KR100404236B1 - Microorganisms for Corpulence or Diabetes Mellitus, or a pharmaceutical composition containing the same - Google Patents

Abstract

In the present invention, by reducing the amount of glucose, sucrose or fructose absorbed in the intestine by converting glucose, sucrose, or fructose into the intestine while entering the intestine, it prevents obesity or diabetes Novel microorganisms and their pharmaceutically effective amounts that make it treatable are disclosed.

Description

Microorganisms for the treatment of obesity or diabetes and pharmaceutical composition containing the microorganisms {Microorganisms for Corpulence or Diabetes Mellitus, or a pharmaceutical composition containing the same}

The present invention converts monosaccharides or disaccharides such as glucose, sucrose and fructose into high molecular weight substances that are not absorbed in the intestine, thereby reducing the amount of monosaccharides or disaccharides absorbed into the body, thereby treating or preventing obesity or diabetes. The present invention relates to a microorganism that represents and a pharmaceutical composition containing the microorganism.

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 Institutes of Health (NIH) report, Clinical guidelines on the identification, evaluation, and treatment of overweight and obesity in adults, 1999, NIH shows that 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. In addition, about 200,000 deaths worldwide are reported annually 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 requires significant personal and financial costs not only for patients and their families but also for society. There are different diabetes types with distinct etiology 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 with adequate production and use of insulin, but with shortcomings in insulin mediated use and glucose 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 treat NIDDM (ie, control of blood glucose levels) can lead to death from cardiovascular disease and other diabetic complications including retinopathy, kidney disease and peripheral neuropathy.

Treatment of NIDDM includes the use of sulfonyl urea and biguanide therapeutic compounds, as well as diet and exercise, as a method of controlling blood glucose levels in NIDDM patients. In addition, metformin and acarbose compounds have also recently been used to treat NIDDM patients. However, in some patients hyperglycemia is not adequately controlled by diet, exercise and the use of such therapeutic compounds. In such cases, the patient must be administered exogenous insulin.

Administering insulin by injection to a patient, in addition to being expensive and painful, can cause various situations or complications that are detrimental to the patient. For example, an insulin response (hypoglycemia) can be caused without error, fasting, irregular exercise, or special cause of insulin dosage. In addition, local 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 it is limitedly used due to the need for surgery, insufficiency of the effect, and high cost. have.

Drug therapies to treat or prevent obesity or obesity-type diabetes are currently the most actively studied, with drugs that reduce blood sugar, inhibit blood sugar absorption, enhance insulin action, or cause loss of appetite. Way. Drugs for the prevention and treatment of obesity and obesity-type diabetes developed to date use a variety of physiological mechanisms.

As drugs that increase the action of insulin, sulfonylureas, metformin, pioglitazone, thiazolidinedione derivatives and the like have been developed. Sulfonylurea acts to stimulate the secretion of insulin from the beta cells of the pancreas, but can cause side effects of hypoglycemia that lowers blood glucose levels below normal.

Metformin is mainly used in insulin-independent diabetics who do not recover from diet or exercise. The drug inhibits the process of hepatic gluconeogenesis in the liver and allows glucose to be stored in muscle and adipose tissue. However, it is known that there are side effects such as vomiting and diarrhea.

Pioglitazone was developed by Tadada Co. of Japan, and plays a role in enhancing insulin action by increasing insulin sensitivity to cells (Kobayashi M. et al ., Diabetes, 41 (4), pp 476-483, 1992 ).

Beta 3-adrenoreceptor inhibitor (BRL-35135) is known as a drug that promotes the breakdown of fat in the body, and currently acts specifically on adipocytes, breaks down fat from adipocytes, releases them to heat, and lowers blood sugar levels.

Pancreatic lipase inhibitors (Orlistat: Roche, Switzerland) are known as inhibitors of fat absorption. They inhibit the action of pancreatic lipase and reduce the absorption of fat or fat-soluble vitamins. There is a possibility of induction.

Appetizers that inhibit appetite mainly affect neurotransmitters (catecholamines) present in the brain, which reduces appetite. However, dexfenfluramine and fenfluramine have side effects of neurotoxicity and valve heart disease, and sibutramine has side effects of increasing heart rate and blood pressure.

Examples of therapeutic agents for inhibiting sugar absorption include α-glucosidase inhibitors (Acarbose: Bayer, Germany), amylase inhibitors, dietary fiber, and polymers. Acarbose is a pseudo-oligosaccharide (glycosaccharide) known to competitively inhibit the action of various α-glucosidases present in the microvilli in the gastrointestinal tract. Ingestion of it in large amounts can cause diarrhea (W. Puls et al ., Front. Horm. Res. 2, 235, 1998).

