STRAIN HAVING ABILITY TO INHIBIT OBESITY AND PHARMACEUTICAL COMPOSITION CONTAINING SAME

Abstract

Provided are gut microbes or cultures having anti-obesity efficacy and a pharmaceutical composition containing them. The gut microbes or cultures have anti-obesity efficacy by lowering fatty acid concentration of the gut fluid contents in a mammalian gastrointestinal tract for the prevention and treatment of obesity and obesity related diseases. This invention demonstrated that said gut microbes having anti-obesity efficacy by lowering fatty acid concentration of the gut fluid contents in the gastrointestinal tract of mammals have the anti-obesity efficacy as much as that of the representative anti-obesity drug, orlistat, without any side effects in animal experiments and clinical trials. Therefore, the gut microbes h can be used to develop universal anti-obesity drugs for obese patients, contributing greatly to the health of mankind.

Claims

1. A gut microbe or culture with anti-obesity efficacy by lowering fatty acid concentration in the gut fluid contents of in a mammalian gastrointestinal tract.

2. According to claim 1, wherein said gut microbe or culture with anti-obesity efficacy can be selected from the groups that include, but are not limited to, Lactobacillus species, Streptococcus species, and Lactococcus species.

3. According to claim 2, wherein said Lactobacillus can be selected from the groups that include, but are not limited to, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus alimentarius, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus pantheris, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus salivarius subsp. salicinius, Lactobacillus mali. and said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis, and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis.

4. According to claim 2, wherein said Lactobacillus species can be selected from the groups that include, but are not limited to, Lactobacillus reuteri JBD301 (KCTC 12606BP), Lactobacillus plantarum JBD302 (KCTC 12918BP), Lactobacillus casei JBD303 (KCTC 12919BP), Lactobacillus casei JBD304 (KCTC 12920BP), Lactobacillus paracasei JBD305 (KCTC 12921BP), Lactobacillus alimentarius JBD306 (KCTC 12922BP), Lactobacillus delbrueckii subsp. Lactis JBD307 (KCTC 12923BP), Lactobacillus pantheris JBD308 (KCTC 12924BP), Lactobacillus fermentum JBD309 (KCTC 12925BP), Lactobacillus rhamnosus JBD311 (KCTC 12926BP), Lactobacillus salivarius subsp. salicinius JBD312 (KCTC 12927BP) and Lactobacillus mali JBD313 (KCTC 12928BP), said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis JBD314 (KCTC 12929BP), and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis JBD315 (KCTC 12930BP).

5. A pharmaceutical composition containing a gut microbe or culture lowering fatty acid concentration in the gut fluid contents of in a mammalian gastrointestinal tract for the prevention or treatment of obesity or obesity-related diseases.

6. The pharmaceutical composition of claim 5, wherein said gut microbe or culture can be selected from the groups that include, but are not limited to, Lactobacillus species, Streptococcus species, and Lactococcus species.

7. The pharmaceutical composition of claim 6, wherein said Lactobacillus can be selected from the groups that include, but are not limited to, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus alimentarius, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus pantheris, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus salivarius subsp. salicinius, Lactobacillus mali. and said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis, and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis.

8. The pharmaceutical composition of claim 6, wherein said Lactobacillus species can be selected from the groups that include, but are not limited to, Lactobacillus reuteri JBD301 (KCTC 12606BP), Lactobacillus plantarum JBD302 (KCTC 12918BP), Lactobacillus casei JBD303 (KCTC 12919BP), Lactobacillus casei JBD304 (KCTC 12920BP), Lactobacillus paracasei JBD305 (KCTC 12921BP), Lactobacillus alimentarius JBD306 (KCTC 12922BP), Lactobacillus delbrueckii subsp. Lactis JBD307 (KCTC 12923BP), Lactobacillus pantheris JBD308 (KCTC 12924BP), Lactobacillus fermentum JBD309 (KCTC 12925BP), Lactobacillus rhamnosus JBD311 (KCTC 12926BP), Lactobacillus salivarius subsp. salicinius JBD312 (KCTC 12927BP) and Lactobacillus mali JBD313 (KCTC 12928BP), said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis JBD314 (KCTC 12929BP), and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis JBD315 (KCTC 12930BP).

