KR20150106075A - Health food and pharmaceutical composition for preventing or improving intestinal disease comprising Bacillus subtilis strain or its culture broth as effective component - Google Patents

Abstract

The present invention relates to a health food and a pharmaceutical composition for alleviating or preventing intestinal diseases containing Bacillus subtilis strains or a culture medium thereof as an effective component and, more particularly to a health food for alleviating or preventing intestinal diseases, a pharmaceutical composition for treating or preventing intestinal diseases, and a constipation medicine containing Bacillus subtilis CBD2 strains (Accession No. KACC91904P) and Bacillus subtilis KMKW4 strains (Accession No. KACC91905P) which are separated from traditional fermented food, and culture mediums of each strain, as an effective component.

Description

TECHNICAL FIELD The present invention relates to a health food and pharmaceutical composition for preventing or ameliorating intestinal diseases, which comprises a Bacillus subtilis strain or a culture thereof as an active ingredient,

The present invention is Bacillus standing subtilis strain or that of the culture solution thereof, a bowel disease prevention or health food for improving and pharmaceutical compositions containing as an active ingredient, and more particularly, standing separated Bacillus from traditional fermented foods subtilis (Bacillus subtilis) A preventive or remedy for enteric disease, bowel disease, prevention or amelioration of intestinal diseases containing CBD2 strain (accession number KACC91904P), Bacillus subtilis KMKW4 strain (accession number KACC91905P) or a culture solution of each strain as an active ingredient And to a therapeutic agent for constipation.

Probiotics have been proposed initially as materials provided by microorganisms that stimulate the growth of other individuals by Lilly and Stillwell, but recently their definition has been limited to beneficial to the host as well as to the colonized resident microbial gun To a formulation or product containing a sufficient number of various microorganisms to provide a health benefit (Schrezenmeir and Michael. 2001 J Clin Nutr 73, 361S-364S). The market for probiotics has been growing as consumers are perceived as health foods. In the food industry, probiotic bacteria are recognized for their resistance to biological barriers such as gastric and bile salts and for their ability to inhibit E. coli bacterial growth in intestinal cells (Servin and Coconnier, 2008 AM J Clin Nutr 73, 361S-364S). As probiotic bacteria, Lactobacillus sp., Bifidobacterium sp., Propioniibacterium , Bacillus sp. Are well known, and intestinal flora and immunity (Ouwehand and Salminen, 2002 Antonie Van Leeuwenhoek 82, 279-289). In addition, sugar hydrolysis and lipid hydrolysis properties are crucial in food, and metabolites expressed by probiotic bacteria can change not only the food matrix but also the composition involved in fermentation Crittenden et al. 2002 Journal of the Science of Food and Agriculture 82, 781-789).

In traditional Korean fermented foods such as kimchi (fermented vegetable products), miso (fermented soybean paste), and kochujang (fermented red pepper paste), the flour is composed of dynamic clusters of bacteria, fungi and yeast. Lim and In attempted to isolate the probiotic lactic acid bacteria from Korean fermented foods (Lim SM and Im DS 2009 J Microbiol Biotechnol 19, 178-186). Bacillus sp.) have been widely known as bacteria that live in large quantities in Korean fermented foods. In particular, his spores, which can germinate in the gastrointestinal tract, are useful as probiotics. In the case of Bacillus in shrimp, digestive enzymes such as amylase, protease and lipase were increased. Bacillus probiotics have been introduced by pharmaceutical companies, offering no harmful effects on human health such as antimicrobial activity, immune function regulation and blood lipid reduction. Lactic acid bacteria for screening probiotic bacteria have been studied. Lactobacillus strains were applied to Spanish fermented sausages in order to add health promoting effect. Recently, lactic acid bacteria have also been considered as oral vectors for therapeutic proteins or DNA vaccines. On the other hand, there is little research on Bacillus probiotic bacteria.

Korean Patent Laid-Open Publication No. 2013-0114942 discloses a novel Bacillus subtilis BCNU 9169 strain and a probiotic composition comprising it as an active ingredient. However, as in the present invention, a strain of Bacillus subtilis or a culture thereof There has been no known therapeutic agent for constipation which is contained as an active ingredient.

SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and the present inventors have found that, after taking a Bacillus subtilis CBD2 strain and a Bacillus subtilis KMKW4 strain isolated from a conventional fermented food in a mouse model, , The excretion frequency of the excretion of the mouse, the weight of the excrement and the wet weight of the excretion were observed and the gastrointestinal tract transport rate and the harmful enzymatic activity of the intestinal tract were examined to examine the degree of constipation treatment. As a result, . Thus, the inventors of the present invention have completed the present invention by confirming that the two species of Bacillus subtilis strains of the present invention are highly effective in preventing or treating constipation.

In order to solve the above-mentioned problems, the present invention relates to a fermented food comprising Bacillus subtilis the present invention provides a health food for preventing or ameliorating bowel disease containing as an active ingredient a subtilis CBD2 strain (Accession No. KACC91904P), a Bacillus subtilis KMKW4 strain (Accession No. KACC91905P), or a culture solution of each of these strains.

The present invention is a standing Bacillus subtilis (Bacillus subtilis) CBD2 strain isolated from a traditional fermented foods (Accession No KACC91904P), Bacillus subtilis standing (Bacillus a subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

The invention also subtilis (Bacillus standing Bacillus isolated from a traditional fermented food subtilis CBD2 strain (Accession No. KACC91904P), Bacillus subtilis Subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

In the present invention, constipation is induced by loferamide after taking a Bacillus subtilis CBD2 strain and a Bacillus subtilis KMKW4 strain isolated from a conventional fermented food in a mouse model, and the excretion frequency of the excretion of the mouse, Weight, and weight of fecal material and investigated gastrointestinal tract transport rate and harmful enzymatic activity of intestinal tract. As a result, it was confirmed that there was an improvement effect compared with untreated control. Thus, the two species of Bacillus subtilis strains of the present invention are highly effective for the prevention or treatment of constipation, so that they can be used as health food for preventing or ameliorating bowel disease, pharmaceutical composition for preventing or treating bowel disease, feed additive, Can be very useful.

Figure 1 shows the effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on fecal parameters in mice. The frequency of excretion (A), the wet weight of excrement (B) and the water content of excrement (C) were evaluated by counting the number of excrement pellets provided within 60 minutes on the first day after the last injection of rofaperide hydrochloride Every n = 8). At the end of the fecal analysis, the mice (n = 5 for each group) were sacrificed and the distal colon removed to determine the number (D) of fecal pellets in the distal colon. Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 had a prophylactic effect on fecal emission changes in the rofelamide-induced constipation mouse model. Data were expressed as means ± SD. One-way ANOVA, different subscripts on the bars indicate significant differences at P <0.05.
Figure 2 shows the effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on GIT. After 24 hours of fasting, the barium sulfate solution was orally administered. At the 15th minute of administration, the longest distance of the barium sulfate solution migration from the pylorus and the length from the pylorus to the terminal ileum were measured (n = 3 for each group). Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 prevent the dysfunction of GIT in a rofeamide-induced constipation mouse model. Data were expressed as means ± SD. One-way ANOVA, different subscripts on the bars indicate significant differences at P <0.05.
Figure 3 shows the effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on harmful enzyme activity. After the final injection of rofemamide hydrochloride, the mice (n = 5 for each group) were sacrificed and the cecum was removed. A cephalic suspension was used for enzyme activity assays such as? -glucosidase (A) and tryptophanase (B). Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 are potentially beneficial as functional probiotics in the prevention of constipation. Data were expressed as means ± SD. One-way ANOVA, different subscripts on the bars indicate significant differences at P <0.05.

According to an aspect of the invention, the invention subtilis (Bacillus standing Bacillus isolated from a traditional fermented food subtilis CBD2 strain (Accession No. KACC91904P), Bacillus subtilis a subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

The Bacillus subtilis subtilis) CBD2 strains or Bacillus subtilis books (Bacillus subtilis KMKW4 strain was deposited on November 8, 2013 at the National Institute of Agricultural Science and Technology ( Bacillus, subtilis CBD2 Accession number: KACC91904P, Bacillus subtilis subtilis KMKW4 strain Accession No .: KACC91905P).