Amylase inhibitors have been developed to prevent metabolic imbalances resulting from excessive nutrient intake by inhibiting the action of amylase, which converts carbohydrates into glucose (Sanchez-Monge R. et al , Eur. J. Biochem., 183, pp 37- 40, 1989).

Dietary fiber intake is one of the easiest ways to reduce the amount of sugar absorbed in the small intestine through a diet with a lot of vegetable fiber, which is to prevent obesity. However, there is a problem that dietary fiber requires a lot of equipment investment and manpower for production, and the productivity of dietary fiber is not so high.

Examples of the high molecular materials include isomaltriotriose, dextran, and furan, although these high molecular substances inhibit the increase in blood glucose levels due to the ingestion of sugar, but have serious side effects. For example, dextran can cause excessive bleeding by delaying blood coagulation time.

However, it does not damage the metabolic balance of the human body among the various drugs as described above, and because it is a natural substance, dietary fiber has been recognized as the most useful obesity prevention or treatment.

Microbial fiber is a genus of GluconobacterGluconobacter sp.), In the AgrobacteriumAgrobacterium sp.), Acetobacter xylinum (Acetobacter xylinum), Acetobacter Hanseni (A. hansenii), Acetobacter pasteerianus (A. pasteurianus),Acetobacter Aceti (A. aceti)., Lysobium genusRhizobium), Genus Alkali genesisAlcaligenes), Salina (Sarcina), Streptococcus thermophilus (Streptococcus thermophilus), Lactococcus Cremoris (Lactococcus cremoris), Lactobacillus helvetica (Lactobacillus helveticus), Lactobacillus vulgaris (L. bulgaricus), Lactobacillus sake (L. sake), Lactobacillus lutery (L. reuteri), Lactobacillus lactis (L. lactis), A subspecies of Lactobacillus del Vulckey (L. delbrueckii subsp.), A variant of Lactobacillus helvetus glucose (L. helveticusglucose var. jugurti), Leukonostock Dextranicum (Leuconostoc dextranicum), Genus Bulgari (Bulgaricus), Campestris (Campestris), The genus SphingomonasSphingomonasIt is made by microorganisms such as).

The dietary fiber produced by these microorganisms is used as a stabilizer, thickener, emulsifier, moisture absorbent, etc. of various foods, as a material for medicines and cosmetics. Guar gum, Locust bean gum, carrageenan, alginate, agar, which are currently dietary fiber refined from microorganisms such as cellulose, xan, acetan and seaweed Etc. are commercialized.

Strains of the genus Lactobacillus have been known for a long time as a major member of the normal microbial flora in the intestine of the human body and are important for maintaining a healthy digestive system and 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 habitats of Lactobacillus strains are the digestive organs of animals ( L. acidophilus, L. intestinalis, L. johnsonii, L. reuteri, etc.), vaginal mucosa ( L. vaginalis, L. gasseri ), food (wine- L. hilgardii). ), Lactic acid bacteria beverage ( L. kefir, L. kefiranofaciens ; cheese -L. casei), vinegar ( L. acetotolerans), oral ( L. oris), yeast ( L. sake , L. homohiochi), fruit juice ( L. kunkeei , L. mali, L. suebicus), fermented sausages 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, constipation or urinary tract infection, the various probiotic activities of Lactobacillus, namely, regulation of immunity, control of blood cholesterol levels, treatment of rheumatism, prevention of cancer, reduction of sensitivity to lactose, atopic dermatitis The importance and interest in the effects on 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". None of the 60 strains belonging to the genus Lactobacillus are known to be toxic to humans.

Recently, studies on the extracellular dietary fiber synthesized by Lactobacillus have been actively conducted, and these strains are very complicated because many genes are involved in the mechanism for making the extracellular fiber, 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, 659-64, 1998; FEMS Microbiol. Rev., Apr 23: 2 153-77, 1999; FEMS Microbiol. Rev., Sep 7: 1-2, 113-30, 1990.)

Acetobacter sp. Is also well known as a microorganism for synthesizing cellulose fiber, and much research has been conducted on the synthesis of cellulose. (Aloni Y., Cohen R., Benziman M., Delmer D, J Biological Chemistry, 258: 4419-4423, 1983; Amikam, Benziman M., Journal of Bacteriology, 171: 6649-6655, 1989; Aschner M., J Bacteriology, 33: 249-252, 1937; Benziman M., Burger Rachamimov H., J Bacteriology, 84: 625-630, 1962; Brown AM, Journal of Polymer Science, 59: 155-169, 1962; Brown AM, Gascoigne JA, Nature, 187: 1010-1012, 1960; Colvin JR , Planta, 149: 97-107, 1980; Delmer DP, Amor Y., Plant Cell, 7: 987-1000, 1995; Delmer DP, Benziman M., Padan E., PNAS USA, 79: 5282-5286, 1982 See Delmer DP, Brown RM Jr., Cooper JB, Lin FC, Science, 230: 822-825, 1985.)

