KR100794701B1 - Microorganisms for Obesity or Diabetes Mellitus - Google Patents

Abstract

The present invention relates to a microorganism for obesity or diabetes prevention and treatment that produces excellent polysaccharides in the intestine with high intestinal viability, and more specifically, does not absorb glucose, sucrose, or fructose into the body. A novel microorganism, a composition containing a pharmaceutically effective amount thereof, and a food composition containing the same as an active ingredient, which can prevent or treat obesity or diabetes by competitively inhibiting the intestinal absorption of low saccharides by converting it into a polymer material. will be.

Dietary fiber, lactic acid bacteria, obesity, diabetes, prevention, treatment

Description

Microorganisms for Obesity or Diabetes Mellitus

1 is a diagram showing the weight change of rats ingested microorganism according to the present invention.

Figure 2 is a chart showing the change in weight gain of rats ingested microorganism according to the present invention.

Figure 3 is a diagram showing the metabolic efficiency change of rats ingested microorganism according to the present invention.

Figure 4 is a diagram showing the change in blood cholesterol of rats ingested microorganism according to the present invention.

Figure 5 is a chart showing the blood glucose level change of rats ingested microorganism according to the present invention.

The present invention relates to a microorganism for obesity or diabetes prevention and treatment that produces excellent polysaccharides in the intestine with high intestinal viability, and more specifically, does not absorb glucose, sucrose, or fructose into the body. A novel microorganism, a composition containing a pharmaceutically effective amount thereof, and a food composition containing the same as an active ingredient, which can prevent or treat obesity or diabetes by competitively inhibiting the intestinal absorption of low saccharides by converting it into a polymer material. will be.

Obesity is still known as a chronic disease that is caused by a combination of factors that have not been clearly identified. Obesity is a cause of hypertension, diabetes, cardiovascular disease, gallstones, osteoarthritis, sleep apnea, respiratory disorders, endometrium, prostate, breast or colon cancer.

According to the 1999 National Institute of Health (NIH) Report (Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults, 1999, NIH), approximately 97 million Americans are obese and obese There are about 15.7 million patients with type 2 diabetes mellitus, one of the diseases associated with the disease. It is also known that about 200,000 people die worldwide each year due to obesity-related diseases (Dan Ferber, Science, 283, pp 1423-1424, 1999).

Diabetes is one of the world's most prevalent chronic diseases that require significant personal and financial costs, as well as patients and their families, and there are different types of diabetes with distinct pathogenesis and pathogenesis. Diabetes mellitus, for example, is characterized by hyperglycemia and diabetes and is a carbohydrate metabolic disorder caused by improper production or use of insulin.                         

Non-insulin-dependent diabetes mellitus (NIDDM) or type II diabetes is diabetes that occurs mainly in adults whose insulin is properly produced and utilized, but which is defective in insulin-mediated glucose and other metabolism in peripheral tissues.

NIDDM is due to three major metabolic abnormalities, resistance to insulin-mediated glucose processing, impairment of nutrient-stimulated insulin secretion and overproduction of glucose in the liver. Failure to control NIDDM, ie control of blood sugar levels, can lead to death from cardiovascular disease and other diabetic complications including retinopathy, kidney disease and peripheral neuropathy.

In the treatment of NIDDM, as a method of controlling blood glucose levels in NIDDM patients, not only diet and exercise but also sulfonylurea drugs and biguanide drugs may be used. In addition, metformin and acarbose compounds have also recently been used to treat NIDDM patients. However, some patients may not be able to adequately control hyperglycemia even by diet, exercise and / or use of the therapeutic compounds described above, requiring administration of exogenous insulin.

In addition to the expensive and painful administration of insulin to the patient by injection, it can cause a variety of situations or complications that are detrimental to the patient. For example, an insulin response (hypoglycemia) can be caused without error, lack of fasting, irregular exercise or special cause of insulin dosage. In addition, local and / or systemic allergic reactions and immunological resistance to insulin may occur.

Prevention or treatment of obesity and diabetes are largely diet-exercise therapy, surgical therapy and drug therapy. Diet-exercise therapy is a therapeutic method through low calorie-low fat intake and physical activity that consumes oxygen, and it is recognized that it is difficult to see the public effect because it must be carried out repeatedly and continuously with patience.

Surgical therapy is a method of physically removing body fat through surgical surgery, which has the advantage of being effective in a short period of time, but its use is limited because of the need for surgery, inability to maintain the effect, and costly lighting. have.

