JP4248985B2 - Lactobacillus fermentum, a preparation for preventing or treating obesity or diabetes, and a food composition comprising the same and a carrier - Google Patents

Description

  The present invention relates to a microorganism having excellent intestinal viability and capable of producing polysaccharides in the intestine with high efficiency to prevent or treat obesity and diabetes, and more specifically flows into the intestine. A novel microorganism capable of preventing and treating obesity or diabetes by converting glucose, sucrose, or fructose into a high-molecular substance that is not absorbed by the body and competitively inhibiting intestinal absorption of low sugars , Compositions (formulations) containing these pharmaceutically effective amounts, and food compositions containing these as active ingredients.

  Obesity is known as a chronic disease that occurs in a complex manner due to various factors that have not yet been clearly investigated. Obesity causes hypertension, diabetes, cardiovascular disease, cholelithiasis, osteoarthritis, sleep apnea, respiratory disorder, endometriosis, prostate cancer, breast cancer, or colon cancer Has been.

  1999 National Health Service (NIH) report (The Evidence Report: Clinical guidances on the identification, evaluation, and treatment of overweight, 1999 and 1999) There are approximately 15.7 million people with Type II diabetes (Mellitus), one of the diseases associated with obesity. In addition, about 200,000 people worldwide die each year from diseases related to obesity (Dan Ferber, Science, 283, pp 1423-1424, 1999).

  Diabetes is one of the world's most prevalent chronic diseases that require personal and financial costs, including patients and their families, each with a distinct etiology and pathogenesis There are different types of diabetes. For example, renal diabetes is characterized by hyperglycemia and diabetes and is due to impaired carbohydrate metabolism caused by inappropriate production and use of insulin.

  Non-insulin dependent diabetes mellitus (NIDDM, type II diabetes) is primarily observed in adults with impaired insulin metabolism and other metabolism in peripheral tissues where insulin is properly produced and utilized. Diabetes mellitus.

  NIDDM is due to three major metabolic abnormalities, such as resistance to insulin-mediated glucose processing, impaired nutrient secretion stimulated insulin secretion, and excessive production of glucose in the liver. Failure to treat NIDDM, ie, control of blood glucose levels, can lead to various diabetic complications including cardiovascular mortality, retinopathy, kidney disease, and peripheral neuropathy.

  For NIDDM treatment, in addition to diet and exercise therapy, oral hypoglycemic drugs such as sulfonylurea and biguanide drugs are used as methods for adjusting blood glucose levels in NIDDM patients. You may do it. Recently, metformin and acarbose compounds are also used for the treatment of NIDDM. However, in some patients, the administration of exogenous insulin may be required because hyperglycemia may not be adequately controlled by diet and exercise therapy and / or the therapeutic drug.

  Insulin administration by injection is expensive and can be painful for the patient and cause various disabilities and complications. For example, an insulin response (hypoglycemia) can be triggered even when there is an incorrect insulin dose, a lack of food, irregular exercise, or no special cause. In addition, local and / or systemic allergic reactions and immunological resistance to insulin may occur.

  Methods for preventing and treating obesity and diabetes are broadly divided into diet / exercise therapy, surgical therapy, and drug therapy. Diet / exercise therapy is a treatment method through physical activity such as low calorie / fat intake and oxygen consumption, but this must be done patiently, repetitively and continuously, so to gain a mass effect It is considered difficult.

  Surgical therapy is a method of physically removing body fat through surgical operation and has the advantage that its effect can be obtained in a short period of time, but it requires a surgical process and does not last long However, its use is limited due to cost issues.

  Therefore, pharmacotherapy for the treatment and prevention of obesity and obesity-type diabetes has been actively studied with drugs that reduce blood glucose levels, inhibit sugar absorption, enhance insulin action, or decrease appetite, and have been developed to date. Drugs for the prevention and treatment of obesity and obesity-type diabetes use various physiological mechanisms.

  Therefore, among the various drugs that are currently developed, dietary fibers that do not damage the metabolic balance of the human body and have the advantage of being natural substances are recognized as the most useful preparations for the prevention and treatment of obesity. Yes.