9. Functional foods containing a gut microbe or culture lowering fatty acid concentration in the gut fluid contents of in a mammalian gastrointestinal tract for the prevention or treatment of obesity or metabolic diseases caused by obesity.

10. The functional foods of claim 9, wherein said gut microbe or culture can be selected from the groups that include, but are not limited to, Lactobacillus species, Streptococcus species, and Lactococcus species.

11. The functional food of claim 10, wherein said Lactobacillus can be selected from the groups that include, but are not limited to, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus alimentarius, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus pantheris, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus salivarius subsp. salicinius, Lactobacillus mali. and said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis, and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis.

12. The functional foods of claim 10, wherein said Lactobacillus species can be selected from the groups that include, but are not limited to, Lactobacillus reuteri JBD301 (KCTC 12606BP), Lactobacillus plantarum JBD302 (KCTC 12918BP), Lactobacillus casei JBD303 (KCTC 12919BP), Lactobacillus casei JBD304 (KCTC 12920BP), Lactobacillus paracasei JBD305 (KCTC 12921BP), Lactobacillus alimentarius JBD306 (KCTC 12922BP), Lactobacillus delbrueckii subsp. Lactis JBD307 (KCTC 12923BP), Lactobacillus pantheris JBD308 (KCTC 12924BP), Lactobacillus fermentum JBD309 (KCTC 12925BP), Lactobacillus rhamnosus JBD311 (KCTC 12926BP), Lactobacillus salivarius subsp. salicinius JBD312 (KCTC 12927BP) and Lactobacillus mali JBD313 (KCTC 12928BP), said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis JBD314 (KCTC 12929BP), and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis JBD315 (KCTC 12930BP).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 and FIG. 2 present the fatty acid concentration in the gut fluid contents (A) and body weight changes (B) of the host administered the said anti-obesity microbes following the method in Example 1.

[0031] FIG. 3 presents the gram staining images (A) and Transmission Electron Microscopy (TEM) images (B) of said anti-obesity microbes Lactobacillus reuteri JBD301 (KCTC 12606BP).

[0032] FIG. 4 presents the phylogenic tree of said anti-obesity microbes Lactobacillus reuteri JBD301 (KCTC 12606BP), identified by the screening by the method in Example 1.

[0033] FIG. 5 presents the capacity of lowering fatty acid concentration in vitro with the said anti-obesity microbes.

[0034] FIG. 6 presents the images of small intestine and large intestine used to measure the fatty acid concentration in the gut fluid contents of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP).

[0035] FIG. 7 presents the fatty acid concentration in the gut fluid contents of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP)(white: control, black: JBD301).

[0036] FIG. 8 presents the fatty acid amount in the gut fluid contents of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP)(white: control, black: JBD301).

[0037] FIG. 9 presents the absorbed amount of dietary fatty acids into the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP)(white: control, black: JBD301).

[0038] FIG. 10 presents the excreted amount of dietary fatty acids from the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP)(white: control, black: JBD301).

[0039] FIG. 11 presents the body weight changes of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 2 (: control, .circle-solid.: JBD301, : orlistat).

[0040] FIG. 12 presents clinical trial design for efficacy of said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 3.

[0041] FIG. 13 presents the assignment of control and experimental for subjects screened in this trial for efficacy of said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 3.

[0042] FIG. 14 presents the result of clinical trial for efficacy of said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 3.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0043] The currently available anti-obesity drugs have side effects or safety problems for long-term use. Previous attempts to develop safe anti-obesity drugs using appetite suppressant or lipase inhibitors have failed although the development is very urgent due to serious abuse problems with anti-obesity drugs.

[0044] Based on the understanding that previous anti-obesity drugs are absorbed into the body and causes various side effects due to the loss of appetite and inhibition of lipase inhibition, we attempted to confirm that symbiotic intestinal microorganisms that can reduce the fat absorption of the host can reduce obesity by reducing calorie intake.

[0045] Thus, in this invention, we conformed that 1) novel anti-obesity microbes were identified by screening intestinal microorganisms in the gastrointestinal tract, 2) novel anti-obesity microbes were colonized in the gastrointestinal tract of administered host, 3) novel anti-obesity microbes lowered the fatty acid concentration in the gut fluid content of host, 4) novel anti-obesity microbes reduced the absorption of dietary fat into the host but increase the secretion from the host, 5) novel anti-obesity microbes reduced the body weight of host.

