KR100794702B1 - Microorganisms for preventing and / or treating obesity or diabetes - Google Patents

Abstract

The present invention relates to a microorganism for the prevention and treatment of obesity or diabetes mellitus, which is excellent in the viability of the intestine, and produces polysaccharides in the intestine with high efficiency, and more specifically, glucose, sucrose, or fructose, which enters the intestine, is not absorbed into the body. A novel microorganism, a composition containing a pharmaceutically effective amount thereof, and a food composition comprising the same as an active ingredient, which are capable of preventing or treating obesity or diabetes by competitively inhibiting the intestinal absorption of low sugars by converting the same into a polymeric material. will be.

Dietary Fiber, Lactobacillus, Obesity, Diabetes, Lactobacillus

Description

Microorganisms which are taken effects in preventing and / or treating Obesity or Diabetes Mellitus}

1 is a diagram verifying the probiotics (probiotics) effect by the microorganism according to the present invention. Where A is Lactobacillus luterie ATCC23272, and N is Lactobacillus permanent NM316.

Figure 2 is a chart showing the weight change of rats ingested in the form of microorganisms in accordance with the present invention. Here, a is the initial weight (g), b is the weight (g) after 40 days of administration, c is the weight gain (g) after 40 days of administration.

Figure 3 is a diagram showing the change in weight gain of rats ingested in the form of lactic acid bacteria fermented milk according to the present invention. Here, a is the initial weight (g), b is the weight (g) after 60 days of administration, c is the weight gain (g) after 60 days of administration.

Figure 4 is a chart showing the change in feed intake of rats ingested microorganism according to the present invention. Here, a is a result of the live cell form administration group of the microorganism of this invention, b is a result with respect to the lactic acid bacteria fermented milk (yoghurt) form administration group.

5 is a chart showing the metabolic efficiency change of rats ingested microorganism according to the present invention. Here, a is a result of the live cell form administration group of the microorganism of this invention, b is a result with respect to the lactic acid bacteria fermented milk (yoghurt) form administration group.                 

Figure 6 is a chart showing the change in blood cholesterol concentration of rats ingested microorganism according to the present invention.

The present invention relates to a microorganism for the prevention and treatment of obesity or diabetes mellitus, which is excellent in the viability of the intestine and produces a high efficiency of the polysaccharide in the intestine, more specifically, glucose, sucrose, or fructose, etc. introduced into the intestine is not absorbed 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 sugars by converting it into a high molecular 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 estimates that approximately 97 million Americans are obese and obese. There are about 15.7 million patients with type II diabetes mellitus, one of the diseases associated with the disease. It is also known that approximately 200,000 people die worldwide each year due to diseases related to obesity (Dan Ferber, Science, 283, p 1423-1424, 1999).

Diabetes is one of the most prevalent chronic diseases in the world that requires 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 (hereinafter referred to as 'NIDDM') or type II diabetes is a diabetes that occurs mainly in adults who have insulin produced and used properly but have defects in insulin-mediated glucose metabolism and other metabolism in peripheral tissues. to be.

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. It can also 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, sulfonylurea drugs and biguanide drugs, which are oral hypoglycemic agents, as well as diet and exercise, may be used. In addition, metformin and acarbose compounds have recently been used to treat NIDDM patients. However, some patients cannot adequately control hyperglycemia even by diet and exercise and / or the use of the therapeutic compounds described above, requiring administration of exogenous insulin.

In addition to the expensive and painful administration of insulin to a 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, fasting, irregular exercise, or special causes 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.

In addition, 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. However, the procedure must be performed, there is no sustainability of the effect, and it is expensive. Usage is limited.

Therefore, drug therapies to treat or prevent obesity or obesity-type diabetes are actively being studied by agents that cause a decrease in blood sugar, inhibition of blood glucose absorption, enhanced action of insulin, and a decrease in appetite. Drugs for the prevention and treatment of obesity-type diabetes are focused on using various physiological mechanisms.

Therefore, among the various drugs currently developed, dietary fiber having the advantage of being a natural substance without damaging the metabolic balance of the human body is recognized as the most useful obesity prevention and treatment agent.