Acetobacter is a strict aerobe, but has the property of growing under extremely low oxygen conditions, and has a property of self-synthesizing cellulose dietary fiber to be supplied with oxygen under conditions of low oxygen. have. According to a study on the amount and rate at which acetobacter converts glucose into cellulose fiber (Brown et al .; Proc. Natl. Acad. Sci. USA, Vol73 (12), 4565-4569), about 400 amol / cell / hr It is said to convert glucose into cellulose at a rate. This is the rate at which 4 × 10 ¹⁵ cells can convert about 200 g of glucose into cellulose fiber in an hour.

It is rarely reported that acetobacters metabolize sucrose, but acetobacters exist in nature that can convert sucrose to glucose (PNAS, 96: pp 14-18, 1999). Acetobacter xylinum is currently approved by the US FDA for the synthesis of acetic acid and sorbose and is classified as generally accepted as safe (GRAS).

As mentioned above, there have been various studies and efforts on the treatment of obesity or diabetes, but the result is still insignificant. Although various compounds mentioned above have been developed for the treatment of obesity or diabetes, side effects are occurring, and they function to forcibly release body fat and protein accumulated in the body, and are groundbreaking agents for inhibiting or treating obesity or diabetes at the source. There is no cure 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 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 saccharides absorbed into the body by containing a pharmaceutically effective amount of the microorganism.

1 is a diagram showing the glucose absorption rate of microorganisms according to the present invention.

Figure 2 is a chart showing the change in blood sugar level when ingesting microorganisms according to the present invention.

Figure 3 is a chart showing the change in energy metabolism efficiency of obese mice ingested microorganism according to the present invention.

Figure 4 is a chart showing the change in energy metabolism efficiency of mice ingested microorganism according to the present invention.

Figure 5 is a chart showing the weight change of the mice ingested the microorganism according to the present invention.

6 is a chart showing the weight change of obese mice induced with drug administration.

7 is a chart showing metabolic efficiency changes in obese mice induced with drug administration.

8 is a phylogenetic diagram of Lactobacillus strain BC-Y009 based on 16S rRNA base sequence.

9 is a phylogenetic diagram of acetobacter strain BC-Y058 based on 16S rRNA base sequence.

The microorganism of the present invention can be grown in the intestine, harmless to the human body, and in the acetobacter genus, gluconobacter genus, Lactobacillus genus or Agrobacterium which convert low sugars into high molecular weight substances that are not absorbed by the body. It is preferable to belong to the strain. Specific examples include acetobacter xylinum (Acetobacter xylinum), Acetobacter Hanseni (A. hansenii), Acetobacter pasteerianus (A. pasteurianus),Acetobacter AcetiA. aceti), Lactococcus Cremoris (Lactococcus cremoris), Lactobacillus helvetica (Lactobacillus helveticus), Lactobacillus vulgaris (L. bulgaricus), Lactobacillus sake (L. sake), Lactobacillus lutery (L. reuteri), Lactobacillus lactis (L. lactis), A subspecies of Lactobacillus del Vulckey (L. delbrueckii subsp.), A variant of Lactobacillus helvetus glucose (L. helveticusglucose var. jugurti) And the like can be used in the composition of the present invention. The microorganisms used in the composition of the present invention are particularly preferably strains of genus Lactobacillus BC-Y009 (KCTC0774BP) and strains of genus Acetobacter BC-Y058 (KCTC0773BP).

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 present 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 compositions 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 which have been lyophilized using a standard carrier can be prepared and then filled into hard gelatin capsules. Alternatively, the suspension or dispersion may be prepared using the microorganism of the invention and any suitable pharmaceutical carrier, such as aqueous gum, cellulose, silicate or oil, and then the dispersion or suspension 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” herein includes all known types of pharmaceutically acceptable coatings that are not degraded by gastric acid so that the coating is retained, but in the small intestine is sufficiently degraded to allow the active ingredient to be released into the small intestine. do.

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 the KH ₂ PO ₄ buffer solution of pH 6.8 Refers to a coating that degrades within 30 minutes in the same artificial intestinal juice.

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. The case where the thickness of the enteric coating of this invention is 5-100 micrometers, Preferably it is 20-80 micrometers showed the satisfactory result as an enteric coating. 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 Remington's Pharmaceutical Sciences, 13th edition, pp. 1689 to 1691 (Mack Publ., Co., 1970), which may include cellulose ester derivatives, cellulose ethers, methylacrylate copolymers of acrylic resins and copolymers of maleic and phthalic acid derivatives.