[0046] Therefore, in the first aspect, the present invention relates to the gut microbes having anti-obesity efficacy by lowering fatty acid concentrations of the gut fluid contents in the gastrointestinal tract of mammals.

[0047] In this invention, said gut microbes can be selected from the groups that include, but are not limited to, Lactobacillus species, Streptococcus species, and Lactococcus species.

[0048] In this invention, said Lactobacillus species can be selected from the groups that include, but are not limited to, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus alimentarius, Lactobacillus delbrueckii subsp. Lactis, Lactobacillus pantheris, Lactobacillus fermentum, Lactobacillus rhamnosus, Lactobacillus salivarius subsp. salicinius, Lactobacillus mali. and said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis, and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis.

[0049] In this invention, said Lactobacillus species can be selected from the groups that include, but are not limited to, Lactobacillus reuteri JBD301 (KCTC 12606BP), Lactobacillus plantarum JBD302 (KCTC 12918BP), Lactobacillus casei JBD303 (KCTC 12919BP), Lactobacillus casei JBD304 (KCTC 12920BP), Lactobacillus paracasei JBD305 (KCTC 12921BP), Lactobacillus alimentarius JBD306 (KCTC 12922BP), Lactobacillus delbrueckii subsp. Lactis JBD307 (KCTC 12923BP), Lactobacillus pantheris JBD308 (KCTC 12924BP), Lactobacillus fermentum JBD309 (KCTC 12925BP), Lactobacillus rhamnosus JBD311 (KCTC 12926BP), Lactobacillus salivarius subsp. salicinius JBD312 (KCTC 12927BP) and Lactobacillus mali JBD313 (KCTC 12928BP), said Streptococcus species can be selected from the groups that include, but are not limited to, Streptococcus lutetiensis JBD314 (KCTC 12929BP), and said Lactococcus species can be selected from the groups that include, but are not limited to, Lactococcus lactis subsp. Lactis JBD315 (KCTC 12930BP).

[0050] In a preferred embodiment of the invention, said gut microbes can be any microbes derived from human gut microbiota or gastrointestinal tract of mammals, can be in mammalian gastrointestinal tract, can lower the fatty acid concentrations in the gut fluid content of gastrointestinal tract, thereby reducing the absorption of dietary fat to the host.

[0051] Preferably, said gut microbes show the following 4 characteristics that can help to solve the side effect problems with the current anti-obesity drugs.

[0052] First, said gut microbes are not absorbed into the body but colonize in the gut and therefore there is no side effects caused by the absorption as in the case of chemical-based anti-obesity drugs. Second, said gut microbes are not acting on central nervous system and therefore there is no side effects caused by abuse or long-term use as in the case of appetite suppression drugs. Third, said gut microbes are not acting on fat digestion and therefore there is no side effects such as diarrhea, fatty stool, indigestion as in the case of lipase inhibition drugs. Forth, said gut microbes are safe and beneficial microorganisms and belongs to GRAS category.

[0053] In another aspect, the present invention relates to pharmaceutical compositions containing a gut microbe or culture with anti-obesity efficacy by lowering fatty acid concentrations in the gut fluid contents of in a mammalian gastrointestinal tract for the prevention or treatment of obesity or metabolic diseases caused by obesity.

[0054] In another aspect, the present invention relates to functional food composition containing a gut microbe or culture with anti-obesity efficacy by lowering fatty acid concentrations in the gut fluid contents of in a mammalian gastrointestinal tract for the prevention or treatment of obesity or metabolic diseases caused by obesity.

[0055] Preferably, said microbes or cultures contained in pharmaceutical or functional food compositions can lower fatty acid concentrations of the gut fluid contents more than 5%, and said gut may include stomach, small intestine and large intestine.

[0056] Preferably, said microbes or cultures may be contained, but not restricted, at dose of 10.sup.310.sup.12 cfu/gram in pharmaceutical or functional food compositions.

[0057] In this invention, said pharmaceutical compositions could be formulated, but not limited to, with more than 1 kind of pharmaceutically acceptable carrier in addition to said microbes or cultures having anti-obesity efficacy by lowering fatty acid concentrations of the gut fluid contents in the gastrointestinal tract.