Therefore, drug therapies for the treatment or prevention of obesity or obesity type diabetes are actively being studied by drugs that cause a decrease in blood sugar, inhibition of blood sugar absorption, enhanced action of insulin and a decrease in appetite. Drugs for the prevention and treatment of obesity type diabetes utilize various physiological mechanisms.

However, among various conventionally developed drugs, dietary fiber is recognized as the most useful obesity prevention and treatment agent because it does not damage the metabolic balance of the human body and is a natural substance.

Conventional microbial dietary fibers include Gluconobacter sp. , Agrobacterium sp. , Acetobacter xylinum , Acetobacter xylinum , A. hansenii . A. pasteurianus , A. aceti , A. aceti , Rhizobium , Alcaligenes , Salina ( Sarcina ), Streptococcus thermophilus , Lactococcus Lactococcus cremoris , Lactobacillus helveticus , Lactobacillus vulgaricus , L. sake , L. reuteri , L. lactis , L. delbrueckii subsp. , L. helveticusglucose var. Jugurti , Leuconostoc dextranicum , L. delbrueckii subsp. Microorganisms such as Bulgaricus , Campestris and Sphingomonas are used.

The dietary fiber produced by these microorganisms is used in various food stabilizers, thickeners, emulsifiers, hygroscopic agents, and medicines and cosmetics.Guar gum is a dietary fiber refined from microorganisms such as cellulose, xan, acetan and seaweed. ), Locust bean gum, carrageenan, alginate, agar and the like have already been commercialized.

Among the microorganisms, the strain Lactobacillus is a major member of the normal microbial flora in the intestine of the human body, and has been known for a long time as being important for maintaining a healthy digestive system and the vaginal environment (Bibel, DJ, ASM News, 54: 661-665, 1988: Reid, G. and AW Bruce, In H. Lappin-Scott (ed.), Bacterial biofilms.Cambridge University Press, Cambridge, England, p. 274-281, 1995; Reid G., AW Bruce, JA McGroarty, KJ Cheng, and JW Costerton, Clin.Microbiol. Rev., 3: 335-344, 1990). In general, the Lactobacillus strains can be found in the digestive organs of animals (L. acidophilus, L. intestinalis, L. johnsonii, L. reuteri, etc.), vaginal mucosa ( L. vaginalis, L. gasseri ), and food (wine- L. hilgardii). ), Lactic acid bacteria drinks ( L. kefir, L. kefiranofaciens ; cheese- L, casei ), vinegar ( L. acetotolerans ), oral ( L. oris ), yeast ( L. sake, L. homohiochi ), fruit juice ( L. kumkeei, L. mali, L. suebicus ), fermented sausage or fish ( L. farciminis, L. alimentarius ).

In fact, many people use health supplements containing strains of the genus Lactobacillus to maintain a healthy gut and prevent urogenital tract infections. Recently, in addition to the prevention of diarrhea and constipation or the prevention of urinary tract infections, the various probiotic activities of Lactobacillus, namely, the regulation of immunity, the control of blood cholesterol levels, the treatment of rheumatism, the prevention of cancer, the reduction of lactose sensitivity, atopy The importance and interest in the effects on sexual dermatitis is increasing.

According to the US Public Health Service guidelines, all 262 Lactobacillus strains currently deposited with the ATCC are not known about the potential risk of causing disease in humans or animals. Are classified as 'Bio-safty Level 1'.

Recently, studies on the extracellular fiber synthesized by Lactobacillus have been actively conducted. Since these genes are involved in the production of extracellular fiber, many genes are involved, and the amount of the synthesized fiber is also very small. (Int. J. Food Microbiol., Mar 3 40: 1-2, 87-92, 1998; Current Opinion in Biotechnology, 10: 498-504, 1999; Current Opinion in Microbiology, 2: 598-603, 1999 ; Appl. Environ.Microbiol., Feb 64: 2, 659-664, 1998; FEMS Microbiol. Rev., Apr. 23: 2 153-77, 1999; FEMS Microbiol. Rev., Sep. 7: 1-2, 113-30, 1990.).

As mentioned above, various studies and efforts have been made to treat obesity or diabetes until now, and until now, various compounds have been developed as drugs for obesity or diabetes, but still have side effects and forcibly release body fat and protein accumulated in the body. Since it is a function that does not inhibit or treat obesity or diabetes fundamentally, there is no groundbreaking therapeutic agent yet.

