KR101228035B1 - New lactobacillus strains and compositions for preventing or treating diabetes comprising the same - Google Patents

Abstract

The present invention is Streptococcus thermophilus ST3 ( Streptococcus thermophilus ST3) (KCTC 11870BP), Lactobacillus rhamnosus LR3 ( Lactobacillus rhamnosus LR3) (KCTC 11868BP) and Lactobacillus paracasei LPC4 ( Lactobacillus paracasei LPC4) (KCTC 11866BP) relates to lactic acid bacteria strains and compositions for the prevention and treatment of diabetes comprising the same, the novel lactic acid bacteria strains and compositions of the present invention As well as excellent hypoglycemic effect as well as histopathological treatment of the size and kidney of Langeshans Island can be useful for preventing or treating diabetes.

Description

Novel Lactobacillus STRAINS AND COMPOSITIONS FOR PREVENTING OR TREATING DIABETES COMPRISING THE SAME

The present invention relates to novel lactic acid bacteria strains and compositions for preventing and treating diabetes comprising the same, and more particularly, to a unique lactic acid bacteria strain and a mixed lactic acid containing composition useful for the prevention and treatment of diabetes, including the same.

Diabetes is a disease that includes a variety of factors that are characterized by high blood sugar levels and metabolic dysfunction, and is a serious chronic metabolic disorder that has a significant impact on the quality of life and health of the patient as well as the health care system. Diabetes is caused by a decrease in the effect of insulin on target cells due to an absolute or relative lack of insulin or an increase in insulin tolerance. Diabetes is classified into types 1 and 2, with the overall prevalence of diabetes being approximately 6% of the population, of which 90% is type 2 diabetes.

Chronic and severe diabetes has serious side effects that have a significant impact on the quality of life of the patient. Serious complications include metabolic abnormalities and infections, and long-term complications include macrovascular complications (e.g. hypertension, dyslipidemia, myocardial infarction, stroke), microvascular complications (e.g. retinopathy, Nephropathy, diabetic neuropathy, diarrhea, neurogenic bladder, impaired cardiovascular reflexes, sexual dysfunction and foot disorders.

Common treatments for diabetes focus on managing lifestyle. Conventional treatments include oral glucose lowering drugs and insulin injections, in addition to exercise, weight control and medical nutrition. Therapies currently used with oral hypoglycemic agents usually involve side effects such as weight gain, hypoglycemia, gastrointestinal disorders, hepatic toxicity and high LDL cholesterol. On the other hand, insulin therapy causes side effects such as weight gain and hypoglycemia.

For example, acarbose, one of many drugs used to treat diabetes, inhibits carbohydrate digestive enzymes, which can break down undigested carbohydrates into carbon dioxide and hydrogen, causing gastrointestinal flatulence. .

In view of the significant side effects associated with conventional therapies, other approaches for the treatment of diabetes with antihyperglycemic activity have been studied by patients and healthcare professionals. No scientific or medical research has been done to find them.

The present invention is to meet the technical needs of the technical field to which the present invention belongs, one object of the present invention can minimize the side effects of oral hypoglycemic agents and enhance the hypoglycemic effect to prevent or treat diabetes It is to provide a novel lactic acid bacteria strains that can be usefully used and compositions for preventing and treating diabetes comprising the same.

One aspect of the present invention for achieving the above object is Streptococcus thermophilus ST3 ( Streptococcus internationally deposited as an accession number KCTC 11870BP to the Korea Collection for Type Culture (KCTC) thermophilus ST3) relates to lactic acid bacteria.

Another aspect of the present invention relates to Lactobacillus rhamnosus LR3 (Lactobacillus rhamnosus LR3) lactic acid bacteria, which has been deposited internationally as Accession No. KCTC 11868BP with the Korea Research Institute of Bioscience and Biotechnology.

Another aspect of the present invention for achieving the above object is Lactobacillus paracaze LPC4 ( Lactobacillus Lactobacillus Internationally deposited as an accession number KCTC 11866BP to the Korea Biotechnology Research Institute microbial resource center paracasei LPC4) related to lactic acid bacteria.

Another aspect of the present invention for achieving the above object is

Streptococcus stand a brush Ruth ST3 (Streptococcus thermophilus ST3) (KCTC 11870BP);

Lactobacillus Lactobacillus rhamnosus LR3) (KCTC 11868BP); And

Lactobacillus casei para LPC4 (Lactobacillus It relates to a composition for preventing and treating diabetes, including paracasei LPC4) (KCTC 11866BP).

The composition for preventing and treating diabetes comprising the mixed lactic acid bacteria of the present invention may further include other lactic acid bacteria and / or pharmaceutically acceptable carriers or excipients.

The novel lactic acid bacterium strains of the present invention and the composition for preventing and treating diabetes comprising the same have an excellent effect of promoting not only an excellent hypoglycemic effect, but also normalizing blood lipid levels and blood pressure, maintaining proper weight, delaying and preventing complications. Since it provides, can be usefully used for the purpose of preventing or treating diabetes. The composition of the present invention can also be usefully used as a composition for the improvement and treatment of obesity through blood sugar control.

1 compares the 16S rRNA sequences of Streptococcus thermophilus ST3 strain (KCTC 11870BP) and Streptococcus thermophilus ^T (ATCC19258) strains.
Figure 2 is a result of random amplified polymorphic DNA (RAPD) analysis of genome DNA of Streptococcus thermophilus ST3 strain.
Figure 3 is a graph showing the results of the evaluation of acid resistance of Streptococcus thermophilus ST3 strain.
Figure 4 compares the 16S rRNA sequences of Lactobacillus rhamnosus LR3 strain (KCTC 11868BP) and Lactobacillus rhamnosus ^T (ATCC7469) strain.
5 is a result of RAPD analysis of genome DNA of Lactobacillus rhamnosus LR3 strain.
6 is a graph showing the results of evaluating acid resistance and bile resistance of the Lactobacillus rhamnosus LR3 strain.
7 is a graph showing the results of evaluation of intestinal fixability of Lactobacillus rhamnosus LR3 strain.
Figure 8 compares the 16S rRNA sequences of Lactobacillus paracasei LPC4 strain (KCTC 11866BP) and Lactobacillus paracasei isolate achiral.
9 is a result of RAPD analysis of genome DNA of Lactobacillus paracasei LPC4 strain.
10 is a graph showing the results of evaluating the bile resistance of Lactobacillus paracasei LPC4 strain.
11 is a graph showing the fasting blood glucose change after the administration of the sample of the Examples and Comparative Examples.
12 is a graph showing changes in blood glucose after meals after administration of samples of Examples and Comparative Examples.
It is the result of the tissue analysis of the Langerhans island of pancreas after sample administration of an Example and a comparative example.
It is the result of the tissue analysis of the kidney after sample administration of an Example and a comparative example.
FIG. 15 is a bar graph showing changes in blood cholesterol concentrations after administration of samples of Examples and Comparative Examples. FIG.