[0058] In this invention, said functional foods are foods supplemented with functional ingredients and include, but not limited to, health food, nutraceuticals, dietary supplement, pharmabiotics. Preferably, said functional ingredients is for prevention and/or treatment of obesity and related metabolic syndrome.

[0059] Said functional foods include, but not limited to, dairy food (milk, soy milk, processed milk), fermented milk (drinking yogurt, set curd yogurt), drink, and soda. Said functional foods can include various supplementary components in addition to the functional ingredients. In the case of the functional food of the present invention, it is preferable to use additional supplements that include, but not limited to, vegetable creamer, skim milk powder, sugar, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3, Vitamins, and minerals such as copper, calcium, iron, magnesium, potassium, and zinc. In addition, said functional foods of the present invention may contain various flavors or natural carbohydrates as additional components as in the case of ordinary beverages. Said flavor agents may include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. Said natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

[0060] Said pharmaceuticals and functional foods in this invention contain said gut microbes having anti-obesity efficacy, and thus the administration of said pharmaceuticals and functional foods reduce the absorption of dietary fat into the host, contributing the prevention and/or treatment of obesity and related metabolic syndromes.

[0061] The technical idea of the present invention for the treatment of obesity can be applied to any gut microbes with anti-obesity efficacy by lowering fatty acid concentrations of the gut fluid contents in the gastrointestinal tract of mammals and will be apparent to those skilled in the art to which the present invention pertains.

EXAMPLES

[0062] The present invention may be better understood with reference to the accompanying examples that are intended for purposes of illustration only and should not be construed to limit the scope of the invention, as defined by the claims appended hereto.

Example 1: Screening for Anti-Obesity Gut Microbes

[0063] Screening for anti-obesity gut microbes had performed as following. Lactobacillus strains were isolated from the feces of healthy lean adult volunteers. Feces of healthy lean adult volunteers were diluted using Man-Rogosa-Sharpe (MRS, Difco.) broth and inoculated onto MRS agar for incubation at 37 C. in anaerobic jar (Gas-Pack anaerobic systems, BBL). After 48 hours, colonies on MRS agar were inoculated into MRS media in 96-well plate for anaerobic incubation at 37 C. The obtained cultures were subjected to high-throughput screening using EnzyChrom FFA Assay Kit (Bio-Assay Systems). MRS broth 250 l with 0.5% (w/v) Brij 58 and 0.25 mM sodium palmitate was added into each well of 96-well plate and 10 l culture was added. After anaerobic incubation for 15 hours at 37 C., the plate was centrifuged at 4,000 rpm for 10 minutes. The amount of fatty acid in the supernatant was determined with EnzyChrom FFA Assay Kit (Bio-Assay Systems) using absorbance at 570 nm.

[0064] Animal experiments were done to assess the anti-obesity efficacy of the candidate strains from the screening. The microbe culture powder was prepared by freeze-drying culture pellet with addition of non-fat skim milk and sugar up to 10%.

[0065] All animal care and use were performed strictly in accordance with the ethical guidelines by the Institutional Animal Care and Use Committee. Six-week-old C57BL/6 female mice (Joongang Experimental Animal Co., Seoul, Korea) were housed at 10 animals per cage with food (10% fat; D12450B; Research Diets Inc., New Brunswick, N.J., USA) and water available ad libitum under a 12-h light/12-h dark cycle at 22 C. and 55% humidity. After 1 week of acclimation, mice were randomly divided into with control or experimental group. Control mice were fed high-fat diet (45% kcal as fat, D12451, Research Diets Inc.) while experimental mice fed high-fat diet supplemented daily with 10.sup.7 CFU of Lactobacillus for 4 weeks. Body weight was monitored weekly.

[0066] After 3-week administration of Lactobacillus, mice were sacrificed under anesthesia. Both total fluid contents from small and large intestine were collected immediately and weighed. The gut fluid contents were collected with known volume of sterile saline. Then, the samples in saline were centrifuged at 10,000g for 30 min. After centrifugation, the supernatant was collected and analyzed for total fatty acid content using the EnzyChrom Free Fatty Acid Assay Kit (Bio-Assay Systems).

