KR20030075716A - Composition containing anti-Helicobacter pylori Bacillus polyfermenticus SCD (KCCM 10104) or polyfermenticin SCD - Google Patents

Abstract

PURPOSE: A composition for the prophylaxis or treatment of gastric diseases containing a Bacillus polyfermenticus SCD strain having an antibacterial effect on a Helicobacter pylori strain or polyfermenticin SCD produced thereby is provided. The composition has excellent antibacterial activity against the H. pylori strain and is thus used in a food, medicines or the like. CONSTITUTION: A preventive or therapeutic agent composition against gastric diseases comprises: as an effective ingredient a B. polyfermenticus SCD strain(KCCM 10104) or polyfermenticin SCD produced thereby. For an example, a B. polyfermenticus SCD strain is inoculated into a TSB broth, precultured for 12hr at 37deg.C, inoculated into a baffle flask and then cultured for 12hr. The toothpaste containing the polyfermenticin SCD contains 65g calcium carbonate, 25g magnesium carbonate, 5g boron and 5g polyfermenticin SCD in 0.1ml peppermint oil, 3ml glycerin and 10ml water.

Description

Bacillus polypermanticus (있는 ａｃｉｌｌｕｓ) Scd strain or polypermanticin (HC-bacterium-containing anti-bacterium-containing anti-bacterium anti-bacterial treatment for the Helicobacter pylori strain polyfermenticus SCD (KCCM 10104) or polyfermenticin SCD}

The present invention relates to a probiotic probiotic having an antimicrobial effect against pathogenic microorganisms and a composition containing the bacteriocin produced by the probiotic. More specifically, the present invention is a bacterium B. polyfermenticus SCD strain (KCCM 10104) having an antimicrobial effect against H. pylori or polyfermenticin produced by the bacterium. It relates to a composition for preventing and treating gastrointestinal diseases containing SCD.

As income levels rise, consumers are interested in health-oriented products, and the demand for functional foods is also increasing. In fact, food development using the functionality and efficacy of probiotic probiotics is recognized as an important field of functional food. Therefore, research to use probiotic probiotics as a functional food has been actively conducted. Probiotic probiotics are products that are produced for the purpose of improving the body function through the intestinal diseases as well as normalizing the intestinal flora by enzymes, organic acids, vitamins and non-toxic antibacterial substances secreted by microorganisms by ingesting live microbial cells , R. Probiotics in man and animals.J. Appl. Bacteriol. 66 : 365-37 (1989)). These probiotic probiotics are associated with stabilizing the intestinal flora, reducing rot production and preventing disease, preventing immunity, and preventing disease, activating immunity, indirect anticancer activity, lowering cholesterol, reducing lactose intolerance, and preventing and preventing constipation. Functionality is recognized (Goldin, BR and Borbach, SL Alterations in fecal microflora enzyme related to diet, age, lactobacillus supplements, and dimethylhydrazine. Cancer 40: 2421-2426 (1977); Gilliland, SE and Walker, DK Factors to consider when selecting a culture of Lactobacillus acidophilus as a dietary adjunct to produce a hypocholesterolemic effect in humans.J. Dairy Sci. 73: 905-911 (1990); Fuller, R. Probiotics in human medicine.Gut 32: 439-442 (1991 Hilton; E., Henry, DI, Phyllis, A., Kenneth, F. and Michael, TB Ingestion of yoghurt containing Lactobacillus acidophilus as prophylaxis for candidal vaginitis.Ann. Int. Med. 116: 353-357 (1992) Shah, N. Lactobacillus acidophil us and lactose intolerance, a review.ASEAN Food J. 9: 47-54 (1994); Matsuzaki, T., Hashimoto, S. and Yokokura, T. Effects on antitumor activity and cytokine production in the thoracic cavity by intrapleural administration of Lactobacillus casei in tumor-bearing mice. Med. Microbiol. Immunol. Berl. 185: 157-161 (1996); Takiguchi, R., Mochizuki, E., Suzuki, Y., Nakajima, I. and Benno, Y. Lactobacillus acidophilus SBT 2062 and Bifidobacterium longum SBT 2928 on harmful intestinal bacteria. J. Int. Microbiol. 11: 11-17 (1997).