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 for 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 typically has a molecular weight of 20,000 to 100,000 Daltons, preferably 80,000 to 130,000 Daltons, hydroxypropyl content of 5 to 10%, 18 to 24% Methoxy content and phthalyl content of 21 to 35%. 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 capable of reducing 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 is administered Route, and subject to 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 for unit administration for human subjects and other mammals, each unit containing 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.

As used herein, the term "composition" does not necessarily mean to be approved as a medicine, but is a concept including a conventional 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 saccharides, but also the non-digestible dietary fiber produced by the microorganisms can control the growth conditions of the intestinal useful bacteria. By improving and stimulating bowel movements, the composition of the present invention functions as a prophylactic and therapeutic agent for 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.

We have isolated and identified new microorganisms that can be used in the compositions of the present invention in the following manner.

Mixture samples from sugar plant sewage and various other areas were inoculated in MRS agar medium and BHS agar medium containing cycloheximide. The resulting colonies were inoculated in MRS and BHS liquid medium, and cultured in a stationary state to select strains that produce a membrane or capsule membrane in the upper layer. The formed membrane was separated and the separated membrane was checked whether it was digested by intestinal digestive enzymes to determine whether non-digestible (or indigestible) polymer material was produced. Among them, strains BC-Y009 and BC-Y058, which produce large amounts of extracellular polysaccharides (dietary fibers), were selected.

Morphological observations of the BC-Y009 and BC-Y058 strains and the similarity of partial sequencing of the 16S rRNA showed high similarity with Lactobacillus and acetobacter, respectively, so that the phenotype and 16S rRNA bases were Based on sequencing analysis, strain BC-Y009 belongs to the genus Lactobacillus, and strain BC-Y058 was identified as the genus belonging to the genus Acetobacter.

As a result of administering Lactobacillus BC-Y009 and Acetobacter BC-Y058 of the present invention to mice that cause obesity and diabetes, the blood glucose level of the mice was reduced by about 70% compared to before administration, whereas in normal mice, It was insignificant.

As a result, the microorganism of the present invention is effective in reducing blood glucose levels, and thus effective in treating and preventing diabetes mellitus, and even if applied to a normal human body, unlike other conventional obesity treatment agents, hypoglycemic symptoms do not occur. Could. When the strains BC-Y009 and BC-Y058 according to the present invention were administered to mice with obesity and diabetes, the feed intake increased by 17 to 24% compared to normal mice, but the weight gain efficiency was significantly reduced compared to the feed intake. This means that when the human ingests the microbial composition according to the present invention, even if the food is sufficiently ingested according to the appetite, there is less concern about obesity or diabetes.

When ingesting the microorganism according to the present invention, the serum lipid content is also lower than that of the control group, so that the microorganism and the microbial composition of the present invention are effective in obesity, diabetes and circulatory diseases (arteriosclerosis, myocardial infarction, etc.). Can be. Furthermore, in the case of normal mice, the group to which the microbial composition of the present invention was administered consumed a little more diet, resulting in a slight decrease in energy efficiency compared to the non-administered control group. No side effects from administration of the agent occurred in mice.

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

Example 1

Microbial Screening of Extracellular Polysaccharides from Soil Samples

To isolate the microorganisms that produce dietary fiber, 10 g of samples taken from the sugar factory sewage and various other areas were taken, crushed, and then dispersed in 90 ml of physiological saline (0.85% NaCl), respectively. After diluting these dispersions with physiological saline 10 ⁻² , 10 ⁻⁴ and 10 ⁻⁶ , these dilutions were diluted with MRS [1% peptone, 1% beef extract, 0.5 containing 1 mg of cycloheximide per 100 ml. % Yeast Extract, 2% Glucose, 0.1% Tween-80, 0.2% Ammonium Citrate, 0.5% Sodium Acetate, 0.01% MgSO _4, 0.005% MnSO ₄ , 0.2% Sodium Phosphate, pH6.5] Agar Medium And BSH (2% glucose, 0.5% peptone, 0.5% yeast extract, 0.27% Na ₂ HPO ₄ , 0.115% citric acid, pH5.0) (see Hestirin and Schramm, J. Gen. Microbiol., 11: 123, 1954) Plated in agar medium and incubated at 30 ℃ for 72 hours.

About 2,000 colonies thus obtained were firstly inoculated into 5 ml MRS liquid medium and BSH liquid medium, and then cultured at 30 ° C. for 72 hours. Strains that produce a membrane in the form of a matrix on top of the liquid medium and strains that form a transparent and encapsulated extracellular polysaccharide were selected. They were inoculated again in 5 ml MRS liquid medium and BSH liquid medium, stirred at 30 ° C. and absorbance at 600 nm. They were diluted with BSH liquid medium to have an absorbance of 0.2, and then 10 ml of the solution was inoculated into 100 ml of BSH liquid medium and left to incubate at 30 ° C. for 72 hours.