An object of the present invention is to increase the amount of low saccharides absorbed into the body in the intestine by absorbing the low-saccharide carbohydrates digested by the digestive enzymes while growing in the intestine to convert into high molecular weight material that is not absorbed into the body To provide microorganisms.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating obesity or diabetes by fundamentally reducing the amount of low sugar absorbed into the body by containing a pharmaceutically effective amount of the microorganism.

Another object of the present invention is to provide a food composition containing the microorganism as an active ingredient.

An object of the present invention is to increase the amount of low saccharides absorbed into the body in the intestine by absorbing the low-saccharide carbohydrates digested by the digestive enzymes while growing in the intestine to convert into high molecular weight material that is not absorbed into the body By providing microorganisms.

The microorganism of the present invention is a microorganism capable of growing in the intestine, harmless to the human body, and converting low saccharides into a high molecular weight substance that is not absorbed in the intestine, and is preferably L. reuteri PRD202.

Another object of the present invention is achieved by providing a pharmaceutical composition for preventing or treating obesity or diabetes by fundamentally reducing the amount of low sugars absorbed into the body by containing a pharmaceutically effective amount of the microorganism.

The microorganisms of the present invention are typically administered in unit form, such as tablets or capsules, obtained by mixing with pharmaceutical carriers and excipients or auxiliary active ingredients selected according to the route of administration. Suitable carriers, excipients and diluents for the compositions of the invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polysilicate Vinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl and propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The microbial composition of the present invention may further comprise lubricants, wetting agents, emulsifiers, and suspending agents, preservatives, sweeteners or flavoring agents. The compositions of the present invention can be prepared into enteric coating formulations using methods known in the art to reach the small intestine after passing through the stomach and rapidly release the active ingredient microorganism into the intestine.

In addition, the microbial composition of the present invention can be prepared in a capsule form using conventional encapsulation methods. For example, pellets containing the microorganisms of the present invention that have been lyophilized using standard carriers can be prepared and then filled into hard gelatin capsules or the microorganisms of the invention and any suitable pharmaceutical carrier, such as aqueous Gum, cellulose, silicate or oil may be used to prepare suspensions, dispersions, and then the dispersions or suspensions may be filled into soft gelatin capsules.

The formulations of the present invention may in particular be provided as enteric coated enteric formulations as oral unit dosage forms. "Enteric coating" as used herein includes all known types of pharmaceutically acceptable coatings that are not degraded by gastric acid so that the coating remains, but in the small intestine is sufficiently degraded to allow the active ingredient to be released into the small intestine. .

The "enteric coating" of the present invention is maintained for at least 2 hours when contacting artificial gastric juice, such as HCl solution of pH 1 at 36 to 38 ℃, preferably after KH ₂ PO ₄ buffer solution of pH6.8 Refers to a coating that degrades within 30 minutes in artificial intestinal juice, such as.

The enteric coating of the present invention is coated in one core in an amount of about 16 to 30, preferably 16 to 20 or 25 mg or less. When the thickness of the enteric coating of this invention is 5-100 micrometers, Preferably it is 20-80 micrometers, the satisfactory result as an enteric coating showed. The material of the enteric coating is suitably selected from known polymeric materials. Suitable polymeric materials are described in L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 3rd edition, 1986, pp. 365-373, H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359, Hagers Handbuch der pharmazeutischen Praxis, 4th edition, vol. 7, pp. 739-742, and 766-778, (Springer Verlag, 1971), and Remingtons Pharmaceutical Sciences, 13th edition, pp. Listed in 1689-1691 (mack Publ., Co., 1970), cellulose ester derivatives, cellulose ethers, methylacrylate copolymers of acrylic resins and copolymers of maleic and phthalic acid derivatives can be included therein.

Preferred enteric coatings of the invention are derived from cellulose acetate phthalate and trimellitate and methacrylic acid copolymers (e.g., at least 40% methylacrylic acid, and especially methylacrylic acid and esters thereof comprising hydroxypropyl methylcellulose phthalate). Copolymer). The Endragit L 100-55 product sold by Rohm GmbH, Germany can be used as the material of the enteric coating of the present invention.

The enteric coating cellulose acetate phthalate of the present invention has a viscosity of about 45 to 90 cP, an acetyl content of 17 to 26% and a phthalate content of 30 to 40%, and a cellulose acetate trimellitate of about 15 to 20 cS And acetyl content of 17 to 26%, trimellityl content of 25 to 35%. Cellulose acetate trimellitate products sold by Eastman Kodak Company may be used as the enteric coating material of the present invention.