Microorganisms that produce conventional microbial dietary fiber include Gluconobacter sp. , Agrobacterium sp. , Acetobacter xylinum , Acetobacter xenlinum , and A. hansenii . , acetonitrile bakteo Pas terrier Taunus (A. pasteurianus), acetonitrile bakteo Oh Shetty (A. aceti), rayijo byum in (Rhizobium), in an alkali to Ness (Alcaligenes), in Salina (Sarcina), Streptococcus write a brush loose (Streptococcus thermophilus ), Lactococcus cremoris , Lactobacillus helveticus , Lactobacillus vulgaricus , L. sake , L. reuteri , L. fermentum , L. lactis , L. delbrueckii subsp. , L. helveticusglucose var. Jugurti , Leuconostoc dextranicum , L. delbrueckii subsp. And using the kusu in Bulgaria (Bulgaricus), Kam-less in paste (Campestris), Sphingomonas in (Sphingomonas) and the like.

The dietary fiber produced by these microorganisms is used as a stabilizing agent, thickener, emulsifier, hygroscopic agent, etc. as a material for medicines and cosmetics. ), Locust bean gum, carrageenan, alginate or agar are already 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 habitat of Lactobacillus strains is the digestive tract of animals (L. acidophilus, L. intestinalis, L. johnsonii, L. fermentum, L. reuteri, etc.), vaginal mucosa ( L. vaginalis, L. gasseri ), food (wine) L. hilgardii ), lactic acid bacteria beverages ( 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 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 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, none of the 262 Lactobacillus strains currently deposited with the ATCC are known about the potential risk of causing disease in humans or animals. Classified as 'Bio-safety Level 1' (Microbiol. Rev., Apr. 23: 2 153-77, 1999; FEMS Microbiol. Rev., Sep. 7: 1-2, 113 -30, 1990.). Recently, studies on 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 fiber synthesized 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)

As described above, various studies and efforts have been made to treat obesity or diabetes until now, and various compounds have been developed to treat obesity or diabetes until now, but there are still side effects and force body fat and protein accumulated in the body. Since it functions to discharge, there is no groundbreaking therapeutic agent to prevent or treat obesity or diabetes at the source.

An object of the present invention is to absorb the low saccharide carbohydrates that are degraded by digestive enzymes while growing in the intestine, and to convert to a high molecular weight material that is not absorbed into the body, thereby greatly reducing the amount of low saccharides absorbed by the body in the intestine To provide microorganisms.                         

Another object of the present invention is 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.

In addition, another object of the present invention to provide a food composition containing the microorganism as an active ingredient.

The present invention relates to a microorganism for the prevention and treatment of obesity or diabetes mellitus, which is excellent in the viability of the intestine, and produces polysaccharides in the intestine with high efficiency, and more specifically, glucose, sucrose, or fructose, which enters the intestine, is not absorbed into the body. A novel microorganism, a composition containing a pharmaceutically effective amount thereof, and a food composition comprising the same as an active ingredient, which are capable of preventing or treating obesity or diabetes by competitively inhibiting the intestinal absorption of low sugars by converting the same into a polymeric material. will be.

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 polysaccharide which is a high molecular weight substance that is not absorbed in the intestine, L.fermentum NM 316 Is preferably.

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 of 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 and the like.

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 a method known in the art to reach the small intestine after passing through the stomach and quickly release the active ingredient microorganism into the intestine.

In addition, the microbial composition of the present invention may be prepared in a capsule form using conventional encapsulation methods. For example, pellets containing the microorganisms of the 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 After preparing a suspension or dispersion using gum, cellulose, silicate or oil, the dispersion or suspension can be filled into soft gelatin capsules.

The formulations of the invention may be provided in particular as enteric coated enteric preparations 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 on 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, 3 edit., 1986, p. 365-373, H. Sucker et al., Pharmazeutische Technologie, Thieme, 1991, pp. 355-359, Hagers Handbuch der pharmazeutischen Praxis, 4 edit., Vol. 7, pp. 739-742, and 766-778, (Springer Verlag, 1971), and Remingtons Pharmaceutical Sciences, 13 edti., 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 air derived from cellulose acetate phthalate and trimellitate and methacrylic acid copolymers such as methylacrylic acid and esters thereof, including at least 40% methylacrylic acid, and especially hydroxypropyl methylcellulose phthalate. It is made from coalescence. 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 cP. Viscosity, 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 can 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, 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. And the daily pharmaceutical effective amount of the microorganism of the present invention is 0.1g.