In addition, for the purpose of preventing or improving constipation, the above-mentioned strain dry powder or strain culture medium of the present invention may be added to foods or beverages. At this time, the amount of the strain dry powder or strain culture solution in the food or beverage may be 0.01 to 15% by weight of the total food, and the health beverage composition may be added in an amount of 0.02 to 5 g, preferably 0.3 to 1 g As shown in FIG.

The health functional beverage composition of the present invention is not particularly limited to the ingredients other than the above-mentioned dry powder of the strain or the culture medium of the strain as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates, &Lt; / RTI &gt; Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tautatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the strain dry powder or strain culture medium of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate, etc.) Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the strain dry powder or strain culture of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination, and the proportion of such additives is not so important, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the strain dry powder or strain culture of the present invention.

In the health food for preventing or ameliorating bowel disease according to an embodiment of the present invention, the food may be a milk product, a fermented milk, a drink, a meat, a sausage, a bread, a chocolate, a candy, a snack, a confection, a pizza, a ram, a gum, Soups, beverages, alcoholic beverages, and vitamin complexes, but the present invention is not limited thereto.

The present invention is a standing Bacillus subtilis (Bacillus subtilis) CBD2 strain isolated from a traditional fermented foods (Accession No KACC91904P), Bacillus subtilis standing (Bacillus a subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

In the pharmaceutical composition for preventing or treating bowel disease according to an embodiment of the present invention, the bowel disease may be any one selected from constipation, acute diarrhea, enteritis, gastroenteritis, abdominal pain and abdominal distension, May be constipation, but is not limited thereto.

The pharmaceutical composition for preventing or treating constipation according to the present invention may contain 0.02 to 80% by weight, preferably 0.02 to 50% by weight, of the above-mentioned strain dry powder or strain culture liquid, based on the total weight of the composition.

The composition comprising the strain dry powder or the culture medium of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the production of pharmaceutical compositions.

The pharmaceutical dosage forms of the strain dry powder or strain culture of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination.

The composition containing the strain dry powder or the culture broth according to the present invention may be formulated into oral compositions, external preparations, suppositories and sterilized preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, May be formulated in the form of injection solutions. Examples of carriers, excipients and diluents that can be contained in the composition including the strain dry powder or the culture medium of the strain include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc. . In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, , Oligosaccharide, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dose of the strain dry powder or strain culture of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for the desired effect, it is preferable to administer the strain dry powder or the culture medium of the present invention at a dose of 0.0001 to 100 mg / kg, preferably 0.001 to 100 mg / kg per day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The strain dry powder or strain culture of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The invention also subtilis (Bacillus standing Bacillus isolated from a traditional fermented food subtilis CBD2 strain (Accession No. KACC91904P), Bacillus subtilis Subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Sample collection and isolation of strain

8 kinds of soybean paste, 7 kinds of soy sauce, 1 kind of chonggukjang, and 6 kinds of kimchi were collected in the traditional fermented foods such as soybean paste, soy sauce, chonggukjang, and kimchi as regional (Kyongnam, Kyungbuk, Jeonnam, Chungnam, Chungbuk). All samples were mixed with 1 g / 9 ml sterile water and ground. The lysis solution was diluted 10-fold and then divided into NA solid medium and cultured at 37 for 1 day to separate the resulting colonies.

Identification of isolated strains

Ten strains isolated from traditional fermented foods were suspended in sterilized water and cultured in nutrient agar (Difco, Detroit, USA) for 24 hours at 37 ° C. The strains were screened and subcultured to select pure strains. The selected strains were assigned to Solgent co., Korea for the identification of microorganisms which can be used as food raw materials, and the results of the analysis were obtained based on the nucleotide sequence of 16s ribosomal DNA.

animal

Four-week-old male BALB / c mice were purchased from Central Lab Animal Inc. (Seoul, Korea). All animals were allowed to adapt to laboratory equipment for a week. Mice were housed at room temperature at a controlled temperature (22 +/- 2 DEG C) and 55 +/- 5% humidity at a 12 hour / 12 hour light / dark cycle. Feed pellets and tap water were freely available. All procedures were subject to animal protocols approved by the Daegu Technopark Bio-Health Convergence Center Institutional Animal Care and Use Committee.