One aspect of the present invention relates to novel lactic acid bacteria strains useful for glycemic control and / or prevention and treatment of diabetes mellitus, which are described in the Korean Collection for Type Culture (KCTC). Deposited.

Streptococcus stand a brush Ruth ST3 (Streptococcus thermophilus ST3) (KCTC 11870BP);

Lactobacillus Lactobacillus rhamnosus LR3) (KCTC 11868BP); And

Lactobacillus casei para LPC4 (Lactobacillus paracasei LPC4) (KCTC 11866BP).

Another aspect of the invention relates to a composition comprising the novel strains of lactic acid bacteria described above, wherein the composition is Streptococcus thermophilus ST3 ( Streptococcus) thermophilus ST3) (KCTC 11870BP); Lactobacillus Lactobacillus rhamnosus LR3) (KCTC 11868BP); And Lactobacillus paracasei LPC4 (KCTC 11866BP).

The novel lactic acid bacteria strains of the present invention rapidly reduce blood glucose levels and also greatly reduce serum cholesterol levels when administered alone or in combination with commercially available diabetes therapeutics. In addition, the histological results show that the novel lactic acid bacteria strains of the present invention reduce the size of the island of Langerhans when administered alone or in combination with a diabetes treatment, and relieve the symptoms of diabetic nephropathy. It provides pathological therapeutic effect. Moreover, the three kinds of lactic acid bacteria strains used in the present invention can suppress the growth of harmful microorganisms in the intestine, thereby reducing side effects such as gastrointestinal disorders caused by oral hypoglycemic agents (eg, acarbose).

Streptococcus thermophilus ST3 ((KCTC 11870BP), Lactobacillus rhamnosus LR3 (KCTC 11868BP), and Lactobacillus paracasei LPC4 (KCTC 11866BP) strains of the present invention are excellent in acid resistance, bile resistance and intestinal fixation Therefore, it is possible to suppress intestinal pathogenic bacteria and provide beneficial effects such as intestinal and branch action.

The composition of the present invention may include a mixture of the three kinds of lactic acid bacteria strains as an active ingredient in an amount of 10 ⁸ to 10 ¹² cfu / g, or a culture having an equal number of live bacteria, based on the total weight of the composition. .

The compositions of the present invention field know, in addition to the above-mentioned lactic acid bacteria Lactobacillus Ruth (Lactobacillus acidophilus ), Lactobacillus plantarum ) , Bifidobacterium breb breve ) , B ifidobacterium lactis), ronggum Bifidobacterium (Bifidobacterium longum ),

Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus helvetikus (Lactobacillus helveticus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus casei (Lactobacillus casei), Lactobacillus del Bruieki (Lactobacillus delbrueckii), Lactobacillus reuteri (Lactobacillus reuteri), Lactobacillus bootsnery (Lactobacillus buchneri), Lactobacillus gasserole (Lactobacillus gasseri), Lactobacillus Jones (Lactobacillus johonsonii), Lactobacillus kefir (Lactobacillus kefir), Lactococcus lactis (Lactococcus lactis), Bifidobacterium bifidium (Bifidobacterium bifidum), Bifidobacterium infantis (Bifidobacterium infantis), Bifidobacterium chimelongum (Bifidobacterium pseudolongumBifidobacterium Thermophile Room (B)ifidobacterium themophilum), Bifidobacterium adolescents (Bifidobacterium adolescentisIt may further comprise one or more probiotic lactic acid bacteria selected from the group consisting of).

The lactic acid bacteria strains may be grown in a general culture method for lactic acid bacteria, recovered in a separation process such as centrifugation, and may be prepared and used in a probiotic form by drying, for example, lyophilization.

The compositions of the present invention may comprise the essential active ingredients described above as well as any additional or optional ingredients useful for nutritional or pharmaceutical uses not described or described herein. Many of these optional ingredients can be used in the compositions of the present invention as long as they are safe and effective for oral administration and are compatible with the essential and other optional ingredients of the compositions of the present invention.

Compositions of the present invention may further comprise conventional pharmaceutically acceptable carriers and / or excipients in addition to the active ingredient, in addition to binders, disintegrants, coatings, lubricants, preservatives, antioxidants, buffers, other pharmaceuticals It may be formulated and formulated with various optional pharmaceutically commonly used ingredients such as active agents, sweeteners, colorants, flavors, flavor enhancers, thickeners and stabilizers, emulsifiers and the like.

The composition of the present invention is a powder, granules, tablets, tablets, capsules using a method known in the art by mixing the three kinds of lactic acid bacteria strains with appropriate carriers, excipients, auxiliary active ingredients, other optional ingredients, etc. It may be formulated in the form of a pill or a liquid.

Excipients usable in the present invention include sugars such as sucrose, lactose, mannitol, glucose, and the like, starches such as corn starch, potato starch, rice starch, partially pregelatinized starch and the like. The binder is a naturally-occurring macromolecular substance such as dexatrin, sodium alginate, carrageenan, guar gum, acacia, agar, tragacanth, gelatin, gluten, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl Cellulose derivatives such as cellulose, ethyl cellulose, hydroxypropyl ethyl cellulose, carboxymethyl cellulose sodium, and polyvinylpyrrolidone, polyvinyl alcohol, polyvinylacetate, polyethylene glycol, polyacrylic acid, polymethacrylic acid and vinyl acetate resin It contains a polymer.

Degrading agents include cellulose derivatives such as carboxymethyl cellulose, carboxymethyl cellulose calcium, low-substituted hydroxypropyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, corn starch, potato starch, rice starch and partially pregelatinized starch. Starch can be used.

Examples of lubricants that can be used in the present invention include talc, stearic acid, calcium stearate, magnesium stearate, colloidal silica, hydrosilicic dioxide, various types of waxes and hydrogenated oils, and the like.

Coating agents include dimethylaminoethyl methacrylate-methacrylic acid copolymer, polyvinyl acetal diethylamino acetate, ethyl acrylate-methacrylic acid copolymer, ethyl acrylate-methyl methacrylate-chlorotrimethylammoniumethyl methacrylate. Water-insoluble polymers such as copolymers, ethyl cellulose, methacrylic acid-ethyl acrylate copolymers, hydroxypropyl methyl cellulose phthalates, hydroxypropyl methyl cellulose acetate succinate, and other polymers such as methyl cellulose, hydroxypropyl methyl cellulose Water-soluble polymers such as polyvinylpyrrolidone and polyethylene glycol, but are not necessarily limited thereto.