  Examples of microorganisms that produce conventional microbial dietary fibers include Gluconobacter sp., Agrobacterium sp., Acetobacter xylinum, Acetobacter hansenii. A. pasteurianus, A. acetici, Rhizobium, Alcaligenes, Sarcina, Streptococcus thermophilus, Streptococcus thermoplas, us・ Lactococcus cremoris, la Lactobacillus helveticus, Lactobacillus bulgaricus, L. sake, L. reuteri, L. fermentum, L. Fermentum, L. Fermentum L. lactis), Lactobacillus delbrucki subspecies (L. delbrueckii subsp.), Lactobacillus helveticus glucose varieties (L. helveticus glucose var. ), Campestris (Ca pestris), and the like genus Sphingomonas (Sphingomonas).

  Dietary fibers produced by these microorganisms are used as stabilizers, thickeners, emulsifiers, hygroscopic agents, etc. for various foods and as raw materials for pharmaceuticals and cosmetics. At present, however, microbial cellulose and xanthan are used. , Acetan, and other dietary fibers purified from seaweed, such as Guar gum, Locust bean gum, Carrageenan, alginate, agar, etc. have already been commercialized. ing.

  Among the microorganisms mentioned above, Lactobacillus strains are the main constituents of normal microbial flora inhabiting the intestines of the human body, and are very important for maintaining a healthy digestive tract and vaginal environment. It has been known for some time (Non-Patent Document 1). In general, the habitat of Lactobacillus strains is the digestive organs of animals (L. acidophilus, L. intestinalis, L. johnsonii, L. reuteri, etc.), vaginal mucosa (L. vaginalis, L. gasseri), food (wine-L Hilgardi), lactic acid bacteria beverages (L. kefir, L. kefiranofaciens; cheese-L, casei), vinegar (L. acetotolerans), oral cavity (L. oris), koji (L. sake, L. homohichi), fruit juice L. kumkeei, L. mali, L. suebicus), fermented sausages or fish (L. farciminis, L. alignentarius) and the like.

  In fact, many people use health aids containing strains of the genus Lactobacillus to maintain a healthy intestine and to prevent urogenital tract infection. Recently, prevention of diarrhea and constipation, or prevention of urinary tract infection and other various bioactive functions of Lactobacillus, such as regulation of immunity, regulation of blood cholesterol level, treatment of rheumatism, prevention of cancer There is a growing importance and interest in reducing the sensitivity to lactose and the effect on atopic dermatitis.

  According to the US Public Health Service guidelines, all of the 262 Lactobacillus strains currently deposited in the US strain depository (ATCC) have potential to cause disease in humans and animals. Classified as “Bio-safety Level 1”, which is recognized as having no danger.

  Recently, research on extracellular dietary fibers synthesized by Lactobacillus has been actively conducted. It is known that the mechanism by which these strains produce extracellular dietary fibers is very complicated because many genes are involved, and the amount of dietary fibers synthesized is extremely small (Non-patent Document 2).

As mentioned above, various researches and efforts have been made to date on the treatment of obesity or diabetes, and various compounds have been developed as therapeutic agents for obesity or diabetes, but they still have side effects and accumulate in the body. However, there is still no epoch-making therapeutic agent that fundamentally suppresses or treats obesity or diabetes.
Bibel, D.C. J. et al. , ASM News, 54: 661-665, 1988: Reid, G .; and A. W. Bruce, In H.M. Lappin-Scott (ed.), Bacterial biofilms. Cambridge University Press, Cambridge, England, p. 274-281, 1995; A. W. Bruce, J. et al. A. McGroarty, K.M. J. et al. Cheng, and J.C. W. Costerton, Clin. Microbiol. Rev. , 3: 335-344, 1990 Int. J. et al. Food Microbiol. Mar. 340: 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

  The object of the present invention is to absorb the carbohydrate of low saccharide decomposed by digestive enzymes while growing in the intestine, and convert it into a high molecular weight substance that is not absorbed in the body, thereby reducing the amount of low saccharide absorbed in the body from the intestine. It is to provide a microorganism that can significantly reduce the amount of the microorganism.

  Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or diabetes by containing a pharmaceutically effective amount of the microorganism and radically reducing the amount of low sugar absorbed in the body. Is to provide.

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

  The present invention relates to a microorganism having excellent intestinal viability and capable of producing polysaccharides in the intestine with high efficiency to prevent or treat obesity and diabetes, and more specifically flows into the intestine. A novel microorganism capable of preventing and treating obesity or diabetes by converting glucose, sucrose, or fructose into a high-molecular substance that is not absorbed by the body and competitively inhibiting intestinal absorption of low sugars , Compositions containing these pharmaceutically effective amounts, and food compositions containing these as active ingredients.

  The microorganism of the present invention is L. fermentum NM316, which is capable of growing in the intestine and converts it into a high molecular weight substance that is harmless to the human body and is not absorbed by the intestinal tract, and is Lactobacillus fermentum NM316. It is preferable.

  Each microorganism of the present invention is usually administered in a unit form such as a tablet or a capsule formed by mixing with a pharmaceutical carrier or excipient selected depending on the administration route, or an auxiliary active ingredient. Carriers, excipients and diluents suitable for the composition of the present invention include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, fine Examples thereof include crystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methyl and propyl hydroxybenzoate, 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 composition of the present invention may be produced as an enteric coated preparation using methods known in the art so that the microorganisms of the active ingredient are rapidly released into the intestine when reaching the small intestine after passing through the gastrointestinal tract. good.

  In addition, the microbial composition of the present invention may be produced as a capsule-shaped composition using a normal encapsulation method. For example, a pellet containing the microorganism of the present invention freeze-dried using a standard carrier may be produced and then filled into a hard gelatin capsule, or the microorganism of the present invention and any suitable pharmaceutical product A suspension or dispersion is prepared using a chemical carrier (eg, aqueous gum, cellulose, silicate, or oil) and then the dispersion or suspension is filled into a soft gelatin capsule. May be.

  The preparation of the present invention is particularly an oral unit dosage form, and may be provided as an enteric-coated enteric preparation. As used herein, an “enteric coating” is any pharmaceutically acceptable that is sufficiently degraded in the small intestine to allow its active ingredient to be released into the small intestine, while not being degraded by gastric acid. Includes known coatings.

The “enteric coating” of the present invention maintains its form for more than 2 hours when an artificial gastric fluid such as a pH 1 HCl solution is contacted at 36-38 ° C., preferably after that KH ₂ PO at pH 6.8. Refers to a coating that degrades within 30 minutes in an artificial intestinal fluid such as a ₄ buffer solution.

  The enteric coating of the present invention is coated on one core in an amount of about 16-30 mg, preferably 16-20 mg or 25 mg or less. The case where the thickness of the enteric coating of the present invention was 5 to 100 μm, preferably 20 to 80 μm, showed satisfactory results as an enteric coating. The material for the enteric coating is appropriately selected from known polymer substances. These polymer substances are described in the literature [L. Lachman et al., The Theory and Practice of Industrial Pharmacology, 3rd edition, 1986, pp. 196 365-373, H.R. Sucker et al., Pharmazeitis Technology, Thiime, 1991, pp. 355-359, Hagers Handbuch der pharmazeutiscnen Praxis, 4th edition, vol. 7, pp. 739-742 and 766-778, (Springer Verlag, 1971), and Remingtons Pharmaceutical Sciences, 13th edition, pp. 196 1689-1691 (Mack Publ., Co., 1970)], and cellulose ester derivatives, cellulose ethers, acrylic resin methyl acrylate copolymers, and maleic acid and phthalic acid derivative copolymers. May be included.