In order to measure the amount of extracellular fiber produced, each culture was centrifuged at 6,000 rpm at 4 ° C. to obtain a microbial precipitate. The membrane adhered to the cells was alkali dissolved in 0.1 N NaOH, left at 80 ° C. for 30 minutes, and centrifuged again at 4 ° C. and 6,000 rpm. Extracellular polysaccharides entangled like white thread were isolated and lyophilized to determine their amount. Among the strains that produced a large amount of extracellular polysaccharides were selected to compare the extracellular polysaccharide production amount (Table 1).

Example 2

Morphological Characteristics and Identification of Selected Strains BC-Y058, BC-Y009

Partial nucleotide sequences of BC-Y009, BC-Y002, BC-Y015, BC-Y026, BC-Y058, BC-Y112, BC-Y130, and BC-Y201 in the strains selected from Example 1 were determined. As a result, BC-Y009, BC-Y002, BC-Y015, BC-Y026 were bacteria of the genus Lactobacillus and BC-Y058, BC-Y112, BC-Y130, BC-Y201 were judged to be microorganisms of the genus Acetobacter.

Among them, BC-Y009 and BC-Y058, which have high amounts of dietary fiber, were inoculated in MRS and BSH liquid media, respectively, and the suspension culture was carried out at 30 ° C. for 72 hours. The culture was centrifuged at 4 ° C., 6,000 rpm to obtain microorganisms, from which nucleic acids were extracted using the CTAB / NaCl method. The nucleic acid extract was amplified and sequenced 16S rRNA by polymerase chain reaction (PCR) using a 16S rRNA consensus primer (16S rRNA consensus primer). BLAST analysis (NCBI, USA) on the determined nucleotide sequences revealed a number of Lactobacillus, including L. hilgardii , A. xylinum , Gluconobacter sp. It showed high sequence similarity with the species and acetobacter species (see Table 2 and Table 3).

Strain BC-Y009 is a gram positive, non-motile, short rod-shaped bacterium with a size of 0.5-3.0 μm. It does not form spores and is anaerobic anaerobic, the growth temperature can be grown between 20 ° C.-37 ° C., pH 2.0-8.0, and the optimum pH range is 4.0-7.0. Milk coagulation, catalase negative, and white colonies were formed in the complex medium. The MRS liquid medium and BSH liquid medium were immersed in the lower layer of the liquid medium in the form of a white capsule, and the turbidity of the liquid medium formed dietary fibers in a clear and transparent medium state, and showed the characteristics of breaking finely when the liquid medium was shaken.

Strain BC-Y058 was Gram-negative bacilli-shaped and existed in single or paired states of 0.6ⅹ0.8 μm. It was inelastic and did not produce spores. Growth was slow and had to be incubated for 5-7 days, and colonies formed small, hard and transparent cellulose pellicles in liquid medium. Ethanol, acetic acid or lactic acid can be used as a substrate and showed catalase positive reaction. Glucose was used to produce acid and failed to grow in Heuer medium.

As described above, the strain BC-Y009 was identified as Lactobacillus sp. BC-Y009 through the phenotype and 16S rRNA sequencing, and the strain BC-Y058 was designated as Acetobacter sp. BC-Y058. On May 30, 2000, he deposited the deposit number KCTC BC-Y009 and KCTC BC-Y058 with KCTC, an international depository institution, KCTC.

Example 3

Degradability of Dietary Fiber by Intestinal Digestive Enzymes

Forcine pancreatin (porcine pancreatin) containing amylase, lipase, protease, nuclease and the like and having 3 × USP activity to determine whether the fiber produced in the intestine by the microorganism is degraded by intestinal digestive enzymes. And 1 g of Sigma Co., Ltd. were added to an aqueous solution (pH7.5) in which 1 g of dietary fiber dry weight was dissolved and suspended. The suspension was incubated at 40 ° C. for 7 days, and the reaction solution was taken once a day to quantify per DNS. As a result, it was confirmed that it was not degraded at all.

Therefore, it was found that the dietary fiber produced by the microorganism of the present invention was not degraded in the intestine.

Example 4

In vivo glucose absorption rate

Lactobacillus BC-Y009, Acetobacter BC-Y002 together with L. acidophilus KCTC3140 and L. hilgardii KCTC3500, strains used as physiologically active bacteria (probiotics). In vivo glucose uptake rates of acetobacter BC-Y058 and E. coli were measured. The measurement results are shown in FIG. 1 and Table 4.