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 divided doses and administered continuously as needed. Thus, the dosage range does not limit the scope of the invention in any way.

The term "composition" in the present specification does not necessarily mean that the drug is approved, but includes a conventional food, such as fermented milk, jelly, pudding, functional foods or even health supplements.

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 the 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 can 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 distributed from various deposit institutions and used in the composition of the present invention, and new microorganisms can be newly separated and used.

In the present invention, lactic acid bacteria that produce polysaccharides are separated from feces, mutant parent strains are selected through their polysaccharide production ability, sugar consumption rate, resistance to gastric acid and bile acids, and then these are mutated using chemicals to produce large amounts of polysaccharides. The resulting NM316 strain was selected. The selected NM316 strain was confirmed to be Lactobacillus permentum, and it was named 'Lactobacillus permanentment NM316' (hereinafter referred to as 'Lactobacillus NM316') and deposited it (KCTC 10458BP).

As a result of confirming whether the polysaccharide produced by the Lactobacillus NM316 strain of the present invention thus selected is degraded by intestinal digestive enzymes, the polysaccharide produced by the Lactobacillus NM316 strain is not degraded by enteric digestive enzymes and finally, It can be seen that it is not absorbed but discharged.

As a result of administering the Lactobacillus fermentum NM316 strain of the present invention to rats in the form of live bacteria and lactobacillus fermented milk, when compared to the control group producing a small amount of polysaccharides, the body weight change was approximately 16.1% in the group of live bacteria. Weight gain was inhibited, and 6.3% of the weight gain was inhibited in the lactobacillus fermented milk form group. In addition, the feed intake was about 10.7% less in the live bacterium-administered group, and about 4.6% more in the lactobacillus fermented milk-form administration group.

Based on the above results, it was calculated in terms of metabolic efficiency expressed in terms of feed intake divided by weight gain, and the control group and the Lactobacillus fermentum NM316 strain of the present invention were roughly measured every other day in the probiotic form administration group. 6.4%, Lactobacillus fermented milk group showed approximately 28.5% higher metabolic efficiency when measured every 12 days. Therefore, when ingesting the microorganism of the present invention, the increase in feed intake was found to have an effect of inhibiting weight gain compared to the control.

In addition, as a result of confirming the change in blood cholesterol when the microorganism of the present invention is ingested, the Lactobacillus permanentment NM316 strain of the present invention was significantly reduced compared to the physiological saline administration group. Therefore, it can be seen that the microorganism of the present invention and the food composition containing the microorganism as an active ingredient are effective in obesity, diabetes and circulatory diseases (arteriosclerosis, myocardial infarction, etc.).

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 this embodiment.

Example 1 Acquire strains that produce large amounts of polysaccharides

Novel microorganisms that can be used in the compositions of the present invention were isolated and improved in the following manner. First, in order to find lactic acid bacteria that produce large amounts of viscous substances, fecal samples were taken from healthy infants who received breast milk for 6 months from Cheongju, Korea. The feces were diluted 10-fold with physiological saline, inoculated with 2% in MRS liquid medium at pH 1.5 and incubated at 37 ° C. for 5 hours. This was plated in MRS medium and lactic acid bacteria selection medium (LBS agar) and incubated for 48 hours at 37 ℃, to obtain 5-30 colonies from each plate. Each colony confirmed each viscosity using a toothpick, and the sticky crack was selected as a viscous substance producing strain. The selected strains were plated twice or more in MRS agar medium and purified. Each colony was isolated from only Lactobacillus by PCR using a primer capable of selectively selecting only Lactobacillus.

Each bacteria isolated from breast-fed infants were cultured in MRS medium at 37 ° C. for 6 hours, centrifuged, washed twice with physiological saline (PBS. PH 7.0) buffer, suspended in the same amount of physiological saline and NTG. (N-methyl-N-nitro-N-nitrosoguanidine) was added to a final concentration of 200 μg / ml and incubated at 37 ° C. for 30 to 120 minutes. The cells obtained by centrifugation were washed twice with physiological saline, and then cultured three times in MRS liquid medium, and the MRS medium containing a high concentration of fructose (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% MgSO 4, 0.01% MnSO 4, 0.05% L-cysteine-HCl) and incubated at 37 ° C. for 48 hours.