The composition of the present invention is enteric-coated so that when formulated for oral use, lactic acid bacteria are killed by gastric acid or bile acid and do not lose their original bioactive function, but are sufficiently decomposed in the small intestine so that the active ingredient is released into the small intestine. Can be. For example, the composition of the present invention may be used lactic acid bacteria double coated with proteins and polysaccharides, and if necessary, triple coated lactic acid bacteria having a nanoparticle coating layer formed on the double coated lactic acid bacteria. The double-coated lactic acid bacteria were subjected to enzyme treatment by adding proteolytic enzymes to the aqueous protein solution, and then fermented by adding glucose, yeast extract, meat extract, ionic components and lactic acid bacteria to the enzyme-treated protein aqueous solution to prepare a single coated lactic acid bacteria. Then, it can be prepared by coating a polysaccharide aqueous solution on a single coated lactic acid bacteria. Triple coated lactic acid bacteria can be prepared by coating nanoparticles on double coated lactic acid bacteria.

The dosage of the strain of lactic acid bacteria as an active ingredient in the composition for preventing and treating diabetes mellitus of the present invention may be appropriately determined in consideration of the purpose of treatment or prevention, the type of patient to be treated or prevented, the symptoms, weight, age or sex of the patient. Can be. In general, for adult patients, at least 1 × 10 ⁶ live cells, preferably 1 × 10 ⁸ to 1 × 10 ¹² live cells, can be administered once or divided as needed.

The particular dosage of each patient depends on a variety of factors, such as age, weight, general state of health, sex, diet, time and route of administration, rate of excretion, combination of medicinal substances and the severity of the particular disease being treated.

The composition for preventing and treating lactic acid bacteria-containing diabetes of the present invention may be administered or ingested as a drug or food to humans or animals. The drug containing the composition for preventing and treating lactic acid bacteria-containing diabetes of the present invention is one embodiment of the present invention, and the food containing the composition for preventing and treating lactic acid bacteria-containing diabetes of the present invention is also another embodiment of the present invention. Examples of such foods include, but are not limited to, yogurt and dairy products.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited by the following examples.

Example

One. Streptococcus Thermophilus ST3  ( Streptococcus thermophilus ST3 ) ( KCTC  11870 BP Identification and Characterization of

1-1. New microbial isolation

Goat milk collected from the ranch was diluted in sterile anaerobic water by serial dilution and 1 ml of each dilution was poured into ManRogosa Sharpe (MRS. BD. USA) solid medium and incubated at 37 ° C. for 3 days under anaerobic conditions. The resulting colonies were picked up again on the Ltobacillus selective medium to which BCP (Bromocresol purple, 0.17g / L) was added to MRS, and then incubated for 3 days under the same conditions. The lactic acid bacteria were selected from the medium for biochemical and molecular biological identification, and then one strain having the best physical properties of the strain was finally selected.

1-2. Identification of microorganisms

1) sugar usability analysis

The sugar availability was confirmed using the API CHL50 kit for the isolated strain. Sugar availability of the isolated strain is shown in Table 1 below. Biochemical properties based on sugar availability were determined by Lactobacillus Lactobacillus. delburueckii spp lactis ) and Streptococcus thermophilus were found to be similar, but the isolated strain was identified as Streptococcus thermophilus ( S. thermophilus ) because the isolate was morphologically coliform.

temperament reaction temperament reaction  Esculin -  Glycerol -  Salincin -  Erythritol -  Cellobiose -  D-Arabinose -  maltose -  L-arabinose -  Lactose +  Ribose -  Melibiose -  D-xylose -  Saccharose +  L-xylose -  Trehalose -  Andonitol -  Inulin -  β-methyl-xyloxide -  Melezitose -  Galactose +  D-rapinose -  D-glucose +  Amidon -  D-fructose +  Glycogen -  D-mannose +  Xylitol -  L-sorbose -  β-gentiobiose -  Rhamnose -  D-turanoose -  Dulcytol -  D-Rixos -  Inositol -  D-tagatose -  Mannitol -  D-fucose -  Sorbitol -  L-fucose -  α-methyl-D-mannoside -  D-arabitol -  α-methyl-D-glucoside -  L-arabitol -  N-acetyl glucosamine -  Gluconate -  Amigalline -  2-keto-gluconate -  Arbutin -  5-keto-gluconate -

2) 16s rRNA  Sympathy

Genome DNA was extracted from the isolated strain and 16s rRNA sequences were analyzed. Using the isolated DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template. PCR products were linked to the pGEM-Teasy vector (Promega, USA). E. coli strain DH5α was transformed and then plated on LB / x-gal / amp plates and incubated overnight at 37 ° C. After screening the recombinant plasmid containing the insert (insert) from the transformant, DNA sequencing was performed. DNA sequence analysis was determined using the method of DNA star Cluster V program streptococcus up a brush loose ^{T (S. thermophilus T) (ATCC19258} ) phase of the strain dongseongreul. 1 shows a comparison of 16s rRNA sequences of Streptococcus thermophilus ST3 strain (KTCC) and Streptococcus thermophilus ^T (ATCC19258) lactic acid bacteria of the present invention. The 16s rRNA sequence of the isolated strain showed 99.5% homology with Streptococcus thermophilus ^T (ATCC19258), confirming that the novel microorganism of the present invention belongs to Streptococcus thermophilus species.

3) RAPD ( Random Amplified Polymorphic DNA ) analysis

Genomic DNA was extracted from the isolated strain, and PCR-RAPD (MyCycler, BIO-RAD, USA) was performed using (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') primer as a template. 2 was stained with Et-Br (Ethidium bromide) and observed with G: BOX (SYNGENE, UK). The band pattern of the isolated strain of the present invention was similar to Streptococcus thermophilus ^T (ATCC 19258), but it was confirmed that it is not the same strain.

Based on the above results, the strain was classified as "Lactobacillus thermophilus ST3 ( Streptococcus). thermophilus ST3), and was deposited on March 2, 2011 to the microbial resource center (KCTC), a depository institution for Korean patent strains, and was assigned accession number KCTC11870BP.

1-3. Acid resistance  evaluation

Acid resistance evaluation was performed in 30 minutes after 1% inoculation of 10% artificial gastric fluid (NaCl 2g, pepsin 3.2g, concentrated hydrochloric acid 5.8ml / 1L, pH = 2.1) of Streptococcus thermophilus ST3 pre-cultured in MRS liquid medium. The viable cell count was measured in MRS solid medium for 2 hours. As a result of the evaluation of acid resistance, the isolated strain of Streptococcus thermophilus ST3 maintained the viable cell number stably even under acidic conditions, and confirmed the acid resistance of the strain.