The hydroxypropyl methylcellulose phthalate used as the enteric coating material of the present invention has a molecular weight of 130,000 Daltons, a hydroxypropyl content of 5 to 10%, a methoxy content of 18 to 24% and a phthalyl content of 21 to 35% Has Cellulose acetate phthalate commercially available from Eastman Kodak Corporation may be used as the material of the enteric coating of the present invention.

The hydroxypropyl methylcellulose phthalate that can be used in the enteric coating of the present invention is HP50 sold by Shin-Etsu Chemical Co. Ltd., Japan, which has a hydroxypropyl content of 6 to 10%, It has a methoxy content of 20 to 24%, a phthalyl content of 21 to 27% and a molecular weight of 84,000 Daltons. Also commercially available from Shin-Etsu Chemical Co., Ltd. as HP55, a product having a hydroxypropyl content of 5 to 9%, a methoxy content of 18 to 22%, a phthalyl content of 27 to 35% and a molecular weight of 78,000 Daltons of the present invention Can be used as coating material.

The enteric coating of the present invention can be prepared using conventional enteric coating methods that spray the enteric coating solution onto the core. Suitable solvents used in the enteric coating process are alcohols such as ethanol, ketones such as acetone, halogenated hydrocarbon solvents such as CH ₂ Cl ₂ and mixed solvents of these solvents may be used. Softeners such as di-n-butylphthalate or triacetin are added to the coating solution at a ratio of 1 to about 0.05 to about 0.3 (coating material macrosoftener). It is appropriate to carry out the spraying process continuously and it is possible to adjust the spraying amount taking into account the conditions of the coating. Spray pressure can be varied and generally satisfactory results are obtained with a spray pressure of about 1 to about 1.5 bar.

As used herein, “pharmaceutically effective amount” means the minimum amount of microorganisms of the present invention that can reduce the amount of low sugars absorbed into the body in the intestine of a mammal, and the amount of microorganisms administered into the body by the composition of the present invention It is adjusted in consideration of the route of administration and the subject of administration.

The composition of the present invention may be administered to the subject individual at least once daily. Unit dosage refers to units that are physically separated, suitable for unit administration for human subjects and other mammals, each unit comprising a suitable pharmaceutical carrier and containing a predetermined amount of the microorganism of the present invention having a therapeutic effect. .

The dosage unit for oral administration of an adult patient preferably contains at least 0.1 g of the microorganism of the present invention, and the composition oral dosage of the present invention is 0.1 to 10 g, preferably 0.5 to 5 g at a time. The pharmaceutically effective amount of the microorganisms of the present invention is 0.1 g / 1 day.

However, the dosage varies depending on the weight of the patient, the severity of the obesity symptoms and the microorganisms and auxiliary active ingredients used. In addition, the total daily dose can be divided into several doses and administered continuously as needed. Thus, the dosage range does not limit the scope of the invention in any way.

In addition, another object of the present invention is achieved by providing a food composition containing the microorganism as an active ingredient.

As used herein, the term "composition" does not necessarily mean to be approved as a medicine, but is a concept including a conventional food, a functional food, or a dietary supplement.

In the case of taking the composition of the present invention periodically, the microorganisms of the present invention in the intestine not only competitively prevent the human body from absorbing sugars, but also non-digestible polysaccharides produced by the microorganisms using these sugars are useful intestinal bacteria. By improving the growth conditions and stimulating the intestinal motility, the composition of the present invention is able to prevent and treat obesity or diabetes.

Hereinafter, the present invention will be described in more detail.

Microorganisms for the prevention and treatment of obesity or diabetes of the present invention ① can be proliferated in the intestine, ② rapidly absorb low sugars into non-digestible or indigestible polymer materials, such as fibrous material, ③ harm to humans or livestock Satisfies the condition Therefore, known microorganisms satisfying the above three conditions can be obtained from various deposit institutions and used in the composition of the present invention, and new microorganisms can be newly separated and used.

The inventors have prepared novel microorganisms that can be used in the compositions of the present invention as follows.

A variety of microorganisms known to produce polysaccharides are distributed from depositing institutions, mutant parent strains are selected through their polysaccharide production capacity, sugar consumption rate, resistance to gastric acid and bile acids, and then mutated using chemicals. PRD202 was selected to produce large quantities of. The selected PRD202 was confirmed to be Lactobacillus ruteri, and was deposited as KCTC by the name Lactobacillus luteri PRD202 (Accession No. KCTC10301BP).