The colonies formed on the plate were picked with a toothpick to identify each of the colonies with increased viscosity, and then cultured by inoculating them again in MRS liquid medium. In order to calculate the yield of polysaccharide production of the culture of the strain culture completed for 24 hours, each supernatant was taken by putting 0.5 ml in each tube and centrifuging for 20 minutes at 13,000 (g), and the same amount of 40% trichloro-acetate (Trichloro- Acetate) was added and stirred, and then, twice the cold (4 ° C) ethanol was added to the supernatant obtained by centrifugation at 13,000 (g) for 20 minutes and mixed. Centrifuge this again at 6,000 (g) for 10 minutes and discard the supernatant carefully, add the initial volume and the same amount of distilled water to suspend the polysaccharide, measure the polysaccharide amount by the phenol-sulfuric acid method, and determine the polysaccharide efficiency (EPS (mg / L) / OD ( 650 nm)) to obtain one strain.

As shown in Table 1, the Lactobacillus fermentum NM316 strain showed more than five-fold polysaccharide-producing ability compared to the control microorganism Lactobacillus luteri ATCC23272.

Polysaccharide Production of Lactobacillus Lutheri ATCC23272 and Lactobacillus Permanent NM316 Strains Strain name Viable Count (cfu / ml) O.D (650nm) ① EPS (mg / L) ② EPS yield (② ÷ ①) Lactobacillus luster ATCC 23272 6.05 X 10 ⁸ 4.38 427 97.4 Lactobacillus Fermentum NM316 7.3 X 10 ⁶ 1.41 714 506.4

Example 2. Characterization and Identification of Lactobacillus NM316 Strain (KCTC 10458BP)

In order to identify the lineage of Lactobacillus NM316 with increased polysaccharide production, the strains were inoculated in MRS liquid medium, respectively, and the cells were left incubated for 12 hours at 37 ° C. 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 16S rRNA consensus primer, the nucleic acid extract was confirmed to be Lactobacillus fermentation.

As a result of comparing sugar availability using API 50 CH Kit (BioMerieux®), the sugar availability for L-arabinose, ribose, galactose, glucose, maltose, lactose, melibiose, fructose was high, and D- Raffinose and Gluconate showed low growth rates.

As described above, by analyzing the phenotype and sequencing of the 16S rRNA gene, the microorganism of the present invention is named as 'Lactobacillus fermentum NM316' strain, and according to the Budapest Treaty on International Approval of Microbial Deposit, KCTC (Korean Collection for Type Culture) (KCTC 10458BP).

Example 3 Degradability of Intestinal Digestive Enzymes of Polysaccharides

In order to confirm the degradation of the intestinal digestive enzymes of the polysaccharides produced by the Lactobacillus NM316 strain of the present invention, the purified polysaccharides were dissolved in phosphate buffer, and the digested enzymes isolated from the small intestine of rats were added according to the concentration. The reaction was carried out for 120 minutes and the results were measured using a trinder kit (cat. 315-500 Sigma, USA) using enzyme colorimetry.

As shown in Table 2, it was found that the polysaccharide produced by the Lactobacillus NM316 strain was not degraded by intestinal digestive enzymes. Therefore, it could be seen that the polysaccharides produced by the Lactobacillus NM316 strain of the present invention are not absorbed into the body but are discharged out of the body.

Degradation of Polysaccharides by Intestinal Digestive Enzymes Type of substrate Substrate concentration (㎍ / ㎖) Enzyme Concentration (µg / mL) ? ? 1250 625 312.5 156.25 78.13 39.06 Fructose 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 4. Confirmation of the probiotic effect of Lactobacillus NM316 strain

To confirm the probiotic effect of Lactobacillus NM316 strain, Escherichia coli (E, coli) was first incubated in a 16 hour LB liquid medium and diluted to a concentration of 1 X 10 ^5-1 X 10 ⁶ . It was soaked in a sterile cotton swab and spread evenly, and then concentrated on a 20-fold lactobacillus NM316 culture supernatant and absorbed 40 μl of fermentation broth into sterilized 8 mm disc paper (Adventech, Japan) and placed on agar plate medium. .