  Preferred enteric coatings of the present invention are derived from cellulose acetate phthalate and trimellitate, and methacrylic acid copolymers such as methylacrylic acid and esters thereof including, for example, 40% or more of methylacrylic acid and hydroxypropylmethylcellulose phthalate in particular. Produced from the copolymer. A product commercially available from Rohm GmbH, Germany, Endrigit L 100-55, 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 45-90 cP, an acetyl content of 17-26%, and a phthalate content of 30-40%, and the cellulose acetate trimellitate has a viscosity of about 15- It has a viscosity of 20 cS and an acetyl content of 17-26% and a trimellityl content of 25-35%. A commercially available product, Eastman Kodak Company, cellulose acetate trimellitate, 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, 5-10% hydroxypropyl content, 18-24% methoxy content and 21-35% Having a phthalyl content of Cellulose acetate phthalate commercially available from Eastman Kodak Company may be used as the material for the enteric coating of the present invention.

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

The enteric coating of the present invention can be produced using a conventional enteric coating method in which an enteric coating solution is sprayed onto the core. Suitable solvents used in the enteric coating step include alcohols such as ethanol, ketones such as acetone, and halogenated hydrocarbon solvents such as CH ₂ Cl ₂ , and a mixed solvent of these solvents may be used. . A softener such as di-n-butyl phthalate or triacetin is added to the coating solution in a ratio of 1: about 0.05 to 0.3 (coating material to softener). The spraying process is preferably performed continuously, and it is possible to adjust the spray amount in consideration of the coating conditions. The spray pressure can be adjusted in various ways, and generally satisfactory results are obtained with a spray pressure of about 1 to 1.5 bar.

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

  The compositions of the present invention can be administered to a subject's solid more than once daily. Unit dose means a physically separated unit that is suitable for unit administration for subjects and other mammals, each unit comprising a suitable pharmaceutical carrier and exhibiting a therapeutic effect. Containing a predetermined amount.

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

  However, dosage is variable depending on the patient's weight, the severity of the obesity symptoms, and the microorganisms and supplementary active ingredients used. Further, the total daily dose can be divided into several times and continuously administered as necessary. Therefore, the range of the dose is not limited by any method.

  The “composition” in the present specification does not necessarily mean only what is permitted as a pharmaceutical product, but is a concept including even normal foods such as fermented milk, jelly, and pudding, functional foods, and health supplements.

  When the composition of the present invention is taken periodically, each microorganism of the present invention forms a group of fungi in the intestine, while competitively preventing the absorption of saccharides by the human body, and utilizing these saccharides The non-digestible polysaccharide produced by the plant improves the habitat conditions of useful intestinal bacteria and stimulates intestinal motility, so that the composition of the present invention can prevent or treat obesity or diabetes. Like that.

Hereinafter, the present invention will be described in more detail. The microorganism for prevention and treatment of obesity or diabetes according to the present invention is (1) capable of growing in the intestine, and (2) rapidly absorbing low saccharides into a fibrous form. It is converted into a non-digestible or indigestible polymer compound such as a substance, and (3) satisfies the condition that it is not harmful to the human body or livestock. Therefore, known microorganisms satisfying the above three conditions can be received from various depository institutions and used in the composition of the present invention, and new microorganisms may be newly separated and used.

  In the present invention, lactic acid bacteria that produce polysaccharides are separated from feces, and the mutant mother strains are selected through experiments on the ability to produce these polysaccharides, the rate of sugar consumption, and resistance to gastric acid and bile acids. The NM316 strain that produces a large amount of polysaccharide by mutation was selected. As a result of examining the selected NM316 strain, it was confirmed that it was Lactobacillus fermentum, and was named “Lactobacillus fermentum NM316” (hereinafter referred to as “Lactobacillus NM316”) and deposited (KTCC). -10458BP).

  As a result of confirming whether the polysaccharide produced by the Lactobacillus NM316 strain of the present invention thus selected is degraded by the intestinal digestive enzyme, the polysaccharide produced by the Lactobacillus NM316 strain is It was found that it was not decomposed by the enzyme and was finally discharged without being absorbed into the body.