Bacteria widely used as probiotic probiotics are Lactobacillus (Lactobacillus)genus,Streptococcus(StreptococcusGenus, Bifidobacterium (Bifidobacterium)genus, Enterococcus(EnterococcusClostridium ButyricumClostridium butyricum), Lactobacillus sporogenes (Lactobacillus sporogenes), Bacillus subtilis (Bacillus subtilis) And Bacillus polyfermentus(B. polyfermenticus) SCD And so on. On the other hand, the properties required as a probiotic probiotic include safety, functionality (survival, fixation, habitat, antimicrobial production ability, immune promoting ability, antigenotoxic activity, inhibitory ability of pathogenic bacteria), technical characteristics (functional characteristics, Stability, bacteriophage resistance, viability during manufacture), and intestinal viability as GRAS (Generally Recognized As Safe) microorganisms.

In recent years, industrial usefulness for probiotic bacteria of the genus Bacillus has been increasing (Park, H.S., Lee, S.H. and Uhm, T.B.Selection of microorganisms for probiotics and their characterization.J. Food Sci.Nutr. 3:433-440 (1998); Green, D.H., Wakeley, P.R., Page, A., Barnes, A., Baccigalupi, L., Ricca, E. and Cutting, S.M. Characterization of twoBacillusprobiotics. Appl. Environ. Microbiol. 65: 4288-4291 (1999); Hoa, N.T., Baccigalupi, L., Huxham, A., Smertenko, A., Van, P.H., Ammendola, S., Ricca, E. and Cutting, S.M. Characterization ofBacillusspecies used fororal bacteriotherapy and bacterioprophylaxis of gastroin testinal disorders. Appl. Environ. Microbiol. 66: 5241-5247 (2000)), Bacillus polyfermenticus, among others called Bispan bacteria (B. polyfermenticusSCD is drawing attention as a probiotic probiotic for food and medicine. Bacillus polyfermentus (B. polyfermenticusSCD is apoptotic bacillus isolated from air by Terakato of Japan in 1933, and it secretes 20 kinds of enzymes and recycles nutrients._One, B₂Synthesize K and spread nutrition. In addition, it digests and absorbs the three major nutrients of the body, carbohydrates, proteins, fats and fibres, and has the function of inhibiting proliferation by lysing pathogenic bacteria such as typhoid bacteria, paratyphoid bacteria, erythrocytes and cholera bacteria. In particular, commercially available lactic acid bacteria and bifidus bacteria should be considered special coating or capsule products due to gastric acid and various enzymes in order to reach the intestines. It has almost lost its activity and shows excellent effects in the treatment of intestinal diseases. Bispan bacillus is a bacterium that has been tested forBacillus) And the Bacillus subtilis (B. subtilis) And Bacillus mesentericus (B. mesentericus), But It does not exist in classification names under the International Naming Convention. However, studies have shown that comparing morphology and biochemical propertiesB. subtilisIt has been identified as very similar to, but Bacillus subtilis (B. subtilisThe difference is that it has the ability to break down lactose and produces more acetic acid and lactic acid from glucose and lactose (Jun, K.-D., Lee, K.-H., Kim, W.-S). and Paik, H.-D.Microbiological identification of medical probiotic bispan strain.Kor. J. Appl.Microbiol.Biotechnol. 28: 124-127 (2000)). And liquid fermentation method for economic production of Bispan bacillus active follicles is revealed (Paik, H.-D., Jun, K.-D., Sung, S.-H., Kim, W.-S., Kim, H.-S. and Lee, B.-C.Liquid cultivation of strains ofBacillus polyfermenticus. United States Patent 6,010,898 (1999).