1-4. Antibiotic resistance

Antibiotic resistance evaluation was performed using the micro-dilution method recommended by the European Food Safety Authority (EFSA), and each antibiotic was purchased from Sigma and Fluka.

Antibiotic concentrates were diluted to concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5 μg / ml using MRS medium, and 100 μl of the diluted solution containing the antibiotics in 96 well plates. Minimal Inhibition Concentration (MIC) was measured by measuring the growth rate after incubating at 37 ° C for 16 hours after inoculating 1% inoculation of Streptococcus thermophilus ST3 cultured in MRS liquid medium. . In order to reduce the error of the experiment, all experiments were composed of three sets, and after taking the average value, the minimum growth inhibition concentration (MIC) value was finally measured. MIC for each strain was determined by the following method. At the same experimental time as the experimental group, 100 μl of the MRS culture medium containing no antibiotics was inoculated with 1% of the strain, and immediately stored at −4 ° C. Based on the OD value of the group that inhibited the growth of the strain at -4 ℃ after 16 hours, the concentration of antibiotics showing an OD value of less than that in the experimental group was defined as MIC. The MIC values for each antibiotic of the Streptococcus thermophilus ST3 strain of the present invention are shown in Table 2 below. Streptococcus thermophilus ST3 inhibited growth at most low concentrations of antibiotics that inhibit cell wall synthesis and protein synthesis.

Antibiotic Minimum growth inhibitory concentration (MIC) Ampicillin (AMP) <0.5 Kanamycin (KAN) <32 Chloramphenicol (CP) <0.5 Vancomycin (VAN) <0.5 Gentamicin (GEN) <8 Tetracycline (TET) <0.5 Neomycin (NM) <64 Streptomycin (ST) <16

2. Lactobacillus Ramnosus LR3  ( Lactobacillus rhamnosus LR3  ( KCTC  11868 BP Identification and Characterization of))

2-1.Isolation of microorganisms

Goat milk collected from the ranch was diluted in sterile anaerobic water by serial dilution and 1 ml of each dilution was poured into ManRogosa Sharpe (MRS. BD. USA) solid medium and incubated at 37 ° C. for 3 days under anaerobic conditions. The resulting colonies were picked up again on a solid medium to which BCP (bromocresol purple, 0.17g / L) was added to MRS, a lactic acid bacterium selection medium, and then cultured again under the same conditions for 3 days. The lactic acid bacteria were selected from the medium for biochemical and molecular biological identification, and then one strain having the best physical properties of the strain was finally selected.

2-2. Identification of microorganisms

1) sugar usability analysis

Lactobacillus strains isolated and screened as described above were analyzed by the API CHL50 kit, and as shown in Table 3, it was identified as having 99.6% biochemical properties similar to Lactobacillus rhamnosus ( Lactobacillus rhamnosus ).

temperament reaction temperament reaction  Esculin +  Glycerol -  Salincin +  Erythritol -  Cellobiose +  D-Arabinose -  maltose +  L-arabinose -  Lactose +  Ribose +  Melibiose -  D-xylose -  Saccharose -  L-xylose -  Trehalose +  Andonitol -  Inulin -  β-methyl-xyloxide -  Melezitose +  Galactose +  D-rapinose -  D-glucose +  Amidon -  D-fructose +  Glycogen -  D-mannose +  Xylitol -  L-sorbose -  β-gentiobiose +  Rhamnose +  D-turanoose -  Dulcytol -  D-Rixos -  Inositol +  D-tagatose +  Mannitol +  D-fucose -  Sorbitol +  L-fucose -  α-methyl-D-mannoside -  D-arabitol -  α-methyl-D-glucoside +  L-arabitol -  N-acetyl glucosamine +  Gluconate +  Amigalline +  2-keto-gluconate -  Arbutin +  5-keto-gluconate -

2) 16s rRNA  Sympathy

Genome DNA was extracted from 1 ml of pure culture of the isolated strain using Accuprep genome extraction kit (Bioneer, Korea). Using the extracted DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template.

PCR products were linked to the pGEM-Teasy vector (Promega, USA) and then E. coli After transformation to DH5α, plated on LB plates containing X-gal and ampicillin and incubated at 37 ° C. for about 16 hours. Recombinant plasmids containing PCR products were isolated from colonies grown on LB plates, followed by DNA sequencing. DNA sequencing was performed using the Cluster V program of the DNA star program. Ramno Seuss ^T (L. rhamnosus ^T Homology with ( ATCC 7469). Figure 4 shows the Lactobacillus rhamnosus Lactobacillus rhamnosus LR3) (KCTC 11868BP) and 16S rRNA sequences of Lactobacillus rhamnosus ^T (ATCC7469) strains. The 16s rRNA sequence of the isolated strain was L. a. Show ramno Versus ^{T (L. rhamnosus T) (ATCC} 7469) and a homology of 99.2%, it was identified as a novel microorganism of the invention Lactobacillus strains belonging to the species ramno Versus.

3) RAPD ( Random Amplified Polymorphic DNA ) analysis

Genomic DNA was extracted from the Lactobacillus rhamnosus LR3 strain, which was then used as a template, and PCR-RAPD (MyCycler, BIO-RAD, USA) was performed using (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') as a primer. PCR products were electrophoresed on 0.8% agarose gel and stained with EtBr and observed with G: BOX (SYNGENE, UK). According to Figure 5, the RAPD band pattern of Lactobacillus rhamnosus LR3 of the present invention is L. Rhamnosus ^T ( L. rhamnosus ^T ) (ATCC 7469) appeared different, and from these results it was confirmed that the two strains are not the same strain.

Based on the above results, the isolated strain was Lactobacillus rhamnosus LR3 ( Lactobacillus). rhamnosus LR3), and was deposited on March 2, 2011 by the microbial resource center (KCTC), a depository institution for Korean patent strains, and was given accession number KCTC11868BP.

2-3. Acid resistance  And My bile  evaluation

Acid resistance evaluation was performed by adding 1% of Lactobacillus rhamnosus LR3 pre-cultured in MRS liquid medium to 10 ml of artificial gastric juice (NaCl 2g, pepsin 3.2g, concentrated hydrochloric acid 5.8ml / 1L, pH = 2.1) and left for 2 hours. . During this period, acid resistance was measured by taking a certain amount in 30-minute increments and smearing in MRS solid medium to measure the number of viable cells.