[0067] FIG. 1 (A) showed the fatty acid concentration (mM) in the gut fluid contents of the host administered the said anti-obesity microbes following the method in Example 1. Compared to the control, experimental group showed clear reduction of fatty acid concentration, particularly G5-1 at 66.8% of the control.

[0068] FIG. 1 (B) showed the body weight changes of the host administered the said anti-obesity microbes following the method in Example 1. Compared to the control, experimental group showed clear reduction of weight gain with the most reduction in G5-1. This indicates that lowering fatty acid concentration in the gut fluid content relates to body weight reduction of the host.

[0069] By screening more than 20,000 strains with the said methods, we were able to find various gut microbes that can lower fatty acid concentration, followed by administration into mice for 12 weeks to measure the fatty acid concentration in the gut fluid contents and body weight changes of the host, as shown in FIG. 2.

[0070] Experiment 1. Identification of Anti-Obesity Gut Microbes

[0071] The anti-obesity gut microbes, G5-1 (JBD301), JBD302-JBD309, JBD311-JBD315, were identified by screening as in Example 1. The taxonomic classification and phylogenetic analysis were based on the 16S rDNA gene and morphological observations. For morphological analysis, freshly cultured Lactobacillus was used for standard light microscopy after Gram staining as well as Transmission Electron Microscopy (TEM). FIG. 3 shows morphology of G5-1 (JBD301), confirming G5-1 as gram (+) Bacillus.

[0072] For 16S rRNA analysis, DNA was extracted from a G5-1 culture. For amplification of the 16s rDNA, PCR was performed in an automated thermal cycler with an initial denaturation at 95 C. for 5 min, followed by 30 cycles at 95 C. for 30 sec, 52 C. for 45 sec, 72 C. for 2 min, and then 72 C. for 10 min. For amplification of the 16s rDNA, universal primers (Sequence 1 and 2) were selected

[0073] [Sequence 1] 27 F: 5-AGA GTT TGA TCC TGG CTC AG-3

[0074] [Sequence 2] 1492R: 5-GGT TAC CTT GTT ACG ACT T-3

[0075] The obtained 16s rDNA sequences (Sequence 3-16) of G5-1 (JBD301), JBD302-JBD309, and JBD311-JBD315 were used to identify homologous sequences by searching the NCBI GenBank, DNA data bank of Japan, European Nucleotide Archive. After confirming novel species of the above microbes, G5-1 (JBD301) were deposited as NCBI GenBank Accession number KJ957189.

[0076] FIG. 4 presents the phylogenic tree of said anti-obesity microbes Lactobacillus reuteri JBD301 (KCTC 12606BP), identified by the screening by the method in Example 1. It was a novel species and deposited to Korean Collection for Type Cultures at Korea Research Institute of Bioscience & Biotechnology as KCTC 12606BP on Jul. 3, 2014.

[0077] Likewise, Lactobacillus plantarum JBD302 (KCTC 12918BP), Lactobacillus casei JBD303 (KCTC 12919BP), Lactobacillus casei JBD304 (KCTC 12920BP), Lactobacillus paracasei JBD305 (KCTC 12921BP), Lactobacillus alimentarius JBD306 (KCTC 12922BP), Lactobacillus delbrueckii subsp. Lactis JBD307 (KCTC 12923BP), Lactobacillus pantheris JBD308 (KCTC 12924BP), Lactobacillus fermentum JBD309 (KCTC 12925BP), Lactobacillus rhamnosus JBD311 (KCTC 12926BP), Lactobacillus salivarius subsp. salicinius JBD312 (KCTC 12927BP) and Lactobacillus mali JBD313 (KCTC 12928BP), Streptococcus lutetiensis JBD314 (KCTC 12929BP), and Lactococcus lactis subsp. Lactis JBD315 (KCTC 12930BP) were deposited to Korean Collection for Type Cultures at Korea Research Institute of Bioscience & Biotechnology on Oct. 13, 2015.

[0078] Experiment 2. Acid Production of Anti-Obesity Gut Microbes

[0079] To test acid production, JBD301 microbe was cultured at 37 C. in MRS broth. The cell culture was inoculated into bottles containing 100 ml of sterile reconstituted skim milk (10%) and glucose (2%). The pH changes of the fermented milk were determined after incubation for 24, 48 and 72 h. The average value with 10 samples per group was indicated as meanstandard deviation, as shown in Table 1.