  As a result of administering the Lactobacillus fermentum NM316 strain of the present invention to the rat in the form of live bacteria and lactic acid bacteria fermented milk, compared to the control group producing a small amount of polysaccharide, About 16.1% of the body weight gain inhibitory effect was shown, and the lactic acid bacteria fermented milk form administration group showed about 6.3% body weight gain inhibitory effect. And in feed intake, it was confirmed that the amount which decreased about 10.7% in the administration group of the living bacteria form, and the amount which increased about 4.6% in the administration group of the lactic acid bacteria fermented milk form were ingested.

  Based on each of the above results, as a result of calculation in terms of metabolic efficiency expressed by a value obtained by dividing the feed intake by the weight gain, in the Lactobacillus fermentum NM316 strain of the present invention compared to the control group In the case of the administration group in the form of viable bacteria, as a result of measurement at an interval of every other day, the result of measurement in a period of about 6.4%, and in the case of the administration group in the form of lactic acid bacteria fermented milk, the period of 12 days was about 28 It showed a metabolic efficiency increased by .5%. Therefore, when the microorganisms of the present invention were ingested, it was confirmed that when the amount of the ingested feed increased, there was an effect of suppressing weight gain compared to the control group.

  Further, as a result of examining changes in blood cholesterol when the microorganism of the present invention was ingested, the Lactobacillus fermentum NM316 strain of the present invention was significantly reduced compared to the physiological saline administration group. Therefore, it turns out that the microorganisms of this invention and the food composition which these microorganisms contain as an active ingredient show an effect in obesity, diabetes, and a circulatory disease (arteriosclerosis, myocardial infarction, etc.).

  The microorganism of the present invention has an excellent intestinal viability and a high-efficiency polysaccharide-producing ability, and has a high low-sugar compound such as butter sugar, sucrose, and fructose while surviving in the intestine. By converting to a molecular weight polysaccharide and reducing the amount of carbohydrate absorbed in the body, it has the effect of treating and preventing obesity or diabetes. In addition, the microorganisms by this invention have effects, such as weight control, an intestinal regulation effect, and cholesterol absorption suppression.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are for explaining the present invention and are not intended to limit the scope of rights of the present invention.
Example 1: Acquisition of a strain producing a large amount of polysaccharides A novel microorganism used in the composition of the present invention was isolated and improved by the following method. First, in order to find lactic acid bacteria that produce a large amount of mucilage, fecal feed was obtained from a healthy infant born in Cheongju, Korea, who fed only breast milk for 6 months. The stool was diluted 10-fold with physiological saline, 2% of the stool was inoculated into MRS liquid medium having a pH of 1.5, and cultured at 37 ° C. for 5 hours. This was spread on MRS medium and lactic acid bacteria selection medium (LBS agar) and then cultured at 37 ° C. for 48 hours to obtain 5 to 30 colonies from each plate. Next, the viscosity of each colony was examined using toothpick branches, and the colonies showing stickiness were selected as a viscous substance-producing strain. The selected strain was applied to MRS agar medium twice or more and purely isolated. For each colony, only Lactobacillus was selected and separated by a PCR method using a primer capable of selectively selecting only Lactobacillus.

Each strain isolated as described above was cultured in MRS medium at 37 ° C. for 6 hours, centrifuged, washed twice with physiological saline (PBS. PH 7.0) buffer solution, and then the same amount of physiology. The suspension was suspended in a saline solution and NTG (N-methyl-N-micro-N-nitrogoguanidine) was added to a final concentration of 200 μg / mL, followed by incubation at 37 ° C. for 30 to 120 minutes. The strain obtained by centrifugation was washed twice with physiological saline, and then again cultured three times with MRS liquid medium. The MRS medium containing 5% fructose [5% fructose, 1% beef extract 1% peptone, 0.5% yeast extract, 0.1% Tween-80 (Tween-80), 0.2% ammonium citrate, 0.5% sodium acetate, 0.01% MgSO ₄ , 0 0.01% MnSO ₄ , 0.05% L-cysteine-HCl] and cultured at 37 ° C. for 48 hours.