Bispan bacteria, which have the characteristics of good probiotics, produce a bacteriocin called polyfermentincin SCD (Lee, K.-H., Jun, K.-D., Kim, W.-S. and Paik, H.- D. Partial characterization of polyfermenticin SCD, a newly identified bacteriocin of Bacillus polyfermenticus.Lett.Appl.Microbiol . 32: 1-6 (2001)), bacteriocin is a proteinaceous agent that exhibits antimicrobial activity against similar species produced by the metabolic activity of bacteria. It is a substance. Since it is composed of protein, when it is ingested by the human body, it is decomposed by the proteolytic enzyme of the digestive organs, and thus, its utility as a natural preservative in foods and the like is increasing in that it is nontoxic and non-residual to the human body. Bacteriocin is not inert at relatively high temperature, has stability at a wide range of pH, and is non-toxic, colorless, and odorless, so it is expected to develop a wide range of applications as a natural preservative to replace a chemical preservative. That is, in addition to improving shelf life in fermented milk, fermented alcoholic beverages, canned food, and refrigerated / frozen products, it extends the life span of red pepper paste, soybean paste, tofu, and lactic acid bacteria fermented products, and prevents rancidity and deterioration of traditional foods such as kimchi, yakju, Takju, etc It is possible to improve the shelf life of fruits and vegetables such as fats and oils and sprouts (Paik, H.-D. and Oh, DW Purification, characterization and comparision of bacteriocins. J. Microbial. Biotechnol. 6: 151-161 (1996)).

Helicobacter pylori (H. pylori) bacteria chronic gastritis, gastric ulcer, duodenal ulcer, gastritis occurrence and physiological characteristics of the bacteria since reported as a cause of peptic ulcers in 1982 by Warren (Warren) and Marshall (Marshall) and pathogenic mechanisms, etc. Many studies are underway to clarify. Helicobacter pylori ( H. pylori ), which lives mainly in the gastric mucosa, is a flying gram-shaped, gram-negative bacterium that has a size of 2 to 7 × 0.4 to 1.2 ㎛. Microaerophilic, the optimum oxygen partial pressure is 2 to 8% and can be incubated at 37 ° C under 10% CO ₂ conditions. It is easy to expect that H. pylori bacteria are acid resistant because they are parasitic bacteria in the gastric mucosa of the strongly acidic stomach, but the optimum growth pH of these bacteria is 7.0-7.4 (Hazell, SL, Lee, A. and Hennessy, W. Campylobacter pyloridis and gastritis: Association to an environment of mucus as importantfactors in colonization of the gastric epithelium. J. Inf. Dis. 153: 658-663 ( 1986); Steer, HW Ultrastructure of cell migration through the gastric epithelium and its relationship to bacteria.J. Clin. Path. 28: 639-646 (1975)).

The most characteristic biological properties of H. pylori bacteria are very potent motility and the ability to produce urease enzymes.

In kinetic experiments using methylcellulose artificial mucus, H. pylori bacteria exhibited motility even at a viscosity 20 times higher than that of E. coli motility (Hazell, SL and Lee, A. Campylobacter pyloridis). ureas, hydrogen ion back-diffusion and gastric ulcers.Lancet 1: 15-17 (1986)). Because of this powerful motility, H. pylori bacteria penetrate the mucous layer, which is difficult to penetrate simple compounds, such as hydrogen ions, in addition to chemicals such as salicylate and ethanol. It is also thought to be able to live freely in the mucous layer and adhere to the junction of gastric mucosal epithelial cells. Bacterial structures associated with motility are flagella. This bacterium is composed of one or five to six bundles of monopolar or bipolar flagella. In particular, they have unique structures called sheaths and terminal bulbs that are not found in other bacterial flagella, and their biological properties are not yet known.

Another characteristic of H. pylori bacteria is its high ability to produce urease (urea aminourelase) (Lee. A. and Hazell, SL Campylobacter pylori in health and disease: An ecological perspective.Microb.Ecol Health Dis. 1: 1-16 (1988)). The production capacity is more than 100 times the production capacity of the Proteus strain , and about 6% of the bacterial protein is known as urease (urease). Urease is an enzyme that breaks down urea into ammonia and carbon dioxide. The bacterium H. pylori can effectively break down urea present in the gastric mucosa, and thus the ammonia produced can counteract the attack by strong hydrochloric acid in the stomach by neutralizing the surrounding environment. It is known.