After 48 hours of incubation at 37 ° C., the size of the clear area formed around the disc was checked to compare the growth inhibition ability. As a result of confirming the growth inhibitory ring of Escherichia coli by the fermentation broth, clear ring formation was confirmed in both strains of Lactobacillus luterie ATCC23272 and Lactobacillus NM316. Therefore, it was confirmed that Lactobacillus NM316 has a probiotic effect inherent in lactic acid bacteria that inhibits the growth of harmful bacteria.

Example 5 Changes in Body Weight, Dietary Amount and Metabolic Efficiency Following Ingestion of Lactobacillus NM316

Animals used in the present invention were SD rats (Sprague Dawley rats) were 5 weeks old males, and each group was composed of physiological saline (PBS) administration group, Lactobacillus luteri ATCC23272 administration group, and Lactobacillus NM316 strain administration group. Was tested. According to the experimental group, the Lactobacillus luterie ATCC23272 and Lactobacillus NM316 strains were administered twice a day, and the total cell weight was 3 x 10 ¹¹ cfu concentration orally when administered once. It shows the change in feed intake of rats in the form of live microorganisms in accordance with the present invention. Here, the rat's weight and feed intake are measured every other day and represent the average of 9 animals.

As shown in Figure 2 and Table 3, after 40 days of administration in this experiment, the weight change of the SD rats began to appear slowly after 12 days from the start of the administration, the measurement after the last 40 days was the control group It was suppressed by about 10.3% compared to the PBS administration group.

Weight Changes in SD Rats During Long-Term Administration 0 day 40 days Weight gain Control (PBS-administered group) 154.09 ± 4.25 390.27 ± 19.63 236.19 ± 19.19 Lactobacillus ruteri ATCC23272 administration group 153.41 ± 4.22 365.50 ± 17.09 212.09 ± 6.31 Lactobacillus percentage NM316 administration group 151.93 ± 4.60 350.03 ± 11.86 198.1 ± 2.46

In addition, it was confirmed that the change in feed efficiency according to the feed intake of SD rats during long-term administration, as shown in Figure 5a, changes in the metabolic efficiency of rats ingested microorganisms in the form of live bacteria according to the present invention as a result of Lactobacillus The total feed intake of the NM316 group was about 10.7% lower than that of the other groups, and the total weight change during the period of administration was also about 16.1% lower. However, the feed efficiency was about 6.4% higher than that of the PBS group. As a result, the Lactobacillus NM316 group was shown to be more effective in inhibiting weight gain even if the rats ingested a large amount of feed than the physiological saline group.

Example 6 Changes in Body Weight, Dietary Amount and Metabolic Efficiency According to Lactic Acid Bacteria Fermented Milk of Lactobacillus NM316 Strain

Animals used in the present invention were SD rats (Sprague Dawley rats) were 5 weeks old males, and each group was administered with physiological saline (PBS), 11 strains of lactobacillus fermented milk (S) using only general starter strains, and Lactobacillus lustery ATCC23272 strain. Lactic acid bacteria fermented milk prepared by addition and Lactobacillus fermented milk prepared by adding Lactobacillus NM316 strain was classified and tested. For Lactic acid bacteria fermented milk administration group except for physiological saline (PBS) administration group, the starter strain Streptococcus thermophilus: Lactobacillus Bulgaricus was inoculated with 7 x 10 ⁶ inoculated cells mixed with a ratio of 5: 2 to prepare a basic starter fermented milk. This was used as the basic starter lactic acid bacteria fermented milk, and the total number of viable cells was 1 X 10 ⁸ . So for the production of lactic acid bacteria fermented milk in Lactobacillus ruteri ATCC23272 and Lactobacillus NM316 strain to separate the culture was administered in addition to the lactic acid bacteria fermented milk The Lactobacillus ruteri ATCC23272 and Lactobacillus administered total gyuncheryang of Bacillus NM316 strain was 4 X10 ¹⁰ cfu, and thus prepared One lactobacillus fermented milk was orally administered twice a day. As shown in b of FIG. 4, the weight and feed intake of the rats were measured at four-day intervals and showed an average value of 10 rats.