In vivo  Experimental design and processing

A random mouse in five weeks divided into four groups, Bacillus subtilis books (Bacilus subtilis CBD2, Bacillus subtilis KMKW4 or PBS (n = 8 for each group) were orally administered once a day for 7 days. Groups of the normal (NOR) and constipated control (CON) groups were orally administered 200 μl PBS; Bacillus subtilis CBD2 (1 x 10 &lt; ⁷ &gt; CFU in 200 [mu] l PBS) was orally administered to the Bacillus subtilis CBD2 (CBD2) group; And Bacillus subtilis KMKW4 (1 × 10 ⁷ CFU in 200 μl PBS) were orally administered to the Bacillus subtilis KMKW4 (KMKW4) group. On the first day after the final administration, mice were injected with perferamide hydrochloride to induce constipation.

Induction of constipation

Constipation was induced by intraperitoneal injection of rofemamide hydrochloride (4 mg / kg body weight, Sigma-Aldrich, St. Louis, MO, USA) in 0.5% Tween 20 (Sigma-Aldrich) in mice of CON, CBD2 and KMKW4 groups for 2 days (Shimotoyodome et al. 2000 Comp .), While the non-constipated NOR group was injected with only 0.5% Tween 20 Biochem Physiok 126,203-211).

Weight and feed intake analysis

Changes in body weight and feed intake of all mice were measured on an electronic scale (CP423S, Sartorius) on day 8 (on the first day after the final administration of Bacillus subtilis) and on day 13 (on the first day after the last injection of ferulumide hydrochloride) AG, Goettingen, Germany). All measurements were performed in triplicate to ensure accuracy.

Measuring excrement parameters

Excretion was assessed by counting the number of excrement pellets produced within 60 minutes on Day 8 (Day 1 after the last administration of Bacillus subtilis) and Day 13 (Day 1 after the last administration of ferulumide hydrochloride) . At the same time, fecal pellet wet and dry weights were also measured (Devries et al. 2010 Front Neurosci 4, 22). The dry weight of the fecal pellet was measured after drying the pellet in a laboratory oven at 70 캜 for 24 hours. The water content of the excrement pellets was calculated as: Excreta water content (%) = (wet weight of excrement pellets - dry weight of excrement pellets) / wet weight of excrement pellets x 100. The mice (n = 5 for each group) were sacrificed and the distal colon removed for counting the number of pelleted excrements in the distal colon. Other mice (n = 3 for each group) were used for gastrointestinal transport rate measurements.

Gastrointestinal tract transportation ( GIT ) Rate measurement

The movement in the gastrointestinal tract was measured according to the method described in the prior art (Miki et al. 2005 Diabetes 54, 1056-1063). Mice (n = 3 for each group) were fasted for 24 hours with free access to water. On the day of the experiment, mice were orally administered with a test solution consisting of 25% barium sulfate (Sigma-Aldrich) suspended in water at 20 占 퐂 / g. The mice were sacrificed after 15 minutes and the gastrointestinal tract was quickly removed. The distance (A) from the pylorus to the farthest point of travel of the barium sulfate solution and the distance from the pylorus to the terminal ileus (B) were measured. The GIT ratio is expressed as a percentage of A to B.

Assay of harmful enzyme activity

The mice were sacrificed and the appendix removed. A 1:10 cecal suspension was prepared in cold 0.1 M phosphate buffer (pH 7.0), and the non-bacterial debris was removed by centrifugation at 6,000 rpm for 10 minutes. The supernatant fraction was used for enzyme activity assays such as? -Glucosidase and tryptophanase.