On the other hand, the current treatment of H. pylori is a tri-therapy and bismuth in combination with amoxicillin or tetracycline together with bismuth and metronidazole. (bismuth), omeprazole, tetracycline, and tetratherapy combined with metronidazole have been reported to show excellent therapeutic effects on H. pylori . The emergence of resistant strains and the permeability of drugs in the stomach and the regrowth of the bacteria that had been inhibited growth after treatment.

The present invention has been made to solve the above problems as a probiotic to prevent gastrointestinal diseases containing Bacillus polyfermenticus SCD strain having an antimicrobial activity against H. pylori strains as an active ingredient and Its purpose is to provide a therapeutic composition.

Still another object of the present invention is to provide a composition containing polypermentin SCD of B. polyfermenticus SCD bacteria production having an antimicrobial activity against H. pylori strains. will be.

An object of the present invention is to partially purify the polypermanticin SCD produced by B. polyfermenticus SCD strain (KCCM 10104) and B. polyfermenticus SCD strain (KCCM 10104). By examining the antimicrobial and its mechanism of action against Helicobacter pylori ( H. pylori ) bacteria to confirm the efficacy and to obtain a composition from it was achieved.

Hereinafter, the configuration and operation of the present invention will be described in detail by way of examples, but the scope of the present invention is not limited to the following examples.

1 is Bacillus polyfermenticus (B. polyfermenticus) Helicobacter pylori by SCD strain (KCCM 10104)H. pyloriA photograph showing the growth inhibition of the strain (KCTC 2948).

2 is spot-picture is also shown that represents the loan (spot-on-lawn) method Poly buffer mentee new (polyfermenticin) SCD of Helicobacter pylori (H. pylori) growth inhibition for (KCTC 2948) strain by -one.

Figure 3 is a graft illustrating the antimicrobial activity of the poly buffer mentee new SCD on the Helicobacter pylori (H. pylori) strain (KCTC 2948) in the logarithmic growth phase (exponential phase).

Figure 4 is a graft illustrating the antimicrobial activity of the poly buffer mentee new (polyfermenticin) SCD for the Helicobacter pylori (H. pylori) strain (KCTC 2948) in the stationary phase (stationary phase).

Figure 5 is an electron micrograph of the control of the logarithmic growth Helicobacter pylori ( H. pylori ) strain (KCTC 2948),

6 is treated with a buffer of poly pylori mentee new SCD logarithmic growth phase Helicobacter (H. pylori) strains electron micrograph of (KCTC 2948),

7 is an electron micrograph of the control station stop Helicobacter pylori ( H. pylori ) strain (KCTC 2948),

8 is an electron photomicrograph of a poly buffer mentee pylori in the logarithmic growth phase to the new treatment SCD Helicobacter (H. pylori) strain (KCTC 2948).

The present invention provides a composition for the prevention and treatment of gastrointestinal diseases containing Bacillus polyfermenticus SCD strain (KCCM 10104) having an antimicrobial effect against H. pylori strains as an active ingredient.

In addition, the present invention is a bacteriocin produced by B. polyfermenticus SCD strain (KCCM 10104), which has an antimicrobial effect against H. pylori strains, containing polyfermentin SCD as an active ingredient. It provides a composition for preventing and treating gastrointestinal diseases.

Hereinafter, the present invention will be described in detail.

By Helicobacter pylori (H. pylori) strains after 10 ⁶ CFU / mL plated in flat, Bacillus poly flops mentee kusu (B. polyfermenticus) SCD strain (KCCM 10104) spot (spot) to 10 μL on top of the logarithmic growth phase 37 When cultured in ℃ it can be observed that H. pylori is the growth of the (H. pylori) strains inhibited. In this case, the organic acid and bacteriocin produced by B. polyfermenticus SCD strain (KCCM 10104) act as a natural antibacterial agent.