Tolerance assessment was performed by inoculating 1% (v / v) of Lactobacillus rhamnosus LR3 precultured in MRS liquid medium into MRS containing oxgall (0%, 0.25%, 0.5%, 0.75%, 1%). After 2 hours left. Thereafter, a predetermined amount was taken at 20 minute intervals and plated on an MRS plate to measure the number of viable cells. Referring to Figure 6, Lactobacillus rhamnosus LR3 in the evaluation of acid resistance and biliary resistance was stable in both conditions, it was confirmed that the excellent acid resistance and bile resistance of the isolated strain.

2-4. Intestinal Settlement Assessment

The evaluation of intestinal fixability of Lactobacillus rhamnosus LR3 was performed using human intestinal epithelial cells, Caco2, and Lactobacillus rhamnosus GG was used as a control. After comparing the Lactobacillus rhamnosus LR3 with Caco2 cell line for 1 hour, the number of viable cells was measured. Referring to Figure 7, Lactobacillus rhamnosus GG known as a good intestinal anchorage strain showed a 75.2% fixability, whereas Lactobacillus rhamnosus LR3 of the present invention showed 86.4% about 10% better intestinal fixability Indicated.

As confirmed above, Lactobacillus rhamnosus LR3 ( Lactobacillus) of the present invention rhamnosus LR3 (KCTC 11868BP)) strain was L. a. Ramno Versus ^{T (L. rhamnosus T) (ATCC} 7469) and is a novel strain showing the difference in the molecular. Lactobacillus rhamnosus LR3 strain of the present invention is excellent in acid resistance and bile resistance is determined to reach the small intestine and large intestine through the stomach after ingestion. Lactobacillus Lactobacillus is known as Lactobacillus Since rhamnosus GG) shows better fixability, it can show a better effect as probiotics.

3. Lactobacillus Paracazei LPC4 ( Lactobacillus paracasei LPC4 ) KCTC  11866 BP Identification and Characterization

3-1. New microbial isolation

Goat milk collected from the ranch was diluted in sterile anaerobic water by serial dilution and 1 ml of each diluted solution was poured into ManRogosa Sharpe (MRS. BD. USA) solid medium and incubated under anaerobic conditions for 3 days. The resulting colonies were picked up again on a solid medium to which BCP (Bromocresol purple, 0.17 g / L) was added to MRS, a lactic acid bacteria selection medium, and then cultured again under the same conditions for 3 days. The lactic acid bacteria were selected from the medium for biochemical and molecular biological identification, and then one strain having the best physical properties of the strain was finally selected.

3-2. Identification of new microorganisms

1) sugar usability analysis

Lactobacillus strains selected and separated as described above were analyzed by the API CHL50 kit, as shown in Table 4 below, Lactobacillus paracasei ( L. paracasei spp paracasei (API50CHL) was found to have a biochemical properties similar to 95.2%.

temperament reaction temperament reaction -  Esculin +  Glycerol -  Salincin +  Erythritol -  Cellobiose +  D-Arabinose -  maltose +  L-arabinose -  Lactose +  Ribose +  Melibiose -  D-xylose -  Saccharose +  L-xylose -  Trehalose +  Andonitol -  Inulin +  β-methyl-xyloxide  Melezitose +  Galactose +  D-rapinose -  D-glucose +  Amidon -  D-fructose +  Glycogen -  D-mannose +  Xylitol -  L-sorbose +  β-gentiobiose +  Rhamnose -  D-turanoose +  Dulcytol +  D-Rixos +  Inositol +  D-tagatose +  Mannitol +  D-fucose -  Sorbitol +  L-fucose -  α-methyl-D-mannoside -  D-arabitol -  α-methyl-D-glucoside +  L-arabitol -  N-acetyl glucosamine +  Gluconate +  Amigalline +  2-keto-gluconate -  Arbutin +  5-keto-gluconate -

2) 16s rRNA  Sympathy

Genome DNA was extracted from 1 ml of pure culture medium of the isolated strain using Accuprep genome extraction kit (Bioneer, Korea). Using the extracted DNA as a template, PCR (MyCycler, BIO-RAD, USA) was performed using a primer F (5'-AGAGTTTGATCCTGGCTCAG3 ') and primer R (5'-AAGGAGGTGATCCAGCC 3') as a template.

PCR products were linked to the pGEM-Teasy vector (Promega, USA). E. coli strain DH5α was transformed and then plated on LB / x-gal / amp plates and incubated overnight at 37 ° C. After screening the recombinant plasmid containing the insert from the transformant, DNA sequencing was performed. DNA sequencing was performed using Lactobacillus isolate of Lactobacillus paracasei using Cluster V method of DNA star program. paracasei subsp. paracasei isolate Akira1) and 16S rRNA sequences of the strains of the present invention were compared and the results are shown in FIG. 8. The 16s rRNA sequence of the novel strain isolated from the present invention is Lactobacillus paracase isolate Achiral ( Lactobacillus) paracasei subsp. paracasei isolate Akira1) showed a homology of 99.7%, it was confirmed that the novel microorganism of the present invention is a strain belonging to the species of Lactobacillus paracasei .

3) RAPD  ( Random Amplified Polymorphic DNA ) analysis

Genome DNA was extracted from purely cultured Lactobacillus paracasei LPC4 and PCR-RAPD (MyCycler, BIO-RAD, USA) was extracted from the isolated DNA using template (GTG) ₅ (5'-GTGGTGGTGGTGGTG 3 ') as a template. PCR products were stained with Et-Br and observed with G: BOX (SYNGENE, UK). Referring to Figure 9, DNA band patterns of the Lactobacillus casei para LPC4 strain isolated in the present invention is Lactobacillus casei ^T p ^(T L. paracasei (ATCC 25302) was shown differently, and as a result it was confirmed that the two strains are not the same strain.

Based on the above results, the strain isolated from the present invention was named Lactobacillus paracasei LPC4 ( Lactobacillus paracasei LPC4), and deposited on March 2, 2011, the microorganism resource center (KCTC), a depository institution for the Korean patent strain, accession number KCTC11866BP. Was granted.

3-3. My bile  evaluation

The biliary resistance of the Lactobacillus paracasei LPC4 strain was inoculated with 1% of Lactobacillus paracasei LPC4 precultured in MRS liquid medium in MRS liquid medium supplemented with different Oxgall concentrations. The viable cell count was measured using a solid medium, and the measurement results are shown graphically in FIG. 10. The Oxgall concentration used was 0%, 0.25%, 0.5%, 0.75%, 1%. As shown in FIG. 10, as a result of evaluating the biliary tract, Lactobacillus paracasei LPC4, which is an isolated strain, can be confirmed that the viable bacterial count is stably maintained at various Oxgall concentrations.