As can be seen in Figure 1 and Table 4, the strains of the present invention was found to be superior in vivo glucose absorption rate compared to other lactic acid bacteria.

Example 5

Intestinal Microbial Concentration and Viability after Feeding Microbial-Containing Feed

Obesity and diabetes-causing mice, C57BL / 6J Lep ^ob ob / ob (hereinafter referred to as "OB mice"), were fasted for 18 hours and lactobacillus BC-Y009, acetobacter BC-Y058 1% (w / w) After ingesting the composition of the present invention (dry weight: 1.0 × 10 ¹³ CFU / g) for 7 days containing the dry weight, the bacterial density in the duodenum, jejunum and colon of these mice was examined. As a control, the bacterial density in the duodenum, jejunum and large intestine of OB mice fed with the microorganism-free feed for 7 days was also investigated.

To determine the number of Lactobacillus bacteria, the duodenum, jejunum and large intestine of the control group and the control group of Lactobacillus were removed. The surface of each organ was washed with physiological saline, and then the contents were dispersed in physiological saline, and then plated on MRS agar medium and incubated at 37 ° C. After 3 days, the number of bacteria was measured with or without floc formation, and the increase and decrease of the bacteria was determined by subtracting the number of Lactobacillus bacteria from the control group (Table 5).

In order to determine the presence of acetobacter, each organ of the mouse was removed, the outside of the organ was washed with physiological saline, the contents were dispersed in physiological saline, inoculated in BSH liquid medium and incubated at 37 ° C. for 3 days. The presence of cellulose-producing acetobacter cells was determined by the presence or absence of a pellicle formed on the upper layer of the liquid medium (Table 6).

The results of Table 5 and Table 6 show that both types of microorganisms can grow in the intestine of mice.

Example 6

Changes in blood sugar levels when ingested BC-Y009, BC-Y058

100 g of mouse feed purchased from Samyang Co., Ltd. mixed 400 g of domestic rice with a carbohydrate ratio of about 60%, followed by kneading with 5 g (1%) dry weight of Lactobacilli BC-Y009 or Acetobacter BC-Y058. Freeze-dried dumplings were prepared to a size that the mice could feed on. This dumpling was provided to mice with water for free feeding.

The mice used in this example were all female OB mice that induced obesity and diabetes. To distinguish between the fungal intake group and the non-intake control group, the acetobacter intake group (OB-058) and the lactobacillus intake group (OB- 009), non-ingestion control (feeding without bacteria feed, OB-con) was separated and bred. The breeding environment was subjected to periodic lighting (lighting until 09:00, shading until 21: 00-09: 00) at 12 hour intervals, and the temperature and humidity were maintained at 20 to 24 ° C. and 40 to 60%, respectively.

In addition, enteric coating preparations were prepared by spray coating the enteric coating composition solution on the prepared dumplings. The enteric coating of the present invention has a weight of about 16 to 30 mg or less at one end of size and is made of a coating having a thickness of 5 to 100 μm. The enteric coating material was appropriately selected from known polymeric materials, including cellulose acetate phthalate and trimellitate or methacrylic acid copolymers (methylacrylic acid and esters thereof, including at least 40% methylacrylic acid, especially hydroxypropyl methylcellulose phthalate). Copolymers derived from) were used.

As the methyl acrylate, Endragit L 100-55 (commercially available from Rohm GmbH, Germany) was used, with a viscosity of about 45 to 90 cP and 17 to 26% acetyl. Cellulose acetate phthalate with a phthalate content of 30 to 40% and a cellulose acetate trimellitate product sold by Eastman Kodak Company (viscosities of about 15 to 20 cS and 17 to 26% acetyl) Content, products having trimellityl content of 25 to 35%).

The enteric coating solution thus prepared was prepared using a conventional coating method, in which the core was sprayed, ethanol and acetone mixed solvents were used, and a softener was added to the coating solution at a ratio of 1 to about 0.05 to about 0.3.

The enteric coating composition of the present invention prepared by the above method was freely ingested by the mouse with water, and the blood glucose level of the mouse to which the composition was administered was measured.

Before measuring blood glucose levels in each group of mice, each mouse was fasted for 18 hours, and then the mice were fed a sufficient amount of food for 60 minutes, and 60 minutes after the removal of the food, the retro-orbital vein was removed. venous plexus) was used to collect blood using capillary tube without anticoagulant to obtain serum.

Blood glucose levels were measured for absorbance at 505 nm with a spectrophotometer (Trinder kit) (Cat. 315-500, Sigma, USA) using enzyme colorimetric method. The blood glucose level for each serum was calculated based on the standard solution. The statistical significance of the experimental results was expressed as the mean ± standard deviation per experimental group and the statistical significance of the mean difference in each group was verified by ANOVA (p <0.02).