One of the antimicrobial agents in the production of B. polyfermenticus SCD strain (KCCM 10104) that inhibits the growth of H. pylori strains is to be identified. At this time, the required polyfermentin SCD is produced in 5 L small fermentation tank (3 L working capacity; Korea Fermenter Co., Korea) and TSB (Difco) is used as a culture medium. B. polyfermenticus SCD bacteria are inoculated in sterile 60 mL TSB medium at 2% (v / v) and incubated at 37 rpm for 160 hours at 160 rpm. The temperature of this culture is adjusted to 37 ℃, pH is maintained at 7.0 ± 0.1 using 3 NH ₂ SO ₄ and 3 N HCl. At this time, the stirring speed and the air supply speed are preferably set to 500 rpm and 1 vvm, respectively. As a bubble remover in culture, 25% LS-300 silicone oil solution (Dow Corning, Seoul, Korea) is used. Obtain the culture medium by time and examine the growth curve in A ₆₆₀ , the bacteriocin activity is confirmed by the spot-on-lawn method after collecting the culture medium by time. At this time, 1 L of the culture medium having the maximum bacteriocin activity was obtained and centrifuged at 8000 × g for 20 minutes in a 4 ° C. centrifuge to obtain a supernatant and used in the following activity experiment.

In order to confirm whether the antimicrobial agent of B. polyfermenticus SCD strain (KCCM 10104) is antimicrobial against the strain of H. pylori, the antimicrobial agent is determined by the above method. Prepare 10 μL of the prepared polyfermentincin SCD as a test sphere, and mix the proteinase K (Sigma Chemical Co., St. Louis, USA) to the concentration of 1 mg / mL in polyfermentincin SCD, and then Prepare 10 μL of the time-reacted solution as one control and 10 μL of the same concentration of proteinase K as another control. The experimental and two control spots were spotted on a plate on which the H. pylori strain was smeared and incubated at 37 ° C. for 6-10 hours.

As a result, H. pylori strain inhibitory effect was confirmed only in the experimental group. On the other hand, anti-helicobacter pylori activity was not found in the polymentincin SCD control and the proteinase K control treated with proteinase K. Therefore, polyfermentin SCD has an antimicrobial action and this substance can be called bacteriocin.

Summarizing the above results, it can be seen that there is an antimicrobial activity against H. pylori bacteria of B. polyfermenticus SCD strain and one of them is bacteriocin.

Helicobacter pylori in certain physiological states(H. pylori)See if there is antibacterial activity against the strain. Pullimentincin SCD recovered as supernatant is partially purified with 75% ammonium sulfate. After that, dilute 400 AU / mL and 1,000 AU / mL in 0.1 M phosphate solution, respectively.(H. pylori)To the bacteria Investigate antimicrobial effects. As a result, when treated with polypermanticin SCD, the number of viable cells decreased with the reaction time in proportion to the concentration. However, if the polymentincin SCD was not treated, the viable cell count was initially changed with little change.⁶Maintain CFU / mL. Especially for Helicobacter pylori in logarithmic growth stages(H. pylori)Helicobacter pylori after 10 hours of incubation when treated with 400 AU / mL polymorphicin SCD(H. pylori)The strain decreased 4.4 log CFU / mL while the Helicobacter pylori of the stationary phase(H. pylori) In bacteria, the number of bacteria decreases by 3.77 log CFU / mL. This results in the polypericincin SCD being the Helicobacter pylori of the logarithmic multiplier.(H. pylori)It can be seen that the antibacterial action against the bacteria more effectively.