TABLE-US-00001 TABLE 1 Time (hr) JBD 301-fermented yogurt pH 24 4.404 0.034 48 4.092 0.059 72 4.048 0.012

[0080] As shown in Table 1, pH reduction by acid production was observed.

[0081] Experiment 3. In Vitro Evaluation of Lowering Fatty Aicd Concentration by Anti-Obesity Gut Microbes

[0082] JBD301 was inoculated onto MRS agar for incubation at 37 C. in anaerobic jar (Gas-Pack anaerobic systems, BBL). After 48 hours, colonies on MRS agar were inoculated into MRS media in 96-well plate for anaerobic incubation at 37 C. The obtained culture was subjected to EnzyChrom FFA Assay Kit (Bio-Assay Systems) as in Example 1. The amount of fatty acid in the supernatant was determined with EnzyChrom FFA Assay Kit (Bio-Assay Systems) using absorbance at 570 nm and the capacity of lowering fatty acid concentration by JBD301 was shown in FIG. 5.

[0083] As shown in FIG. 5, fatty acid concentration in the media of JBD301 group was 55% of the control, which is statistically significant (p=0.01). Also, fatty acid concentrations in the media of other JBD group, JBD302-JBD309, JBD311-JBD315 was also lowered than that of the control group.

[0084] Experiment 4. In Vivo Evaluation of Lowering Fatty Acid Concentration in the Gut Fluid Content by Anti-Obesity Gut Microbes

[0085] Lactobacillus colonize in the small intestine, where fatty acids, digestion products of lipid, are mainly absorbed in the body. The decrease in the amount of fatty acids in the small intestine where most of the fatty acid is absorbed reduces the amount of fat that can be absorbed by the human body, thus reducing the caloric intake of the host. To confirm this, the concentration and amount of fatty acids in the intestinal fluid (gut fluid content) were measured.

[0086] Six-week-old C57BL/6 female mice (Joongang Experimental Animal Co., Seoul, Korea) were housed at 7 animals per cage with food (10% fat; D12450B; Research Diets Inc., New Brunswick, N.J., USA) and water available ad libitum under a 12-h light/12-h dark cycle at 22 C. and 55% humidity. After 1 week of acclimation, mice were randomly divided into with control or experimental group. Control mice were fed high-fat diet (45% kcal as fat, D12451, Research Diets Inc.) while experimental mice fed high-fat diet supplemented daily with 10.sup.7 CFU of JBD301 for 3 weeks. After 3-week administration of Lactobacillus, mice were sacrificed under anesthesia. Both total fluid contents from small and large intestine were collected immediately and weighed. The gut fluid contents were collected with known volume of sterile saline. Then, the samples in saline were centrifuged at 10,000g for 30 min. After centrifugation, the supernatant was collected and analyzed for total FFAs content using the EnzyChrom Free Fatty Acid Assay Kit (Bio-Assay Systems), as shown in FIGS. 7 and 8.

[0087] FIG. 6 shows the images of small intestine (a, b) and large intestine (c, d) used to measure the fatty acid concentration in the gut fluid content.

[0088] FIG. 7 presents the fatty acid concentration in the gut fluid contents of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP). The fatty acid concentration in the small intestine of JBD301 group was 69% of the control, which is statistically significant difference (p=0.022), while that in the large intestine was not much different (p=0.158).

[0089] FIG. 8 presents the fatty acid amount in the gut fluid contents of the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP). The fatty acid amount in the small intestine of JBD301 group was 59% of the control, which is statistically significant difference (p=0.01), while that in the large intestine was not much different (p=0.153).

[0090] Experiment 5. In Vivo Evaluation of Lowering Fatty Acid Absorption and Increasing Fatty Acid Excretion by Anti-Obesity Gut Microbes

[0091] To evaluate lowering of fatty acid absorption in the host after administration of JBD301, mice were fed high-fat diet supplemented with JBD301 for 3 weeks and then 14C-radiolabeled triolein was orally administered as in Experiment 4. Blood was collected at times (2h96 h), mixed with liquid scintillation cocktail, and radioactivity from 14C was measured with Microplate Scintillation Counter (PerkinElmer), as shown in FIG. 9.