The blood glucose level measurement result is shown in FIG. 2. As can be seen in Figure 2, the blood sugar level was high in the OB-con group was about 500 mg / dl, while the blood sugar level of the OB-058 group blood glucose levels of normal C57BL / 6J mice (hereinafter referred to as "Lean group") It can be seen that the blood sugar level is similar to. In addition, it can be seen that the administration of acetobacter BC-Y058 and Lactobacillus BC-Y009 reduced the blood glucose levels of the mice by about 70% and 53%, respectively (Table 7).

Example 7

Changes in Body Weight and Diet and Consequent Metabolic Efficiency of BC-Y058 and BC-Y009

Mice were divided into OB-058 group, OB-009 group, OB-con group, Lean-009 group, Lean-058 group, and Lean-con group, and acetobacter BC-Y058 and Lactobacillus BC-Y009 were administered according to the group. While the body weight of the mouse was measured at weekly intervals. In addition, the weight of the food consumed by the mouse along with the change in the weight of the mouse was measured, and the change in metabolic efficiency between groups was investigated.

The weight change of mice was obviously the change in weight between species with different genetic characteristics, but there was no difference in weight change in the group with the same genetic characteristics. As can be seen in Table 8, the 7-week weight change was increased by about 47% with or without acetobacter BC-Y058 or Lactobacillus BC-Y009 for OB mice and BC for Lean mice as well. -Y058, BC-Y009 administration resulted in about 27% increase in body weight. However, as shown in Table 9 and Table 10, the intake of feed according to the fungal administration was increased by 17 to 24% in the OB mouse group, and about 15 to 23% in the Lean mouse group.

In other words, ingesting the microorganism-added feed according to the present invention was found to cause weight gain as much as the case of ingesting the microbial non-added feed. These results suggest that acetobacter BC-Y058 or Lactobacillus BC-Y009 inhibits the increase in postprandial blood sugar level, and thus it is considered that more feed is ingested as a compensation action. This difference in metabolic efficiency can be attributed to the glucose consumption of BC-Y058 or BC-Y009 administered and the effect of dietary fiber produced by BC-Y058 or BC-Y009.

According to the following equation, the change in energy efficiency according to the feed intake is calculated and shown in Table 10.

As can be seen from the table, when the microorganisms were administered to both OB mice and normal mice (Lean mice), the energy metabolic efficiency was about 75 to 85% compared to the non-microbial administration (Fig. 3, Fig. 4). .

Example 8

Changes in Body Weight and Diet and Consequent Metabolic Efficiency of Obese Mice with BC-Y058 or BC-Y009

Prior to ingesting Acetobacter BC-Y058 and Lactobacillus BC-Y009, mice were injected with goldthioglucose (Cat. A-0632, Sigma, USA) at 1 g / kg per horse to induce obesity In addition, only mice induced with obesity were selected by measuring body weight change over 3 to 4 weeks. For the accuracy of the experiment, mice with relatively large or small weight gain were excluded.

For female C57BL / 6J mouse species, the breeding environment and conditions were applied in the same manner as mentioned in Example 6. According to the species to be ingested were classified into BC-Y058 group, KCTC3140 group, KCTC3500 group, BC-Y009 group.

The weight change of the mouse according to the administered bacteria is shown in Figure 6. Figure 6 shows that the rate of weight gain decreases when ingesting Acetobacter BC-Y058 and Lactobacillus BC-Y009.

In addition, as shown in Table 11 and Figure 7, in the case of KCTC3140, KCTC3500 consumed sugar but does not produce dietary fiber, the energy efficiency in the obesity-induced mice is higher than the control group did not administer the cells. However, mice fed the BC-Y009 and BC-Y058 producing dietary fiber showed relatively less energy efficiency. In particular, the energy efficiency of BC-Y058 was reduced by 55% compared to the control group, resulting in normal mouse levels. It showed close energy efficiency (see Table 12).

Example 9

Changes in Body Weight and Diet and Normal Metabolic Efficiency of Normal Mice after Ingestion of BC-Y058 or BC-Y009

The mice used in this experiment were all female C57BL / 6J mouse species, and the breeding environment and conditions were applied in the same manner as mentioned in Example 6. The feed prepared in Example 6 was divided into KCTC3500 group, KCTC3140 group, BC-Y058 group, BC-Y009 group, and the control group which did not administer the bacterium according to the strains of the mice ingested in the experiment. Unlike the case of Example 8, the weight change of the mouse according to the administered bacteria was not seen when ingesting Acetobacter BC-Y058 and other strains. In addition, as shown in Table 12, it can be seen that the difference in energy efficiency is not significant regardless of the presence or absence of dietary fiber in the case of normal mice.