Experimental Example 1: Bacillus polyfermentus ( B. polyfermenticus ) Determination of activity of SCD (KCCM 10104)

Bacillus polyfermentus (used in the present invention)B. polyfermenticus) SCD bacteria (KCCM 10104) were activated by three to four passages after being distributed by Binex Co., Ltd. and preserved at -70 ℃ by glycerol stock method, strain for experiment Subcultures were used once at. Bacillus polyfermentus (B. polyfermenticus) As a culture medium of the SCD strain (KCCM 10104) was used TSB medium (Difco Laboratories, Detroit, USA). Helicobacter pylori(H. pylori) Strain (KCTC 2948) was used at the Daejeon Gene Center and used for Helicobacter pylori(H. pylori) DSM 4867 Strains and Helicobacter pylori(H. pylori) DSM 9691 The strain was distributed from DSM, a strain depositing institution in Germany, and used for the clinical strain Helicobacter pylori.(H. pylori) HD 223 Strains, Helicobacter pylori(H. pylori) HD 489 Strains, Helicobacter pylori(H. pylori) HD 496 Strains are sold at Seoul National University. The medium was used after sterilizing Brain Heart Infusion (Difco) at 121 ° C. for 15 minutes and cooling to 50 ° C., followed by the addition of 10% bovine calf serum.

B. polyfermenticus SCD (KCCM 10104) was inoculated into TSB broth in a 10 mL test tube, pre-incubated for 12 hours at a stirring speed of 150 rpm at 37 ° C, and then again baffle of 500 mL. working volume: 100 mL) and incubated for 12 hours. Helicobacter pylori ( H. pylori) bacteria were incubated in a 10% CO ₂ incubator at a temperature of 37 ℃.

After the Helicobacter pylori (H. pylori) smear of 10 ⁶ CFU / mL in the bacterial culture plate by the above method, the logarithmic growth phase of the culture by the method of the Bacillus poly flops mentee kusu (B. polyfermenticus) SCD bacteria ( 10 μL of KCCM 10104) was spotted thereon and incubated at 37 ° C. As a result, as shown in Table 1, the antimicrobial inhibitory power was confirmed.

Antimicrobial against various Helicobacter pylori (H. pylori) bacteria Name Determination of antimicrobial activity by spot-on-lawn method B. polyfermenticus SCD (+: excellent effect) Polyfermenticin SCD H. pylori (KCTC 2948) + 400 AU / mL H. pylori DSM 4867 + 800 AU / mL H. pylori DSM 9691 + 400 AU / mL H. pylori HD 223 (clinical strain) + 800 AU / mL H. pylori HD 489 (clinical strain) + 800 AU / mL H. pylori HD 496 (clinical strain) + 600 AU / mL

1 shows in particular Helicobacter pylori(H. pylori) It is a photograph showing the antibacterial inhibitory activity against the strain (KCTC 2948).

Experimental Example 2: Bacillus polyfermentus ( B. polyfermenticus ) Determination of the activity of polyfermenticin SCD produced by SCD (KCCM 10104)

Polyfermentin SCD was produced in 5 L small fermentation tank (3 L working capacity; Korea Fermenter Co., Korea) and TSB (Difco) was used as a culture medium. B. polyfermenticus SCD bacteria were inoculated in sterile 60 mL TSB medium at 2% (v / v) and incubated at 37 rpm for 160 hours at 160 rpm. The temperature of the main culture was adjusted to 37 ℃, pH was maintained at 7.0 using 3 NH ₂ SO ₄ and 3 N HCl. At this time, the stirring speed and the air supply speed were 500 rpm and 1 vvm, respectively. 25% LS-300 silicone oil solution (Dow Corning, Seoul, Korea) was used to remove the foam during the culture. The growth curve was examined by obtaining the culture medium by time and A ₆₆₀ was measured. The bacteriocin activity was confirmed by spot-on-lawn method after collecting the culture medium by time.

1 L of the culture medium having the maximum bacteriocin activity was obtained by the above method, and centrifuged at 8000 × g for 20 minutes in a 4 ° C. centrifuge to obtain a supernatant.