The polysaccharide produced by the Lactobacillus luteri PRD202 strain of the present invention thus selected was not degraded by the intestinal digestive enzyme, and thus, the polysaccharide produced by the Lactobacillus luster PRD202 was degraded by the intestinal digestive enzyme and absorbed into the body. It can be seen that it is not discharged. In addition, as a result of measuring the titer of beta-glucuronides of the microorganism of the present invention, it can be seen that the microorganism of the present invention does not produce beta-glucuronides at all, so that there is no harmful effect due to beta-glucuronides. there was.

As a result of administering the Lactobacillus ruteri PRD202 of the present invention to rats, the body weight was reduced by about 6.5% and the feed intake was about 9.3% less. The metabolic efficiency was calculated and the control and Lactobacillus of the present invention were calculated. The Bacillus Lutheri PRD202 differed by about 5.6%. Therefore, the microorganism of the present invention can be seen that the effect on weight control.

As a result of confirming the change in blood cholesterol when the microorganism of the present invention is ingested, the Lactobacillus luteri PRD202 of the present invention was significantly reduced compared to the physiological saline administration group. Therefore, it can be seen that the microorganisms and microbial compositions of the present invention are effective in obesity, diabetes and circulatory diseases (arteriosclerosis, myocardial infarction, etc.).

In addition, it was confirmed that the hypoglycemic symptoms lower than the normal blood glucose level due to the administration of the microorganism of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention to this embodiment.

Example 1: Measurement of Low Sugar Consumption Rate and Polysaccharide Production Capacity of Lactobacillus Lusteri ATCC 23272

To measure the sugar consumption rate of the Lactobacillus ruteri ATCC 23272 strain, Lactobacillus bacteria stored in an 80 ° C. cryogenic freezer were inoculated into a 10 ml cap tube and activated in a 37 ° C. incubator for 24 hours, and then the activated strain was 100 ml of MRS. Medium (2% glucose, 1% beef extract, 1% peptone, 0.5% yeast extract, 0.1% Tween-80), 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% MgSO4, 0.005% MnSO4, 0.05 % L-cysteine-HCl) and sampled at 3 hour intervals to measure growth rate, yield and sugar consumption rate. As a result, Lactobacillus ATCC 23272 strains showed growth rate of 0.071 OD / hr, yield of 0.082 Ys / x and The glucose consumption rate was 1.21 g / L / hr.

 To determine the polysaccharide-producing ability of the Lactobacillus ruteri ATCC 23272, the strain was incubated for 24 hours in an MRS medium containing 0.5 ml in a tube and centrifuged at 13,000 g for 20 minutes, and a supernatant was taken and an equivalent amount of 40% trichloroacetate was added. After stirring vigorously, 2 times cold (4 ° C) ethanol was added to the supernatant obtained by centrifugation again at 13,000 (g) for 20 minutes, followed by centrifugation at 6,000 (g) for 10 minutes to discard the supernatant. The same amount of distilled water was added to suspend the polysaccharides and the polysaccharides were measured by the phenol-sulfuric acid method.

To test the gastric and duodenal transit ability of Lactobacillus ruteri ATCC 23272, a hydrochloric acid artificial gastric juice of pH 1.5 was prepared and inoculated with 10 ⁶ concentrations, and the survival rate after 2 hours (initial inoculation bacterial count (cfu / ml) / viable cell count after culture) (cfu / ml)), the survival rate after 2 hours in pseudo gastric juice (pH 1.5) was 25.5%, the survival rate after 9 hours in bile was 667%, and EPS production was 670 mg / L.

Example 2: Acquired the mutant strain of the present invention to produce a large amount of polysaccharides

In order to obtain a mutant strain that produces a large amount of dietary fiber, Lactobacillus ruteri ATCC 23272 obtained by incubating the bacteria at 37 ° C. for 6 hours in a modified MRS medium and then centrifuged was precipitated with physiological saline (PBS. PH 7.0) buffer. After washing, NTG (N-methyl-N-mitro-N-nitrogoguanidine) was added to a final concentration of 200 μg / ml, and cultured at 37 ° C. for 30 minutes to kill 99.9% of the strain.

The mutant group thus obtained was composed of high concentrations of fructose containing MRS (5% fructose, 1% beef extract, 1% peptone, 0.5% yeast extract, 0.1% Tween-80), 0.2% ammonium citrate, 0.5% Sodium acetate, 0.01% MgSO4, 0.01% MnSO4, 0.05% L-cysteine-HCl) medium was incubated for 48 hours at 37 ℃.

