46) were classified by species level to confirm the presence or absence of the intestines. As a result, it was confirmed that the ingested lactic acid bacteria remained. Table 11 is a table showing the residual levels in feces of the administered three microorganisms.

Confirmation of the residual lactic acid bacteria administered (Species level)% Species P1 P2 P3 P4 P5 P6 P7 L. paracasei
( LMT 1-30) 0 0 0 0.03 0 0 0.1 L. plantarum
( LMT 1-48) 0 0 0 0 0.22 0 0.02 P. acidilactici
( LMT 2-46) 0 0 0 0 0 0.01 0.01

Korea Research Institute of Bioscience and Biotechnology KCTC13023BP 20160513 Korea Research Institute of Bioscience and Biotechnology KCTC13024BP 20160513 Korea Research Institute of Bioscience and Biotechnology KCTC13025BP 20160513

<110> Mey-tox inc. <120> Microorganism capable of reducing body fat and use thereof <130> PN116027KR <160> 17 <170> KopatentIn 2.0 <210> 1 <211> 1166 <212> DNA <213> Lactobacillus paracasei LMT1-30 <400> 1 aacagatgct aataccgcat agatccaaga accgcatggt tcttggctga aagatggcgt 60 aagctatcgc ttttggatgg acccgcggcg tattagctag ttggtgaggt aatggctcac 120 caaggcgatg atacgtagcc gaactgagag gttgatcggc cacattggga ctgagacacg 180 gcccaaactc ctacgggagg cagcagtagg gaatcttcca caatggacgc aagtctgatg 240 gagcaacgcc gcgtgagtga agaaggcttt cgggtcgtaa aactctgttg ttggagaaga 300 atggtcggca gagtaactgt tgtcggcgtg acggtatcca accagaaagc cacggctaac 360 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggatt tattgggcgt 420 aaagcgagcg caggcggttt tttaagtctg atgtgaaagc cctcggctta accgaggaag 480 cgcatcggaa actgggaaac ttgagtgcag aagaggacag tggaactcca tgtgtagcgg 540 tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg gtctgtaact 600 gacgctgagg ctcgaaagca tgggtagcga acaggattag ataccctggt agtccatgcc 660 gtaaacgatg aatgctaggt gttggagggt ttccgccctt cagtgccgca gctaacgcat 720 taagcattcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 780 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 840 ttgacatctt ttgatcacct gagagatcag gtttcccctt cgggggcaaa atgacaggtg 900 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 960 acccttatga ctagttgcca gcatttagtt gggcactcta gtaagactgc cggtgacaaa 1020 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1080 tgctacaatg gatggtacaa cgagttgcga gaccgcgagg tcaagctaat ctcttaaagc 1140 cattctcagt tcggactgta ggctgc 1166 <210> 2 <211> 1159 <212> DNA <213> Lactobacillus plantarum LMT1-48 <400> 2 taataccgca taacaacttg gaccgcatgg tccgagcttg aaagatggct tcggctatca 60 cttttggatg gtcccgcggc gtattagcta gatggtgggg taacggctca ccatggcaat 120 gatacgtagc cgacctgaga gggtaatcgg ccacattggg actgagacac ggcccaaact 180 cctacgggag gcagcagtag ggaatcttcc acaatggacg aaagtctgat ggagcaacgc 240 cgcgtgagtg aagaagggtt tcggctcgta aaactctgtt gttaaagaag aacatatctg 300 agagtaactg ttcaggtatt gacggtattt aaccagaaag ccacggctaa ctacgtgcca 360 gcagccgcgg taatacgtag gtggcaagcg ttgtccggat ttattgggcg taaagcgagc 420 gcaggcggtt ttttaagtct gatgtgaaag ccttcggctc aaccgaagaa gtgcatcgga 480 aactgggaaa cttgagtgca gaagaggaca gtggaactcc atgtgtagcg gtgaaatgcg 540 tagatatatg gaagaacacc agtggcgaag gcggctgtct ggtctgtaac tgacgctgag 600 gctcgaaagt atgggtagca aacaggatta gataccctgg tagtccatac cgtaaacgat 660 gaatgctaag tgttggaggg tttccgccct tcagtgctgc agctaacgca ttaagcattc 720 cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg cccgcacaag 780 cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac cttaccaggt cttgacatac 840 tatgcaaatc taagagatta gacgttccct tcggggacat ggatacaggt ggtgcatggt 900 tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt 960 atcagttgcc agcattaagt tgggcactct ggtgagactg ccggtgacaa accggaggaa 1020 ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat 1080 ggatggtaca acgagttgcg aactcgcgag agtaagctaa tctcttaaag ccattctcag 1140 ttcggattgt aggctgcaa 1159 <210> 3 <211> 1429 <212> DNA <213> Pediococcus acidilactici LMT2-46 <400> 3 ttaattgatt atgacgtgct tgcactgaat gagattttaa cacgaagtga gtggcggacg 60 ggtgagtaac acgtgggtaa cctgcccaga agcaggggat aacacctgga aacagatgct 120 aataccgtat aacagagaaa accgcctggt tttcttttaa aagatggctc tgctatcact 180 tctggatgga cccgcggcgc attagctggt tggtgaggta acggctcacc aaggcgatga 240 tgcgtagccg acctgagagg gtaatcggcc acattgggac tgagacacgg cccagactcc 300 tacgggaggc agcagtaggg aatcttccac aatggacgca agtctgatgg agcaacgccg 360 cgtgagtgaa gaagggtttc ggctcgtaaa gctctgttgt taaagaagaa cgtgggtgag 420 agtaactgtt cacccagtga cggtatttaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt atccggattt attgggcgta aagcgagcgc 540 aggcggtctt ttaagtctaa tgtgaaagcc ttcggctcaa ccgaagaagt gcattggaaa 600 ctgggagact tgagtgcaga agaggacagt ggaactccat gtgtagcggt gaaatgcgta 660 gatatatgga agaacaccag tggcgaaggc ggctgtctgg tctgtaactg acgctgaggc 720 tcgaaagcat gggtagcgaa caggattaga taccctggta gtccatgccg taaacgatga 780 ttactaagtg ttggagggtt tccgcccttc agtgctgcag ctaacgcatt aagtaatccg 840 cctggggagt acgaccgcaa ggttgaaact caaaagaatt gacgggggcc cgcacaagcg 900 gtggagcatg tggtttaatt cgaagctacg cgaagaacct taccaggtct tgacatcttc 960 tgccaaccta agagattagg cgttcccttc ggggacagaa tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttattac 1080 tagttgccag cattcagttg ggcactctag tgagactgcc ggtgacaaac cggaggaagg 1140 tggggacgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 atggtacaac gagttgcgaa accgcgaggt ttagctaatc tcttaaaacc attctcagtt 1260 cggactgtag gctgcaactc gcctacacga agtcggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc atgagagttt 1380 gtaacaccca aagccggtgg ggtaaccttt taggagctag ccgtctaag 1429 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcatggtgcc ttcgctga 18 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tggcatctct gtgtcaacca tg 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 caagaacagc aacgagtacc g 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtcactggtc aactccagca c 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgggttctag ccagcagagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 accaccagag accgttatgc 20 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 agtgggcttt gccacaa 17 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggtgatttca tcgaattcca 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 aacgacccct tcattgac 18 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tccacgacat actcagcac 19 <210> 14 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cctacgggng gcwgcag 17 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gactachvgg gtatctaatc c 21 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 agagtttgat cmtggctcag 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggttaccttg ttacgactt 19