4. Animal testing using type 1 diabetes model

1. Preparation of Lactic Acid Bacteria Powder

Fermentation medium for the culture of lactic acid bacteria strains contained in the composition of the present invention is made of 1% -10% (w / v) aqueous solution by mixing or mixing skim milk powder and soybean isolated protein, respectively, Streptococcus thermophilus ST3 ( Streptococcus thermophilus For ST3) (KCTC 11870BP) and Lactobacillus rhamnosus LR3 (KCTC 11868BP), a solution of proteinase (Delvolase; DSM, Netherlands) in the range of 0.01% -1% (w / w) relative to protein was added. Enzyme treatment. Lactobacillus casei para LPC4 (Lactobacillus For the lactic acid species of paracasei LPC4) (KCTC 11866BP), the enzyme treatment step was omitted.

After enzymatic treatment, glucose, lactose, fructose or sucrose in the range of 0.1% -5% (w / v), yeast extract in the range of 0.1% -5% (w / v), 0.1% Ionic components of soypeptone in the range -5% (w / v), ammonium citrate in the range of 0.01% -0.1% (w / v), sodium acetate, dipotassium poststate, magnesium sulfate, manganese sulfate, sodium chloride Lactic acid bacteria culture medium was prepared by adding and dissolving. The prepared lactic acid bacteria culture medium was sterilized using a heat exchanger.

Lactobacillus spawn precultured in the culture medium component was inoculated into the culture medium at a concentration of 0.1% -1% (v / v) and maintained at 37 ° C. and pH 5.0-6.0 for 8 to 18 hours depending on the lactic acid species. Fermentation was carried out. After completion of the lactic acid bacteria culture lactic acid bacteria were separated and concentrated using a continuous centrifuge (Separator). The isolated lactic acid cells were lyophilized by adding a lyoprotectant and the like, and the lactic acid bacteria were pulverized using a grinder to have a constant particle size, and then, the lactic acid bacteria were prepared as raw materials.

The original lactic acid bacteria obtained in the previous step was determined to have the following viable cell count:

Streptococcus thermophilus ST3 lactic acid bacteria (1X10 ¹¹ CFU / g or more)

Lactobacillus rhamnosus LR3 lactic acid bacteria (1X10 ¹¹ CFU / g or more)

Lactobacillus paracasei LPC4 lactic acid bacteria (1 × ^{10 11} CFU / g or more).

2. Animal and Experimental Design

In this experimental example, an anti-diabetic agent and three kinds of lactic acid bacteria of the present invention (Lactobacillus paracaze LPC4, Lactobacillus rhamnonus LR3, Streptococcus) against streptozotocin-induced type 1 diabetes in vivo model Co-administration of Cocus thermophilus ST3) was evaluated in comparison with various controls.

Male Sprague-Dawley species (SD) rats (6 weeks after birth, 200-230 g per subject) were acclimated to the experimental environment for 1 week, and then randomly divided into 6 groups of 8 animals in each group as shown in Table 5 below. Example 1 After administration of streptozotocin to induce diabetes, live cells of the lactic acid bacterium strain of the present invention was administered, Example 2 after administration of streptozotocin to induce diabetes, the present invention The dead cells of the lactic acid bacteria strains of the group was administered. In Example 1 and Example 2, a live cell mixture or a dead cell mixture containing all three kinds of lactic acid bacteria strains of the present invention was administered. In the group of Example 3 , acarbose and a mixture containing the three kinds of lactic acid bacteria obtained above were administered. Surviving or dead Lactobacillus paracasei LPC4, Lactobacillus rhamnosus LR3 And Streptococcus thermophyllus ST3, and then rats were treated with 1 × ^{10 8} Probiotics of cfu / head were administered orally once a day and acarbose was administered at a dose of 50 mg / kg.

Comparative Example 1, which is a control group, is a normal group corresponding to a sham group, and only saline of the same amount and root was administered during the experiment. Comparative Example 2 is a diabetic control group in which experimental diabetes was induced by administering streptozotocin, and Comparative Example 3 is a control group each administered only acarbose, which is an oral diabetes treatment, after diabetes was induced by administering streptozotocin.

The experimental animals were acclimated to the environment for one week after arriving at the laboratory, and only animals that exhibited no special symptoms during this period were used in the experiment. During the acclimation period, animals were raised at 22 ± 2 ° C, 55 ± 10% relative humidity, light / dark cycles at 12/12 hours, and were freely available for commercial rodent feed and water.

In this example, the rats were fasted for 16 hours to induce diabetes, and 50 mg / kg of streptozotocin dissolved in 0.01M citric acid buffer (pH 7.4) was injected into the tail vein of the rat. Streptozotocin is known to cause experimental diabetes by selectively attacking beta cells of the Langerhans islet in the pancreas. In order to confirm the induction of diabetes, the rats were fasted for 16 hours, and after 48 hours, blood glucose levels were measured using a blood glucose meter (ACCU-CHEK Active: Roche, Germany) to have a blood glucose level of 300 mg / dl or more. Only mice were considered diabetic mice and used in the experiment. Body weights were measured immediately prior to streptozotocin administration and weighted just before weekly fasting 30 days after sample administration.

division Display Experimental sample animal Comparative Example 1 Sham - Normal Comparative Example 2 Control - Diabetes Induction (STZ) Example 1 Probiotic Lactobacillus (live cells) Diabetes Induction (STZ) Example 2 Probiotic Lactobacillus (Death Cells) Diabetes Induction (STZ) Comparative Example 3 Acarbose Akabos Diabetes Induction (STZ) Example 3 Mixture Acarbose + 3 kinds of lactic acid bacteria Diabetes Induction (STZ)

3. Weight and Blood Sugar Analysis

3-1. Weight measurement

To determine whether there was a weight loss characteristic of type 1 diabetes, diabetic-induced mice by streptozotocin were weighed every 6 days until 27 days, and are shown in Table 6 below. As can be seen from Table 6, body weight was increased only in the false control group (Comparative Example 1), consistent with the characteristics of type 1 diabetic rats, but not in the groups in which experimental diabetes was induced by streptozotocin. The average body weight of the individual rats in the false control group was heavier than the average body weight of the individual mice of the diabetic control group of Comparative Example 2, which suggested that diabetes affected weight loss. In addition, two weeks after the measurement, there was no significant difference in fasting blood glucose levels among the experimental groups, and after 27 days, the fasting blood glucose concentrations among the experimental groups were 450 mg / dl in the diabetic control group of Comparative Example 2 and acarbose. In the group of Example 3, which was administered in combination with the lactic acid bacteria mixture, the difference between the groups increased significantly to 350 mg / dl ( p <0.05). Thus, it is demonstrated that the lactic acid bacteria and acarbose mixture may have a therapeutic effect on weight loss and blood glucose concentration increase. Composition comprising three kinds of lactic acid bacteria of the present invention can be found to be effective in improving weight loss due to diabetes.