  The viscosities of the colonies formed on the flat plate were examined using toothpick branches, and colonies with increased viscosity were selected, and then again inoculated into MRS liquid medium and cultured. In order to calculate the polysaccharide production yield of the strain culture solution cultured for 24 hours, 0.5 mL was put in each tube, centrifuged at 13,000 (g) for 20 minutes, and the supernatant was collected. After the same amount of 40% trichloroacetate was added and stirred, the supernatant obtained by centrifuging again at 13,000 (g) for 20 minutes was added and mixed with 2 chilled ethanol (4 ° C.). . After centrifuging it again at 6,000 (g) for 10 minutes and discarding the supernatant while paying attention, add the same volume of distilled water as the first volume to suspend the polysaccharide and then phenol. -The amount of polysaccharide was measured by the sulfuric acid method, and polysaccharide efficiency [EPS (mg / L) / OD (650 nm)] was measured to obtain one strain.

  As shown in Table 1, Lactobacillus fermentum NM316 strain shows 5 times or more polysaccharide production ability as compared with Lactobacillus reuteri ATCC23272 which is a control microorganism.

Example 2: Characteristics and identification of Lactobacillus NM316 strain (KCTC 10458BP) In order to grasp the strain of Lactobacillus NM316 with improved polysaccharide production, each strain was inoculated into MRS liquid medium and allowed to stand at 37 ° C for 12 hours. And cultured. The culture broth was centrifuged at 6,000 (g) at 4 ° C. to obtain microorganisms, and nucleic acids were extracted therefrom using the CTAB / NaCl method. As a result of determining the base sequence of the 16S rRNA gene from the nucleic acid extract using a 16S rRNA consensus primer (16S rRNA consensus primer), it was confirmed that the microorganism of the present invention was Lactobacillus fermentum.

  In addition, as a result of comparing the sugar availability using API 50 CH Kit (Bio Merieux Co.), the sugar availability for L-arabinose, ribose, galactose, glucose, maltose, melibios, fructose was high, In addition, D-raffinose and gluconate showed low growth rates.

  As described above, through the analysis of the phenotype and the base sequence of the 16S rRNA gene, the microorganism of the present invention is named as 'Lactobacillus fermentum NM316 strain', which is named KCTC based on the Budapest Treaty on International Approval in the Deposit of Microorganisms. Deposited (Korean Collection for Type Culture) (KTCC 10458BP).

Example 3: Degradability of polysaccharides by intestinal digestive enzymes In order to examine the degree of degradation of the polysaccharides produced from the intestine by the intestinal digestive enzymes by the Lactobacillus NM316 strain of the present invention, purified polysaccharides were phosphorylated. After dissolving in acid buffer solution, digestive enzyme separated from rat small intestine was added according to concentration and reacted for 120 minutes, and the result was used as a result of the Trinder kit (Cat. 315-500 Sigma, USA). ).

  As shown in Table 2, it was found that the polysaccharide produced by Lactobacillus NM316 strain was not degraded by intestinal digestive enzymes. Therefore, it was found that the polysaccharide produced by the Lactobacillus NM316 strain of the present invention is excreted outside the body without being absorbed into the body.

Example 4 Confirmation of Probiotic Effect of Lactobacillus NM316 Strain To confirm the probiotic effect of Lactobacillus NM316 strain, E. coli was first cultured in LB medium for 16 hours, and 1 × 10 ⁵ to 1 × Dilute to a concentration of 10 ⁶ . This was wetted with a sterilized cotton swab and uniformly spread, and then the Lactobacillus NM316 culture supernatant was diluted 20-fold, and 40 μL of the fermentation broth was absorbed into a sterilized 8 mm disc paper (ADVANTEC, Japan). Placed on agar plate.

  Next, after culturing at 37 ° C. for 48 hours, the size of the transparent region formed around the disc was examined in order to compare its growth inhibitory ability. As a result of confirming the Escherichia coli growth inhibition ring by the fermentation broth, formation of a transparent ring could be confirmed in both Lactobacillus reuteri ATCC23272 and Lactobacillus NM316. Therefore, it was confirmed that Lactobacillus NM316 strain has a probiotic effect unique to lactic acid bacteria that suppresses the growth of harmful bacteria.