0.5 ml of the culture solution was added to each tube and centrifuged at 13,000 g for 20 minutes, the supernatant was collected, stirred by addition of an equal amount of 40% trichloroacetate, and then again centrifuged at 13,000 (g) for 20 minutes. Add cold (4 ℃) ethanol, mix it, and centrifuge again at 6,000 (g) for 10 minutes, then discard the supernatant carefully, add the first volume and the same amount of distilled water to suspend the polysaccharides and measure the amount of polysaccharides by phenol-sulfuric acid method. Polysaccharide efficiency (EPS (mg / L) / OD (650 nm)) was measured.

The strain having high polysaccharide efficiency was mutated again in the same manner to obtain a strain having high polysaccharide production efficiency.

In the first generation mutant strains, strains with increased polysaccharide production than the original strains were obtained and mutated again in the same manner to obtain second generation mutant strains. As a result, the Lactobacillus Lutheri PRD202 (hereinafter referred to as PRD202) showing high EPS (exopolysccharide) production efficiency was obtained, and PRD202 showed a polysaccharide production capacity of about 7 times that of Lactobacillus Luteri ATCC23272 as shown in Table 1. .

Polysaccharide Production of Lactobacillus Luteri ATCC 23272 and Lactobacillus Luteri PRD202 Strain name O.D (650nm)? ① EPS (mg / L)? ② EPS yield (② ÷ ①) Lactobacillus and Lutheri ATCC23272 5.15 673.5 130.8 Lactobacillus luster PRD202 2.10 1266.5 602.8

Example 3: Characterization and Identification of PRD202 Strains

In order to determine the genetic difference between the PRD202 microorganism and the parent strain having a large amount of polysaccharide production, the strains were inoculated in MRS liquid medium, respectively, and cultured at 37 ° C. for 12 hours. The culture was centrifuged at 4 ° C., 6,000 (g) to obtain microorganisms, from which the nucleic acids were extracted using the CTAB / NaCl method. As a result of determining the nucleotide sequence of the 16S rRNA gene using the nucleic acid extract 16S rRNA consensus primer (16S rRNA consensus primer), it was confirmed that the microorganism of the present invention is a Lactobacillus luterie ATCC23272 variant strain.

The growth rate of the microorganism of the present invention was lower than that of the parent strain, and the strains were less precipitated during the cultivation of the culture. As a result of comparison of sugar availability using the API 50 CH Kit (BioMerieux), the parent strain was αmethyl-D. While the growth was poor with -glucoside as a carbon source, the Lactobacillus luteri PRD202 of the present invention did not use it at all.

As described above, the microorganism of the present invention was named Lactobacillus reuteri PRD202 through phenotyping and sequencing of the 16S rRNA gene, and on July 12, 2002, the deposit number KCTC was assigned to KCTC, an international depository institution. Deposited to 10301BP.

Example 4 Degradability of Intestinal Digestive Enzymes of Polysaccharides

In order to determine whether the polysaccharide produced in the intestine by the microorganism PRD202 of the present invention is degraded by the intestinal digestive enzyme, the purified polysaccharide is dissolved in phosphate buffer, and the digestive enzyme separated from the small intestine of the rat is concentrated by concentration. The reaction was carried out for 120 minutes and measured using a trinder kit (cat. 315-500 Sigma, USA) using enzyme colorimetry.

As a result of the measurement, as shown in Table 2, the polysaccharide produced by PRD202 was not degraded by intestinal digestive enzymes. Therefore, it can be seen that the polysaccharide produced by the PRD202 of the present invention is decomposed by intestinal digestive enzymes and released into the body rather than absorbed into the body.

Degradation of Polysaccharides by Intestinal Digestive Enzymes ? Type of temperament Substrate concentration (mg / ml) Enzyme and concentration (ug / ml) 1250 625 312.5 156.25 78.13 39.06 fruit sugar 6.85 172.52 ^* 144.39 196.71 125.32 78.92 45.00 Polysaccharides 5 0.51 0.48 0.80 0.38 0.29 0.19

Example 5 Expression of Beta-glucuronides from Lactobacillus Luteri PRD 202

Beta-glucuronides produced by intestinal microorganisms delay the degradation of carcinogenic aromatic compounds such as benzopyrene and prevent the toxins produced in the body from being decomposed in the liver, thus delaying detoxification. Inflicted.