3-2. Blood glucose measurement before meals

Blood glucose was collected from the tail vein after fasting for 16 hours once every 6 days, and measured by preprandial blood glucose, and is shown graphically in FIG. 11. Referring to FIG. 11, the blood glucose concentration rapidly increased except for the diabetes control group of Comparative Example 2 15 days after the samples were administered to all treatment groups. The treatment effect of lactic acid bacteria against diabetes began to appear 15 days after the administration of lactic acid bacteria to diabetic rats, and the combination of the acarbose mixture and the acarbose mixture of Example 3 and the group administered with acarbose of Comparative Example 3 after 21 and 27 days The blood glucose concentration of one group was significantly reduced both compared to the diabetic control of Comparative Example 2. The blood glucose concentration of the group administered only the lactic acid bacteria live cells of Example 1 and the group administered only the lactic acid bacteria killing cells of Example 2 was also reduced compared to the diabetic control of Comparative Example 2. The therapeutic effect of lactic acid bacteria in relation to blood glucose concentration was higher in the group administered only acarbose of Comparative Example 3 and the group administered in combination of a lactic acid bacterium mixture and acarbose of Example 3 than the group administered only live cells of Example 1 lactic acid bacteria. Serum cholesterol concentration was lower in the group administered only the lactic acid bacteria live cells of Example 1 than the diabetic control of Comparative Example 2. Taken together, these results, it can be seen that the lactic acid bacteria strains of the present invention can reduce the blood glucose level and serum cholesterol concentration.

3-3. Post-prandial blood sugar measurement

Post-prandial blood sugar was measured after diabetic rats were fasted for 12 hours, followed by oral administration of test samples and feeding for 1 hour. In order to examine the change in blood glucose concentration according to the administration of the test sample, the blood glucose concentration was measured three times for 3 hours at 1 hour intervals. In the diabetic control group of Comparative Example 2, the blood glucose level was increased after meals and maintained high for 3 hours after meals. In the group administered only the lactic acid bacteria live cells of Example 1 and the group administered only the lactic acid bacteria killing cells of Example 2, the post-prandial blood sugar concentration increased to a similar level as the diabetic group, but the concentration rapidly decreased until 3 hours after the meal. On the other hand, in the group administered only the acarbose of Comparative Example 3 and the group of the lactic acid bacterium mixture of the example and acarbose in combination, the postprandial blood sugar level was significantly lower than that of the control group of Comparative Example 2 ( p <0.05), and during 3 hours after eating A blood glucose level change of 200 mg / dl was maintained. These results demonstrate that the lactic acid bacteria of the present invention and compositions containing the same can reduce postprandial blood sugar levels.

4. Histopathology of the Pancreas

Rats are anesthetized and lethal and blood samples are drawn. Then, after perfusion fixation, the pancreas is excised and fixed in 10% formalin. The fixed pancreas is thinly sliced and then fixed in 10% formalin at room temperature for 24 hours and then washed with tap water for 1 hour. The washed tissue is loaded into an automatic tissue processor (Hypercenter XP, Shandon, London). After dehydration, clearing, and infiltration in an autologous tissue treatment device, the tissue is fixed in paraffin, and then histologic sections are cut using a microtome. After paraffin removal and dehydration, tissue sections are observed by light microscopy with hematoxylin and eosin staining (H & E staining).

Histology Comparison of Langerhans Islands

Considering type 1 diabetes, blood glucose is not controlled by destroying beta cells constituting the islet of Langerhans islet in the pancreas, so blood glucose is not controlled, and the size of Langerhans is observed using hematoxylin and eosin staining. The measurement results are shown in FIG. 13. Arrows in FIG. 13 indicate the Langerhans islets of the pancreas. Referring to FIG. 13, the size of the Langerhans island of the diabetic control group of Comparative Example 2 (C and 4D of FIG. 13) was significantly reduced when compared to the false control group (A and B of FIG. 13). The size of Langerhans islet in the group administered only the lactic acid bacteria live cells of Example 1 (E and F of FIG. 13) and the group administered only the lactic acid bacteria killing cells of Example 2 (G and H of FIG. 13) was compared to the diabetic control group of Comparative Example 2. Increased compared to that. The size of the Langerhans islands of the group administered only the acarbose of Comparative Example 3 (FIGS. 13 I and J) and the group of the lactobacillus mixture of Example 3 combined with the acarbose (K and L of FIG. 13) was the diabetic control group of Comparative Example 2. Increased compared to

Considering the above results, the size of the Langerhans island was increased when a lactic acid bacterium, acarbose, or a mixture thereof was administered to streptozotocin-induced diabetic rats. Although the post-prandial blood sugar concentration, which is an important indicator of diabetes, increased similarly to that of the diabetic control group of Comparative Example 2, it rapidly decreased until 3 hours after eating. The blood glucose level of the group administered only with acarbose of Comparative Example 3 and the combination of lactic acid bacterium mixture and akabose of Example 3 was lower than that of other groups even after a meal, and moreover, acarbose inhibited the function of carbohydrate digestive enzymes and thus blood glucose concentration. It is consistent with the findings of Leonhardt et al. In particular, the low blood glucose concentration increase rate of the group administered only acarbose of Comparative Example 3 and the group administered in combination with the lactobacillus mixture of Example 3 and acarbose was considered to be due to the synergistic effect of the lactic acid bacteria in the control of postprandial blood sugar concentrations. In all groups except the diabetic control group of Comparative Example 2, the blood glucose level was reduced to a certain level by 3 hours after eating, indicating that even the administration of lactic acid bacteria may have a therapeutic effect on diabetes.

Recovery of type 1 diabetes could be determined by the size of the Isle of Langerhans, which secretes insulin. As shown in FIG. 13, the size of the island of Langerhans in all treatment groups was increased compared to the size of the island of Langerhans in the diabetic control group of Comparative Example 2. Among the treatment groups, the therapeutic effect related to the size recovery of the island of Langerhans was determined by the lactic acid bacteria mixture of Example 3 and the acarbose group administered only the lactic acid bacteria live cells of Example 1 and only the lactic acid killing cells of Example 2. Higher than the group. Moreover, co-administration of acarbose with lactic acid bacteria showed a better therapeutic effect with respect to recovery of insulin secretion function than acarbose alone administration.