β-glucosidase activity was assayed in 2 ml of a reaction mixture (suspended excrement sample) containing 800 μl of 2 mM p-nitrophenyl-β-D-glucopyranoside (Sigma-Aldrich) and 200 μl of enzyme solution did. The reaction was incubated for 30 min at 37 [deg.] C, and 1 ml of 0.5 N NaOH was added to stop. The reaction mixture was then centrifuged at 3,000 rpm for 10 minutes. Enzyme activity was measured by absorbance monitoring at 405 nm (An et al. 2011 Lipids Health Dis 10, 116).

The tryptophanase activity was determined by adding 200 ul of complete reagent solution (2.75 mg of pyridoxal phosphate in 100 ml of 0.05 M potassium phosphate buffer, pH 7.5, 19.6 mg of disodium EDTA dihydrate and 10 mg of bovine serum albumin) Of 20 mM tryptophan (Sigma-Aldrich) and 100 [mu] l of enzyme solution. The reaction was incubated for 1 hour at 37 ° C and then stopped by adding ₂ ml of a color reagent solution (14.7 g p-dimethylaminobenzaldehyde in 52 ml H ₂ SO ₄ and 948 ml 95% ethanol). The reaction mixture was then centrifuged at 3,000 rpm for 10 minutes. Enzyme activity was measured by absorbance monitoring at 550 nm.

Statistical analysis

Data were expressed as means ± SD. Differences between groups were analyzed by one-way analysis of variance (ANOVA) according to Duncan's multi-range test. The difference between the means was considered statistically significant at p <0.05. All analyzes were performed using SPSS / Win software (Version 12.0, SPSS Inc, Chicago, IL, USA).

Example  1. Identification and enzymes of isolated strains Secretory function  Isolation of Excellent Strain

After isolating the strain from the conventional fermented food, 11 strains which were well grown and had a clear colony shape were selected and identified. All of them were found to be Bacillus sp. Microorganisms. Among them, 4 kinds of edible microorganisms were Bacillus subtilis Bacillus subtilis were named Bacillus subtilis CBD2, Bacillus subtilis CBKW, Bacillus subtilis KGC and Bacillus subtilis KMKW4. Among them, Bacillus subtilis having excellent growth and enzyme activity CBD2 and Bacillus subtilis KMKW4 were selected and used in the present invention.

Example  2. Weight and feed conditions

To determine if body weight and feed intake were associated with treatment with Bacillus subtilis CBD2, Bacillus subtilis KMKW4 and loperamide hydrochloride treatment, weight and feed intake changes were monitored in all mice. As can be seen in Table 1, the body weight of the mice was significantly different between Day 8 (the first day after the last administration of Bacillus subtilis) and Day 13 (the first day after the last injection of piperazide hydrochloride) I did. Feed intake at the same time did not change in all experimental groups. Such results indicate that treatment with Bacillus subtilis CBD2, Bacillus subtilis KMKW4 and rofelamide did not induce any changes in body weight and feed intake.

Effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on body weight and feed intake in mice The money-
meters Before loperamide injection After loperamide injection NOR CON CBD2 KMKW4 NOR CON CBD2 KMKW4 Body weight (g) 22.08 + - 0.83 22.89 ± 0.75 21.89 ± 0.96 22.24 + - 0.80 22.49 + - 1.23 24.12 + - 0.67 22.72 ± 1.04 22.98 + 0.78 Food intake (g / day) 4.94 0.11 4.95 + 0.03 5.06 ± 0.18 4.77 ± 0.22 5.29 ± 0.02 4.76 ± 0.25 5.05 + - 0.12 5.18 ± 0.11