Therefore, in order to confirm the production of beta-glucuronides by the microorganism of the present invention in the intestine, hydrolysis of PNPG (para-nitrophenyl-beta-D-glucuronide) (Cat. N-1627, Sigma. USA) The absorbance measurement method was used.

First, the PRD202 was incubated in 300 ml of MRS medium at 37 ° C. for 24 hours, and then, the culture medium was centrifuged to recover the bacteria and washed with physiological saline (PBS), followed by GUS buffer (100 mM sodium phosphate-2.5). 50-400 μl of cell extract obtained by resuspending in 10 ml of mM EDTA [pH 6.0]), pulverizing and removing the residues of the cell walls, was added to 500 μl of GUS buffer containing 12.5 mM PNPG, and then 5, 15, 30 After reacting in a 37 ° C. water bath for 1 minute, the reaction was stopped using 1 M Na ₂ CO ₃ and absorbance was measured at a wavelength of 405 nm (OD405). The titer of beta-glucuronides (1 unit) was measured using a standard sample (Cat. No. G-8162, Sigma. USA), the comparison is shown in Table 3.

As a result of measuring the titer of beta-glucuronides of Lactobacillus luteri PRD202, the microorganism of the present invention does not produce beta-glucuronides at all, and as a result, the problem of the harmfulness caused by beta-glucuronides is I could not see.

Titer Determination of Beta-glucuronides Amount Response time (O.D. = 405 nm) 5 minutes 15 mins 30 minutes Standard concentration (beta-glucuronides) 1 unit 0.235 0.397 0.527 4.3 unit 0.540 1.054 1.715 8.7 unit 0.854 1.765 2.451 17.5 unit 1.516 2.436 2.533 Cell extract concentration 50 μl 0.011 0.016 0.013 100 μl 0.001 0.018 0.003 200 μl 0.050 0.048 0.050 400 μl 0.114 0.125 0.120

Example 6 Changes in Body Weight and Diet, and therefore Metabolic Efficiency upon Ingestion of Lactobacillus Luteri PRD202

The animals used for the measurement were SD rats (Sprague Dawley rats), and 5 week old males were used. The animals were classified into 9 groups of physiological saline (PBS), Lactobacillus luteria ATCC23272, and PRD202. According to the group, Lactobacillus luteri ATCC23272 and PRD202 were administered twice a day, and once administered, the cell mass was orally administered at a concentration of 3 × 10 ¹¹ cfu. Rat weight and feed intake were measured at two-day intervals and averaged 9 animals.

As a result of administration for 30 days, the weight change of the SD rats was shown gradually after 12 days as shown in Table 4 and FIG. 1, and the final 30 days of measurement showed that the body weight was suppressed by about 6.5% compared to the control PBS group.

Weight Changes in SD Rats During Long-Term Administration ? 0day 10day 20day 30day Control? (PBS? 147.1 ± 6.3 207.3 ± 4.2 273.1 ± 4.8 328.4 ± 6.9 Lactobacillus luster ATCC23272? 151.3 ± 3.2 207.6 ± 9.2 270.4 ± 7.2 320.4 ± 7.1 PRD202? 150.5 ± 6.0 200.4 ± 11.3 260.0 ± 7.5 307.3 ± 10.9 (Unit: g)

In addition, when comparing the change in feed intake as shown in Table 5 and Figure 2 was found to consume about 9.3% less than the PBS administration group. As a result, the metabolic efficiency of the feed was calculated, and the difference between the PBS-administered group and the PRD202-administered group was about 5.6%, and the metabolic efficiency of PRD202 was high (FIG. 3).

Changes in Feed Intake of SD Rats During Long-Term Administration ? 10day 20day 30day Control? (PBS? 188.8 ± 5.8 213.2 ± 10.8 231.6 ± 9.3 Lactobacillus ruteri ATCC23272 177.3 ± 10.5 202.7 ± 11.4 216.0 ± 9.9 PRD202? 176.7 ± 10.9 195.1 ± 16.8 210.0 ± 9.2 (Unit: g)

As a result, the microorganism of the present invention absorbs low saccharides and converts them into polysaccharides, which has a great effect on the treatment and prevention of obesity, and it can be seen that there is an effect of reducing food intake due to satiety caused by polysaccharides formed in the body. .

Example 7 Changes in Blood Cholesterol upon Ingestion of Lactobacillus Luteri PRD202

Ingestion of PRD202 according to the present invention, the change in blood cholesterol was analyzed to determine whether the effect on the reduction of circulatory diseases (arteriosclerosis, myocardial infarction, etc.) as well as diseases such as diabetes and obesity.