5. Histological Analysis of the Kidney

Rats were anesthetized at the end of the experiment and killed by anesthesia to collect blood samples from the abdominal artery, and serum was separated from the blood samples to analyze serum lipids using a FUJIFILM DRI-CHEM 4000i biochemical analyzer (Kanagawa, Japan). . In order to confirm the presence of diabetic nephropathy as a complication of diabetes, the cortex and outer medullar and inner medullar of the kidney are stained. FIG. 14 shows the results of this tissue analysis, in which arrows indicate interstitial expansion or dilated tubule.

In diabetic nephropathy, the enlargement of the glomerular basement membrane, the enlargement of the tubular basement membrane, the expansion of the mesangials, the expansion of the interstitial, the tubular ) Various histological changes such as dilation, nodular glomerulosclerosis, afferent and efferent arteriolar hyalinosis.

In this example, no histological changes occurred in the cortex of the diabetic control group of Comparative Example 2 (D, E, and F of FIG. 14), but in the medulla, histological changes such as swelling of the interstitium and dilation of tubules were observed. . In Example 1 (groups administered only with lactic acid bacteria live cells), Example 2 (groups of heated killer cells), Comparative Example 3 (groups administered with acarbose only), and Example 3 (groups administered with a combination of lactic acid bacteria mixture and acarbose), Histological changes such as swelling and enlargement of the tubules are almost gone. Therefore, as shown in FIG. 14, the therapeutic effect of diabetic nephropathy was higher in the group administered with only the lactic acid bacteria live cells of Example 1 and the group treated with only the acarbose of Comparative Example 3 than the group administered with only the lactic acid bacteria killed cells of Example 2. Found to be high. Unlike the diabetic control group of Comparative Example 2, Example 1 (lactic acid bacteria-only group), Example 2 (heated killer cell group), Comparative Example 3 (acarbose-only group), Example 3 (lactic acid bacteria mixture and In the group treated with acarbose), little histopathological changes such as swelling of the interstitium and expansion of the tubules occurred. Therefore, it can be confirmed that the novel lactic acid bacteria strains of the present invention and a mixture of acarbose and lactic acid bacteria are useful for the treatment of diabetic nephropathy.

6. Analysis of Blood Cholesterol Levels in Diabetic Rats

Serum cholesterol and serum lipids are known to cause atherosclerosis and may affect the development of diabetic nephropathy, a complication of diabetes. Serum cholesterol levels were measured by collecting blood samples from the abdominal vein of rats 27 days after the administration of the samples.

In the diabetic control group of Comparative Example 2, the serum cholesterol concentration was 88 mg / dl, and in the group treated with acarbose of Comparative Example 3, 86.7 mg / dl, there was no significant difference between the two groups. This is consistent with Leonhardt et al.'S report that acarbose does not affect serum cholesterol levels. On the other hand, the serum cholesterol level of the lactic acid bacteria live cell administration group of Example 1 was 69 mg / dl, which is about 20% lower than the diabetic control group of Comparative Example 2, and the lactic acid bacteria reduced the increase of serum cholesterol concentration induced by streptozotocin. It can be confirmed that it can (see Fig. 15).

Taken together, the surviving or dead Lactobacillus paracasei LPC4 and Lactobacillus rhamnosus LR3 have therapeutic effects on diabetes. And the lactic acid bacteria mixture of Streptococcus thermophilus ST3 and the acarbose and lactic acid bacteria mixture have a synergistic effect on the treatment of these diseases.

The present invention can be practiced with various modifications and changes without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are merely for explaining preferred embodiments of the present invention and should not be construed as limiting the present invention. It is intended that the scope of protection of the present invention be defined by the appended claims, and that the various modifications and variations are intended to be included within the scope of the present invention.

Korea Biotechnology Research Institute KCTC11870BP 20110302 Korea Biotechnology Research Institute KCTC11868BP 20110302 Korea Biotechnology Research Institute KCTC11866BP 20110302

Claims (11)

Streptococcus ST3 ( Streptococcus internationally deposited as Korean Collection for Type Culture (KCTC) Accession No. KCTC 11870BP thermophilus ST3) lactic acid bacteria.
Lactobacillus rhamnosus LR3 (Lactobacillus rhamnosus LR3) Lactobacillus internationally deposited as Korean Collection for Type Culture (KCTC) Accession No. KCTC 11868BP.
Lactobacillus LPC4 ( Lactobacillus LCT4), an international deposit deposited with the Korean Collection for Type Culture (KCTC), KCTC 11866BP. paracasei LPC4) lactic acid bacteria.
Streptococcus thermophilus ST3 (KCTC 11870BP);
Lactobacillus rhamnosus LR3 (KCTC 11868BP); And
Lactobacillus paracasei LPC4 (Lactobacillus paracasei LPC4) (KCTC 11866BP) comprising a composition for preventing and treating diabetes.
The composition for preventing and treating diabetes of claim 4, wherein the three kinds of lactic acid bacteria strains are double coated with proteins and polysaccharides or triple coated with proteins, polysaccharides and nanoparticles.
The method according to claim 4, wherein the composition comprises a culture having the same number of live bacteria or containing a mixture of the three types of lactic acid bacteria strains in an amount of 10 ⁸ to 10 ¹² cfu / g, as an active ingredient, based on the total weight of the composition. Diabetic prevention and treatment composition comprising a.
The method of claim 4, wherein the composition is Lactobacillus Lactobacillus acidophilus ), Lactobacillus plantarum ) , Bifidobacterium breb breve ) , B ifidobacterium lactis), ronggum Bifidobacterium (Bifidobacterium longum ), Lactobacillus salivarius ), Lactobacillus brevis , Lactobacillus helveticus ( Lactobacillus) helveticus ), Lactobacillus fermentum ), Lactobacillus paracasei ), Lactobacillus casei ), Lactobacillus delbrueckii ), Lactobacillus reuteri ), Lactobacillus buchneri ), Lactobacillus gasseri ), Lactobacillus johonsonii ), Lactobacillus kefir ), Lactococcus lactis ), Bifidobacterium ( Bifidobacterium) bifidum ), Bifidobacterium infantis ), Bifidobacterium pseudolongum), Bifidobacterium standing room writing brush (B ifidobacterium themophilu m), Adolfo sentiseu Bifidobacterium (Bifidobacterium Adolescent is a ) composition for preventing and treating diabetes, further comprising at least one lactic acid bacteria selected from the group consisting of.
The composition for preventing and treating diabetes of claim 4, wherein the composition further comprises a pharmaceutically acceptable carrier or excipient.
The composition for preventing and treating diabetes of claim 4, wherein the lactic acid bacteria strains are freeze-dried to be prepared in the form of probiotics.
A drug comprising the composition for preventing and treating diabetes according to any one of claims 4 to 9.
Food comprising a composition for preventing and treating diabetes according to any one of claims 4 to 9.

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