Example 5: Lactobacillus NM316 body weight, change in food intake, and accompanying change in metabolic efficiency The animal used in the present invention is an SD rat (Sprague Dawley rat) (5 weeks old, male), physiological saline The group was roughly divided into a water (PBS) administration group, a Lactobacillus reuteri ATCC 23272 administration group, and a Lactobacillus NM316 strain administration group, and each group was composed of 11 rats before the experiment. For each experimental group, Lactobacillus reuteri ATCC23272 and Lactobacillus NM316 strains were administered twice a day, and the total amount of cells at the time of each administration was 3 × 10 ¹¹ cfu, and the oral administration method was adopted. . FIG. 4a shows a change in the amount of feed intake of a rat ingesting the microorganism according to the present invention in the form of viable bacteria. Here, the body weight and feed intake of rats were measured every other day, and the average value of 9 animals was shown.

  As shown in FIG. 2 and Table 3, as a result of administration for 40 days according to this experiment, the body weight change of SD rats gradually appears after 12 days from the administration disclosure date, and the measured value on the 40th day of the final day is Compared with the PBS group of the control group, a result of about 10.3% reduction was obtained.

  In addition, changes in feed efficiency accompanied by changes in feed intake of SD rats during long-term administration were examined. As shown in FIG. 5a, metabolism of rats ingesting microorganisms according to the present invention in the form of viable bacteria. As a result, it can be confirmed that the total feed intake of the Lactobacillus NM316 administration group is approximately 10.7% lower than the other groups, and the total body weight change during the administration period is about the same. It was confirmed that it was lowered by about 16.1. However, the feed efficiency was about 6.4% higher than that of the physiological saline (PBS) administration group.

As a result, it can be seen that the Lactobacillus NM316 administration group is more effective in suppressing body weight gain, even if it consumes a larger amount of feed than the physiological saline administration group.
Example 6: Lactobacillus NM316 strain Lactobacillus fermented milk intake body weight, change in food amount, and accompanying change in metabolic efficiency The animal used in the present invention is SD rat (Sprague Dawley rat) (5 weeks old, Male), physiological saline (PBS) administration group, lactic acid bacteria fermented milk (S) administration group using only general starter strains, lactic acid bacteria fermented milk administration group produced by adding Lactobacillus reuteri ATCC23272 strain, and Lactobacillus NM316 The lactic acid bacteria fermented milk administration group produced by adding the strain was classified, and each group was composed of 11 rats before the experiment. Here, the lactic acid bacteria fermented milk administration group excluding the physiological saline is inoculated with 7 × 10 ⁶ cells mixed with a starter strain of Streptococcus thermophilus and Lactobacillus bulgaricus in a ratio of 5: 2. The basic starter fermented milk was produced and used as the basic starter lactic acid bacteria fermented milk, and the total number of viable bacteria in the fermented milk was about 1 × 10 ⁸ . Lactobacillus reuteri ATCC23272 and Lactobacillus NM316 strains are separately cultured and added to lactic acid bacteria fermented milk and administered to the lactic acid bacteria fermented milk thus produced. At this time, administration of Lactobacillus reuteri ATCC23272 and Lactobacillus NM316 strains The total amount of the microbial cells thus produced was 4 × 10 ¹⁰ cfu, and the lactic acid bacteria fermented milk produced in this way was orally administered twice a day. As shown in FIG. 4b, the body weight and feed intake of the rats were measured every 4 days and showed the average values for 10 animals.