As seen in Examples 8 and 9, changes in body weight and diet and subsequent metabolic efficiency when ingested with Acetobacter BC-Y058 and Lactobacillus BC-Y009 are effective only on abnormal obese mice induced with drug administration. It did not show any change in normal mice.

Example 10

Distribution of fat and its changes with BC-Y058 or BC-Y009

When ingesting the microorganism according to the present invention, the change in serum lipids, especially 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. .

Lipid analysis was carried out using enzyme colorimetric method as in Example 6, TG-glycezyme-V (榮 硏 化學, Japan), HDL-zyme-V (榮 硏 化學, Japan), Cholestezyme-V (榮 硏 化學, Japan), LDL cholesterol (Cat. 61532, BioMeriux, France) and the like to measure the absorbance with a standard solution at 505-570nm, to calculate the amount of each fat present in the serum.

As shown in Table 13, the lipid content before administration of the feed did not show a difference in the obese mouse group, and also showed no difference between the normal mouse groups. However, when acetobacter BC-Y058 and Lactobacillus BC-Y009 were ingested, the lipid content changed after 7 weeks, as shown in Table 14.

Obese mice fed with microorganisms maintained a similar level of lipid distribution as at the beginning of the experiment. However, non-ingested controls showed an increase in overall lipid content in the blood and normal mice regardless of microbial intake. There was no significant change in lipid content.

Therefore, ingestion of acetobacter BC-Y058 and Lactobacillus BC-Y009 does not affect normal lipid metabolism, but rather prevents lipid metabolism of abnormal metabolic state and thus reduces circulatory diseases (arteriosclerosis, myocardial infarction, etc.) Could be predicted.

As described above, the configuration and preferred embodiments of the present invention have been described in the present specification, but the scope of the present invention should not be construed as being limited to the described embodiments, and a person skilled in the art may describe the present specification within the scope of the present invention. Modifications may be made.

The microorganism of the present invention, while living in the intestine, converts carbon compounds such as monosaccharides and disaccharides into high molecular weight substances that are not absorbed in the small or large intestine, thereby reducing the amount of carbon compounds absorbed into the body, Necessary energy is supplied from the body fat and protein accumulated in the body to suppress obesity and diabetes. In addition, by producing dietary fiber in the intestine to be discharged to the body along with substances harmful to the body, it shows the effects of intestinal action, cholesterol absorption suppression, colon cancer prevention, appendicitis prevention.

Claims (8)

Lactobacillus sp. BC-Y009 (KCTC-0774BP) to produce polysaccharide material. Acetobacter sp. BC-Y058 (KCTC-0773BP) to produce polysaccharide material. Selected from the group consisting of the bacteria Acetobacter sp. And Lactobacillus sp., Which are characterized by growing in the intestine and converting the intestinal low-saccharides into polysaccharides that are not absorbed by the body. An anti-obesity agent comprising a pharmaceutically effective amount of a microorganism and a carrier. According to claim 3, wherein the microorganism is Acetobacter xylinum (Acetobacter xylinum), Acetobacter BC-Y058 (Acetobacter BC-Y058), Acetobacter Hansenii, Acetobacter pasteurianus, Acetobacter Acetobacter aceti, Lactobacillus BC-Y009, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus sakeus Lactobacillus sake, It is selected from the group consisting of Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus delbrueckii subsp., And Lactobacillus helveticusglucos var. Jugurti. An agent for the prevention and treatment of obesity, characterized in that. The preparation for preventing and treating obesity according to claim 4, wherein the composition comprising the pharmaceutically effective amount of the microorganism and the carrier is coated with an enteric coating material. Selected from the group consisting of the bacteria Acetobacter sp. And Lactobacillus sp., Which are characterized by growing in the intestine and converting the intestinal low-saccharides into polysaccharides that are not absorbed by the body. A pharmaceutical for preventing diabetes, comprising a pharmaceutically effective amount of a microorganism and a carrier. According to claim 6, wherein the microorganism is Acetobacter xylinum (Acetobacter xylinum), Acetobacter BC-Y058 (Acetobacter BC-Y058), Acetobacter Hansenii (Acetobacter pasteurianus), Acetobacter Acetobacter aceti, Lactobacillus BC-Y009, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus sakeus Lactobacillus sake, It is selected from the group consisting of Lactobacillus reuteri, Lactobacillus lactis, Lactobacillus delbrueckii subsp., And Lactobacillus helveticusglucos var. Jugurti. Diabetic prophylactic agent. 8. The antidiabetic agent according to claim 7, wherein the composition comprising the pharmaceutically effective amount of the microorganism and the carrier is coated with an enteric coating material.