Example  3. Fecal release characteristics

In order to test the prophylactic effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on excretion frequency, excrement weight and wet weight of excrement in the Loperamide-induced constipation mouse model, Bacillus subtilis CBD2 and Bacillus subtilis Tylaris KMKW4 was orally administered once a day for 7 days. Then, loperamide hydrochloride was injected twice a day for 5 days to induce constipation in mice. (4 mg / kg) decreased the excretion frequency, the wet weight of the feces and the water content of the excrement to 42.4 ± 20.1%, 40.4 ± 18.5% and 33.1 ± 11.6%, respectively, of the NOR group (FIGS. 1A, C). Two types of Bacillus subtilis (Bacillus subtilis CBD2; Bacillus subtilis KMKW4) were compared. Bacillus subtilis CBD2 prevented excretion frequency, wet weight of excrement, and water content of excrement to 173.8 ± 32.9%, 126.7 ± 14.1% and 128.6 ± 22.4%, respectively, of CON group. Similarly, Bacillus subtilis KMKW4 prevented the excretion frequency and water content of excretions to 157.1 ± 60.7% and 124.1 ± 31.3%, respectively, in the CON group. However, the wet weight of the excrement was not changed by Bacillus subtilis KMKW4 when compared to the CON group. At the end of the fecal analysis, the mice (n = 5 for each group) were sacrificed and the distal colon removed to determine the number of feces in the distal colon. The number of excrement pellets in the distal colon was increased in the CON group; The values were 138.1 ± 31.0% of the NOR group. Increases in the number of distal colonic pellets in the CON group were prevented by Bacillus subtilis CBD2 and Bacillus subtilis KMKW4; The values were 68.9 ± 21.1% and 68.9 ± 17.2%, respectively, of the CON group (FIG. 1D).

Example  4. Gastrointestinal tract transportation rate

To determine the effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on gastrointestinal tract, GIT was measured in mice (Figure 2). After the final injection of rofemamide hydrochloride, the mice were fasted for 24 hours. On the day of the experiment, the barium sulfate solution was orally administered. At 15 minutes of administration, the length from the pylorus to the farthest point of travel of the barium sulfate solution and the length from the pylorus to the terminal ileus were measured. Loperamide hydrochloride reduced GIT time; The value was 68.2 ± 9.1% of the NOR group. Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 significantly accelerated GIT (p &lt;0.05); The values were 145.8 ± 27.6% and 140.6 ± 23.5% of the CON group, respectively.

Example  5. Harmful enzymatic activity of intestinal tract

Subsequently, the effect of Bacillus subtilis CBD2 and Bacillus subtilis KMKW4 on harmful enzyme activity in a model of rofferaemide-induced constipation mice was tested (Fig. 3). After the final injection of rofemamide hydrochloride, the mice were sacrificed and the cecum was removed. A cephalic suspension was used for enzyme activity assays such as β-glucosidase and tryptophanase. Β-glucosidase and tryptophanase activated by ferulumide hydrochloride; The values for the NOR group were 176.0 ± 35.2% and 123.7 ± 18.0%, respectively. Bacillus subtilis CBD2 maintained similar β-glucosidase and tryptophanase activities as the NOR group; The values were 109.7 ± 21.7% and 102.4 ± 12.3% of the NOR group, respectively. Similarly, Bacillus subtilis KMKW4 retained? -Glucosidase and tryptophanase activity at 141.4 ± 30.5% and 98.6 ± 12.8%, respectively, of the NOR group.

Agricultural Biotechnology Research Institute KACC91904P 20131108 Agricultural Biotechnology Research Institute KACC91905P 20131108

Claims (5)

Separated from Bacillus subtilis standing traditional fermented foods (Bacillus subtilis CBD2 strain (Accession No. KACC91904P), Bacillus subtilis subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient. The food according to claim 1, wherein the food is selected from the group consisting of dairy products, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, Or a pharmaceutically acceptable salt thereof. Bacillus subtilis CBD2 (Accession No. KACC91904P), Bacillus subtilis KMKW4 (Accession No. KACC91905P) isolated from traditional fermented foods, or a culture containing the culture medium of each of these strains as an active ingredient A pharmaceutical composition for preventing or treating a disease. [Claim 5] The pharmaceutical composition according to claim 3, wherein the bowel disease is any one selected from constipation, acute diarrhea, enteritis, gastroenteritis, abdominal pain and abdominal distension. Separated from Bacillus subtilis standing traditional fermented foods (Bacillus subtilis CBD2 strain (Accession No. KACC91904P), Bacillus subtilis Subtilis KMKW4 strain (Accession No. KACC91905P) or a culture solution of each of these strains as an active ingredient.

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