At this time, Bacillus polyfermentus(B. polyfermenticus)Antibacterial material of bacteria production is Helicobacter pylori(H. pylori) Strain (KCTC 2948), Helicobacter pylori(H. pylori) DSM 4867 Strains, Helicobacter pylori(H. pylori) DSM 9691 Strains, Helicobacter pylori(H. pylori) HD 223 Strains, Helicobacter pylori(H. pylori) HD 489 Strains, Helicobacter pylori(H. pylori) HD 496 In order to confirm whether the antimicrobial agent as well as the strain is bacteriocin, 10 μL of polyperthinecin SCD prepared by the above-described method was prepared as a test sphere, and the proteinase K ( Sigma Chemical Co., St. Louis, USA) was mixed to a concentration of 1 mg / mL, 10 μL of the solution reacted for 3 hours at 37 ℃ in one control, 10 μL of the same concentration of proteinase K Another control was prepared.

Helicobacter pylori in the experimental and two control groups(H. pylori) Strain (KCTC 2948), Helicobacter pylori(H. pylori) DSM 4867 Strains, Helicobacter pylori(H. pylori) DSM 9691 Strains, Helicobacter pylori(H. pylori) HD 223 Strains, Helicobacter pylori(H. pylori) HD 489 Strains, Helicobacter pylori(H. pylori) HD 496 The strains were spotted on a plate on which they were plated and incubated at 37 ° C. for 10 hours.

As a result, Helicobacter pylori only in each experimental section(H. pylori) Strain (KCTC 2948), Helicobacter pylori(H. pylori) DSM 4867 Strains, Helicobacter pylori(H. pylori) DSM 9691 Strains, Helicobacter pylori(H. pylori) HD 223 Strains, Helicobacter pylori(H. pylori) HD 489 Strains, Helicobacter pylori(H. pylori) HD 496 Strain inhibitory effects were confirmed, and inhibitory effects were not seen in the polyfermentincin SCD controls and proteinase K controls treated with proteinase K. Figure 2 is a Helicobacter pylori(H. pylori) It is a photograph showing the inhibitory effect on the strain (KCTC 2948). From the above results, it was confirmed that the polyfermentin SCD had an antibacterial action and that the substance was bacteriocin.

Experimental Example 3: Helicobacter pylori according to physiological characteristics H. pylori ) Investigation of growth inhibition (KCTC 2948)

The supernatant recovered in Example 1 was partially purified with 75% ammonium sulfate, and the purified polypermanticin SCD was diluted to 400 AU / mL and 1,000 AU / mL in 0.1 M phosphoric acid solution, respectively. Chic Helicobacter Pylori(H. pylori) For strain (KCTC 2948) Antimicrobial effect was measured. As a result, when treated with polypermanticin SCD, the number of viable cells decreased with reaction time in proportion to the concentration. However, if the polymentincin SCD was not treated, the viable cell count was almost unchanged.⁶CFU / mL was maintained.

Also, Helicobacter pylori in logarithmic multipliers(H. pylori) Treatment of strains (KCTC 2948) with 400 AU / mL of polyfermentin SCD resulted in a decrease of 4.4 log CFU / mL after 10 hours, while the Helicobacter pylori at stationary phase(H. pylori) Strains (KCTC 2948) reduced 3.77 log CFU / mL. This results in the polypermanticin SCD being the Helicobacter pylori of the logarithmic multiplier.H. pylori) The bacterium (KCTC 2948) could be judged to have a more effective antimicrobial effect (Fig. 3, 4). That is, Helicobacter pylori of logarithmic multiplier(H. pylori)The bacterium (KCTC 2948) was more sensitively inhibited against polypermanticin SCD than the stationary cells.

Experimental Example 4: Helicobacter pylori (H. pylori) Electron Microscope Observation (KCTC 2948)

Helicobacter pylori (H. pylori) For morphological observation by the antimicrobial effect of polyperthinecin SCD against the bacteria (KCTC 2948) was observed by scanning electron microscope (Scanning electron microscope, S-4200 FE-SEM, Hitachi, Japan), the method was as follows. Helicobacter pylori of logarithmic multiplier, stopH. pylori) The bacteria (KCTC 2948) were centrifuged at 8000 x g for 20 minutes to obtain pure cells, followed by immobilization with 25% glutaraldehyde for 12 hours and centrifugation. For bacteriocin addition, 400 AU / mL of polyfermentin SCD was added for 10 hours, followed by centrifugation to immobilize with 25% glutaraldehyde for 12 hours, washed three times with 0.1 M phosphate solution. Dry in 60-100% ethanol. The dried sample was fixed on a slide glass and then re-dried. A photograph was obtained by ion-spatter coating in a reduced pressure evaporator for 30 minutes before observation.