[0092] FIG. 9 confirmed that the radioactivity of the blood in JBD301 group was decreased at all times compared to the control group. Particularly, it decreased down to 62% compared to that of the control, showing significant difference at 4 hours after intake of radiolabeled food (p=0.048).

[0093] Also, feces were collected to evaluate the excretion amount of fatty acid from the host after administration of JBD301. Fecal samples at times were added to Solvable (PerkinElmer), mixed for 1 hour at 60 C., and bead homogenized samples were centrifuged at 13,000 rpm for 5 minutes. Then, the supernatant was added with 1/10 (v/v) H.sub.2O.sub.2, mixed for 10 minutes at 50 C. and centrifuged at 13,000 rpm for 5 minutes. The obtained supernatant was added with liquid scintillation cocktail and 14C radioactivity was measured with Microplate Scintillation Counter (PerkinElmer).

[0094] FIG. 10 shows the excreted amount of dietary fatty acids from the host administered the said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP). The radioactivity of the feces in JBD301 group was increased for 3 days compared to the control group. Particularly, it increased up to 177% compared to that of the control, showing significant difference (p=0.037).

Example 2. In Vivo Evaluation of Weight Control Efficacy by Anti-Obesity Gut Microbes

[0095] All animal care and use were performed strictly in accordance with the ethical guidelines by the Institutional Animal Care and Use Committee. Six-week-old C57BL/6 female mice (Joongang Experimental Animal Co., Seoul, Korea) were housed at 10 animals per cage with food (10% fat; D12450B; Research Diets Inc., New Brunswick, N.J., USA) and water available ad libitum under a 12-h light/12-h dark cycle at 22 C. and 55% humidity. After 1 week of acclimation, mice were randomly divided into with control or experimental groups. Control mice were fed high-fat diet (45% kcal as fat, D12451, Research Diets Inc.), JBD301 group fed high-fat diet supplemented with 10.sup.7 CFU of JBD301, and orlistat group fed high-fat diet supplemented with orlistat (Xenical, Roche) 50 mg/kg diet. Body weight changes were monitored for 4-week administration as shown in FIG. 11.

[0096] As shown in FIG. 11, there was clear weight reduction in diet-induced obese mice after daily administration of JBD301 at 110.sup.7 CFU and the degree of anti-obesity efficacy by Lactobacillus JBD301 was as much as that of mice fed anti-obesity drug, orlistat.

Example 3. Clinical Trial to Evaluate Weight Control Efficacy by Anti-Obesity Gut Microbes

[0097] Phase 2 clinical trials were performed to validate the efficacy of JBD301 as anti-obesity microbes (Clinical Research Information Service of Korea as KCT0000452). This is to determine whether the intake of JBD301 in overweight or obese subjects is effective in reducing body weight or body fat. The clinical trial design of JBD301 is shown in FIG. 12.

[0098] Participating subjects were recruited according to clinical trial protocols approved by the IRB. Study participants were enrolled for the study if they satisfied the following criteria: 1) written informed consent, 2) men or women 25-65 years of age, and 3) body mass index of 25-35 kg/m.sup.2. The exclusion criteria included the following: subjects who received antibiotics within 12 weeks prior to the first visit; subjects with current use of medications for body weight reduction (lipase inhibitors, anti-depressants, appetite suppressants), diuretics, contraceptives, steroids, and hormones; subjects who were involved in commercial diet programs or were under a diet formula within 12 weeks prior to the first visit; subjects with uncontrolled hypertension or diabetes; subjects with current medical conditions including cardiac, renal, hepatic, neurovascular, thyroid or parathyroid disorders/diseases; subjects with medical conditions including depression, schizophrenia, psychosis, alcohol abuse or drug abuse; subjects diagnosed with cancer within the past 5 years; subjects with hyper-allergic reactions to components in the testing food; subjects who were planning to reduce body weight by food and exercise during the clinical trial period; and subjects that were pregnant or breast-feeding.