For the analysis of fat, the blood of rats under test was collected by tail blood collection at a weekly interval. Lipid analysis was performed using enzyme colorimetric method and the amount of cholesterol in serum was calculated by measuring the absorbance with standard solution at 505-570 nm using Cholestezyme-V (Shinyang Chemical).

As shown in Table 6 and FIG. 4, the total cholesterol level in blood was significantly decreased (P <0.005) as compared with the physiological saline administration group (P <0.005) on the 28th day of administration of PRD202. Therefore, the microorganism of the present invention was expected to be effective in reducing circulatory diseases (arteriosclerosis, myocardial infarction, etc.).

Changes in blood cholesterol levels in SD rats during long-term administration 0day 7day 14day 21day 28day Control (saline-administered group) 87.99 ± 2.11 109.13 ± 7.38 84.98 ± 6.73 87.66 ± 5.30 82.46 ± 3.94 Lactobacillus ruteri ATCC23272 82.81 ± 3.50 107.87 ± 4.65 85.3 ± 9.17 83.11 ± 9.96 74.38 ± 7.36 PRD202 administration group 82.98 ± 2.64 105.85 ± 9.26 82.95 ± 3.71 80.66 ± 8.04 68.01 ± 4.47 ^* *: When comparing the control group with the control group on day 28? PD202? P <0.005, (unit: mg / dl)

Example 8 Changes in Blood Glucose Levels after Ingestion of Lactobacillus Luteri PRD202

The blood glucose level of the rat was measured to identify problems such as hypoglycemia due to glucose uptake by the microorganisms of the present invention.

Blood was collected from rats at weekly intervals for blood glucose measurements and serum was isolated. Blood glucose levels were measured for absorbance at a wavelength of 505 nm using a enzyme kit (Cat. 325-500, Sigma, USA). The blood glucose level for each serum was calculated based on the standard solution. The experimental results were expressed as mean ± standard deviation per experimental group. The blood glucose level measurement result is shown in FIG. 5.

As can be seen in FIG. 5, blood glucose levels were similar to those of normal rats. Therefore, it could be seen that there is no fear of showing hypoglycemia lower than normal blood glucose levels due to the administration of the microorganism of the present invention (Table 7).

Changes in Blood Glucose Levels in SD Rats During Long-Term Administration 0day 7day 14day 21day 28day Control (saline-administered group) 129.8 ± 5.5 98.2 ± 8.8 127.1 ± 8.4 114.9 ± 7.9 107.8 ± 10.7 Lactobacillus ruteri ATCC23272 126.6 ± 5.2 106.9 ± 8.8 129.2 ± 8.4 105.8 ± 4.1 101.7 ± 2.5 PRD202 administration group 125.3 ± 11.5 111.8 ± 12.7 121.0 ± 9.5 121.6 ± 9.6 106.9 ± 10.1

While the configuration and preferred embodiments of the present invention have been described herein, the scope of the present invention is not to be construed as being limited to the described embodiments, and those skilled in the art will be provided with the present specification within the scope of the present invention. Modifications of the disclosure may be made.

The microorganism of the present invention has excellent intestinal viability and high-efficiency polysaccharide production ability, while surviving in the intestine, by converting low-saccharide carbon compounds into high molecular weight polysaccharides to reduce the amount of carbon compounds absorbed into the body, obesity and Has a therapeutic and prophylactic effect of diabetes. In addition, there is no harmful effect of beta-glucuronides, and it shows the effects of weight control, intestinal action, and cholesterol absorption inhibition.

Claims (7)

Lactobacillus lusteri (KCTC-10301BP) to produce polysaccharide material. A pharmaceutical preparation for the prevention of obesity, comprising a pharmaceutically effective amount of Lactobacillus luterie (KCTC-10301BP) and a carrier to produce a polysaccharide substance. The anti-obesity preparation according to claim 2, wherein the anti-obesity preparation is coated with an enteric coating material. A food composition containing Lactobacillus ruteri (KCTC-10301BP), which produces a polysaccharide substance, as an active ingredient. The food composition of claim 4, wherein the food composition has a weight loss effect. A pharmaceutical preparation for the prevention of diabetes comprising a pharmaceutically effective amount of a Lactobacillus luterie (KCTC-10301BP) and a carrier for producing a polysaccharide substance. The antidiabetic preparation according to claim 6, wherein the antidiabetic preparation is coated with an enteric coating material.

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