  As a result of administering the fermented milk produced above for 60 days, as shown in FIG. 3 and FIG. 4, the weight change of the SD rat gradually appears after 44 days, and the measured value on the 60th day of the final day is Compared with the yogurt administration group as a control group, the body weight decreased by about 2.6%. Regarding the change in feed intake of SD rats during long-term administration, it was found that the Lactobacillus NM316 administration group ingested about 4.6% more food than the starter yogurt administration group, and the change in body weight during the administration period Was also found to be suppressed by about 4.3%. Based on the above results, as shown in FIG. 5b, when the feed efficiency was calculated, the value was about 5.2% higher than the yogurt administration group.

Example 7: Change in cholesterol upon ingestion of Lactobacillus NM316 strain When Lactobacillus NM316 strain according to the present invention was ingested, not only diseases such as diabetes and obesity but also onset of cardiovascular diseases (arteriosclerosis, myocardial infarction, etc.) In order to confirm whether there is an inhibitory effect on the rate, changes in blood cholesterol levels were analyzed.

  For the analysis of fat, the blood of the rat used in the experiment was collected using the tail blood collection method at the end of the experiment and then used for the analysis. Fatty analysis uses an enzyme colorimetric method, and cholesterol content in serum was calculated by measuring absorbance with a standard solution at 505 to 570 nm using Cholestezyme-V (Shinyang Chemical, Korea). .

  As shown in FIG. 6 and Table 5, the total blood cholesterol level of Lactobacillus NM316 strain started to decrease from the point of 40 days after the administration disclosure date, compared to the physiological saline administration group, and rather decreased from before administration. It was. Therefore, it can be predicted that the microorganism of the present invention has an effect of reducing the incidence of cardiovascular diseases (arteriosclerosis, myocardial infarction, etc.).

  As described above, the configuration and preferred embodiments of the present invention have been described herein. However, the scope of the present invention should not be construed as being limited to the embodiments described above. It is judged that a person skilled in the art can implement the content described in this specification by modifying it within the scope of rights of the present invention.

3 is a diagram for verifying probiotic effects by microorganisms according to the present invention. Here, A represents Lactobacillus reuteri ATCC 23272, and N represents Lactobacillus fermentum NM316. It is a graph which shows the mode of the weight change of the rat which ingested the microorganisms by this invention with the form of a living microbe. Here, a represents initial body weight g, b represents body weight g after administration for 40 days, and c represents body weight gain g after administration for 40 days. It is a graph which shows the mode of the weight change of the rat which ingested the microorganisms by this invention with the form of lactic-acid-bacteria fermented milk. Here, a represents initial body weight g, b represents body weight g after 60 days of administration, and c represents body weight gain g after 60 days of administration. It is a graph which shows the change of the feed intake of the rat which ingested the microorganisms by this invention. Here, a is the result for the administration group in the form of viable microorganisms of the present invention, and b is the result for the administration group in the form of lactic acid bacteria fermented milk (yogurt). It is a graph which shows the change of the metabolic efficiency of the rat which ingested the microorganisms by this invention. Here, a is the result for the administration group in the form of viable microorganisms of the present invention, and b is the result for the administration group in the form of lactic acid bacteria fermented milk (yogurt). It is a graph which shows the mode of the blood cholesterol level change of the rat which ingested the microorganisms by this invention.

Claims (7)

Lactobacillus fermentum NM316 (KTCC-10458BP), which produces a polysaccharide material. A preparation for the prevention and treatment of obesity comprising a pharmaceutically effective amount of Lactobacillus fermentum NM316 (KCTC-10458BP) and a carrier to produce a polysaccharide substance. The obesity prevention / treatment preparation according to claim 2, wherein the obesity prevention / treatment preparation is covered with an enteric coating substance. A food composition comprising, as an active ingredient, Lactobacillus fermentum NM316 (KCTC-10458BP) that produces a polysaccharide substance. The food composition according to claim 4, wherein the food composition is a jelly, pudding, powder, capsule, or fermented milk. A preparation for diabetes prevention / treatment comprising a pharmaceutically effective amount of Lactobacillus fermentum NM316 (KCTC-10458BP) and a carrier for producing a polysaccharide substance. 6. The obesity prevention / treatment preparation according to claim 5, wherein the diabetes prevention / treatment preparation is covered with an enteric coating substance.