[0099] The subjects were instructed to maintain their usual diet with no intake of probiotics, antibiotics or agents known to affect body weight. The enrolled subjects were simply randomized for parallel assignment. The study consisted of daily administration of 1 capsule of either Lactobacillus JBD301 (10[9] CFU) or placebo for 12 weeks without any dietary restriction. In a capsule of 450 mg, major ingredient, either JBD301 powder or non-dairy coffee creamer, is 12% while other excipients include 40% skim milk, 40% sucrose and 8% ascorbic acid.

[0100] FIG. 13 presents the assignment of control and experimental for subjects screened in this trial for efficacy of said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 3. The total 40 subjects were enrolled and 37 subjects finished the trial. The study participant's baseline characteristics were shown in Table 2.

TABLE-US-00002 TABLE 2 Group Variable L. reuteri JBD301 Control P value All participants 18 (48.6) 19 (51.4) 0.103 Sex, male 7 (38.9) 13 (68.4) Sex, female 11 (61.1) 6 (31.6) Age mean SEM 39.4 2.7 42.1 3.1 0.578 Height mean SEM 165.0 2.4 169.0 2.3 0.233 Weight mean SEM 79.6 3.4 80.1 3.0 0.822 BMI mean SEM 29.0 0.7 27.9 0.5 0.150

[0101] The primary outcome from this study was body weight in obese adults. The adipose tissue area and body fat mass of each subject were measured using computerized tomography (CT) and Dual-Emission X-ray Absorptiometry (DEXA), respectively. For the clinical data, the statistical analysis was performed using procedures in SAS (Version 9.2) and MedCalc (Version 11.6.0). Depending on the normality of the underlying data, the Mann-Whitney U test and the Wilcoxon Signed Rank test were used to perform statistical analyses.

[0102] The Mann-Whitney U test was used to compare the outcome variables. The percentage change in outcome variables at 12 weeks (%) was calculated by the following formula: ((value at 12 weeksvalue at 0 week)/value at 0 week)100. Without any dietary restrictions or additional exercise, changes in the body weight from baseline to endpoint were 0.31% (0.21 kg) in the Lactobacillus JBD301 group and 1.77% (1.45 kg) in the placebo group, resulting in a 1.46% (1.24 kg) between-group difference. A Mann-Whitney U test showed that there was a statistically significant difference in the percentage change of weight between the Lactobacillus JBD301 and the placebo group (P=0.026) as well as in the BMI (P=0.036) from the 0 week assessment to the 12 weeks assessment.

TABLE-US-00003 TABLE 3 (Unit: %) L. reuteri JBD301 Control Mean Mean % change in Variable n SEM n SEM P value Total abdominal Fat 18 1.64 4.26 19 3.45 4.23 1.000 area (cm.sup.2) Body fat mass (g) 18 0.38 2.09 19 3.41 1.17 0.053 Weight (kg) 18 0.31 0.71 19 1.77 0.40 0.026* BMI (kg/m.sup.2) 18 0.32 0.76 19 1.65 0.46 0.036* Hip circumstance (cm) 18 0.20 0.45 19 0.76 0.31 0.126 HbA1c (%) 18 1.61 1.02 19 1.39 1.16 0.118 *P values were obtained from Mann-Whitney U test, significant difference if P value is 0.05.

[0103] The Wilcoxon Signed Rank test was used to compare the percentage differences in outcome variables for the two treatment groups. The percentage differences in outcome variables were calculated by the following formula: (value of experimental/value of placebo)100. Wilcoxon Signed Rank test confirmed that there was a statistically significant difference in the pairwise comparison of the percentage between 97.94% of JBD301 group (n=18) and 100% of control group (n=19)(P=0.028).

TABLE-US-00004 TABLE 4 (Unit: %) 0 week 12 week P value % control in body weight 99.45 4.26 97.94 4.21 0.028* *P values were obtained from Wilcoxon signed rank test, significant difference if P value is 0.05. Values are mean SEM.

[0104] FIG. 14 presents the result of clinical trial for efficacy of said anti-obesity Lactobacillus reuteri JBD301 (KCTC 12606BP) following the method in Example 3. FIG. 14 confirms that there was a statistically significant difference in body weight of JBD301 group from the control group in clinical trial. Body weight or body fat is significantly affected by factors that often change due to external environmental factors such as food intake or exercise habits. Considering this, the difference between the control and experimental groups is very important.