As a result of administering the prepared fermented milk for 60 days, as shown in FIG. 3 and Table 4, the weight change of SD rats began to gradually appear after 44 days, and the measurement at the last 60 days was compared with that of the control yogurt group. The body weight was about 2.6% lower. In the long-term administration of the rats, the Lactobacillus NM316 group consumed about 4.6% more feed than the starter yogurt group, and inhibited 4.3% of body weight change during the administration period. appear. As shown in b of FIG. 5, based on the above results, the feed efficiency was calculated to be about 5.2% higher than the yogurt group.

These results were also shown to be effective in inhibiting weight gain even if a large amount of feed intake as in the case of probiotics.

Changes in Body Weight of SD Rats Prolonged Administration of Lactic Acid Bacteria 0 day 60 days Weight gain Control (PBS-administered group) 153.65 ± 6.11 413.47 ± 14.02 259.83 ± 16.44 S ^* 154.19 ± 6.61 420.3 ± 19.33 266.11 ± 17.15 S + Lactobacillus NM316 strain administration group 154.7 ± 7.27 409.17 ± 19.48 254.47 ± 15.22

S ^* : Fermented milk made using starter microorganism

Example 7 Changes in Blood Cholesterol upon Ingestion of Lactobacillus NM316 Strains

Ingestion of the Lactobacillus NM316 strain according to the present invention, the change in blood cholesterol was analyzed to determine whether the reduction effect on the incidence of circulatory diseases (arteriosclerosis, myocardial infarction, etc.) as well as diseases such as diabetes and obesity.

For the analysis of fat, the blood of rat under test was collected by blood collection using tail blood sampling at the end of the experiment. The analysis of fat was performed using enzyme colorimetry and Cholestezyme-V (Shinyang Chemical, Korea). The cholesterol content in serum was calculated by measuring the absorbance along with the standard solution at 505-570 nm using the light and the like.

As shown in Figure 6 and Table 5, the total cholesterol level in the blood of the Lactobacillus NM316 strain compared to the physiological saline administration group, began to decrease from the 40 days after the start of administration was shown to decrease more than before administration. Therefore, the microorganism of the present invention was expected to be effective in reducing the incidence of circulatory diseases (arteriosclerosis, myocardial infarction, etc.).

Changes in Blood Cholesterol Levels in SD Rats Prolonged Administration with Probiotics ? 0 day 40 days Control (saline administration group) 87.86 ± 4.25 75.18 ± 5.67 Lactobacillus ruteri ATCC23272 administration group 87.58 ± 8.75 69.23 ± 4.36 Lactobacillus permanent NM316 strain administration group 95.37 ± 9.87 70.94 ± 5.11

As described above, although the configuration and preferred embodiments of the present invention have been described herein, the scope of the present invention should not be construed as being limited to the embodiments described above, those skilled in the art to see within the scope of the present invention Modifications of the contents described in the specification may be made.

The microorganism of the present invention has excellent intestinal viability and high efficiency of polysaccharide production ability, and thus, in the intestine, it converts low-saccharide compounds such as glucose, sucrose, or fructose into high molecular weight polysaccharides to reduce the amount of carbohydrates absorbed into the body. By giving, it is effective in the treatment and prevention of obesity and diabetes. In addition, the microorganism according to the present invention has the effect of weight control, intestinal action and cholesterol absorption.

Claims (7)

Lactobacillus fermentum NM316 (KCTC-10458BP) to produce polysaccharide material. A pharmaceutical preparation for the prevention of obesity, comprising a pharmaceutically effective amount of Lactobacillus fermentum NM316 (KCTC-10458BP) and a carrier to produce a polysaccharide substance.        The agent for the prevention of obesity according to claim 2, wherein the agent for the treatment of obesity is coated with an enteric coating material.       A food composition containing Lactobacillus fermentum NM316 (KCTC-10458BP), which produces a polysaccharide substance, as an active ingredient.      5. A food composition according to claim 4, wherein the food composition is jelly, pudding, powder, capsule, or fermented milk. delete delete

Images