Claims (14)

Lactobacillus paracasei LMT1-30 (Accession No. KCTC13023BP). The microorganism according to claim 1, which has antibacterial activity against Enterobacter cloacae causing obesity. The microorganism according to claim 1, having an acid resistance, wherein the acid resistance is viable in an MRS medium at pH 2.5 and 37 DEG C for 2 hours or more. The microorganism according to claim 1, wherein the biliary acid tolerance is capable of surviving at 37 DEG C for at least 2 hours in an MRS medium containing 0.3% bile acid. The microorganism according to claim 1, having an activity of inhibiting the differentiation of lipid precursor cells into adipocytes. The microorganism according to claim 5, wherein the lipid precursor cell is 3T3-L1. The microorganism according to claim 1, which inhibits the accumulation of fat in adipocytes. The microorganism according to claim 1, which inhibits the expression of at least one gene selected from the group consisting of PPARv, C / EBPa, FAS, and FABP4 among adipocytes. The microorganism according to claim 1, which reduces one or more selected from the group consisting of body weight and volume of adipose tissue in the body when administered to a subject. The microorganism according to claim 1, wherein the microorganism reduces, when administered to the subject, one or more selected from the group consisting of blood triglycerol, and leptin content. A composition for use as a food product for reducing fat content in adipocytes, which comprises a microorganism which is Lactobacillus paracasei LMT1-30 (Accession No. KCTC13023BP) as an active ingredient. A composition for use as a food product for reducing fat content in a subject, which comprises as an active ingredient a microorganism which is Lactobacillus paracasei LMT1-30 (Accession No. KCTC13023BP). CLAIMS 1. A method for reducing fat content in adipocytes comprising contacting a microorganism that is Lactobacillus paracasei LMT1-30 (Accession No. KCTC13023BP) with adipocytes, Lt; / RTI &gt; A method for reducing the fat content of a subject, comprising administering to the subject a microorganism that is Lactobacillus paracasei LMT1-30 (Accession No. KCTC13023BP), said subject being a non-human mammal / RTI &gt;

Images