Helicobacter pylori without polypersistin SCD (H. pylori) The bacterium (KCTC 2948) appeared in the form of a bacillary (FIGS. 5, 7), but the Helicobacter pylori of the logarithmic growth stage (H. pylori) (KCTC 2948) was observed shrinkage phenomenon as a result of treatment with polymentincin SCD (Fig. 6). This phenomenon has been reported to occur due to the formation of pores in the cell membrane, disrupting the proton motive force, inhibiting amino acid migration, and releasing amino acids accumulated in the cells out of the cells (Abee, T. Pore-forming bacteriocins of gram-positive bacteria and self-protection mechanisms of prodction organisms.FEMS Microbiol. Lett. 129: 1-10 (1995)).

While the Helicobacter pylori in stopH. pylori) The bacterium (KCTC 2948) was found to be converted to coccoid (spherical) form from bacillary (spinning) form as a result of treatment with polymentincin SCD (Fig. 8). Helicobacter pylori (H. pylori) The transition from the rod form to the spherical form of KCTC 2948 Helicobacter pylori (H. pylori) Fungi (KCTC 2948) The surrounding environment is said to occur in an unsuitable state. In particular, it is known that the conversion to spherical form is accelerated by long-term incubation, exposure to air, influence on temperature, depletion of media components, and contact with antibiotics (Lee, H.-S. and Choe, T.- B. Morphologic conversion of Helicobacter pylori from bacillary to coccoid by environmental stress.Kor. J. Microbiol. Biotechonl. 25: 240-247 (1997)).

Preferred embodiments of the present invention are as follows.

Example 1 Preparation of Toothpaste Including Polyfermentin SCD

65 g of calcium carbonate, 25 g of magnesium carbonate, 5 g of borax and 5 g of polymentycin were put in a mortar and mixed well. Add 20 g of this to the evaporating dish and add 0.1 mL of peppermint oil and mix well. This is called tooth powder. Meanwhile, 10 mL of water was added to 3 mL of glycerin, and the mixture was diluted lightly. Diluted glycerin dilutions were added little by little in the powder to clear. The toothpaste prepared as mentioned above was put in the bottle with wide mouth, and it stopped and preserved.

Example 2: Preparation of Yoghurt Comprising Polypermanticin SCD

1000 mL of Seoul milk delivered was poured into fermentation vessels and inoculated with Lactobacillus bulgaricus , which is a lactic acid bacterium. Thereafter, the temperature was 40 ° C. and the fermentation was carried out for 10 to 12 hours. When the fermentation is over and the sour taste is appropriate, some of the tangled yogurt is removed from the fermentation vessel and cooled in a refrigerator. Yoghurt containing polyfermentin SCD was added to 5 g of polyfermentin SCD.

Example 3: Bacillus polyfermentus ( Bacillus polyfermenticus ) Preparation of Gastrointestinal Disease Prevention and Treatment Including SCD (KCCM 10104) or Polyfermentin SCD

Conventional gastrointestinal prophylaxis and treatment A gastrointestinal drug was prepared by mixing 2 g of polyfermentin SCD with respect to 100 g of carrier.

In addition, the gastrointestinal drug containing B. polyfermenticus SCD bacteria (KCCM 10104) is lyophilized by adding B. polyfermenticus SCD bacteria (KCCM 10104) to a conventional vacuum freeze dryer. Prepared by encapsulation.

Example 4 Preparation of Chewing Gum Containing Polyfermentin SCD

120 g of gum base, 315 g of sugar, 35 g of starch syrup, 35 g of glucose, 1.6 g of emollient, 2.7 g of emulsifier, 0.4 g of chlorophyll, 20 g of paracinite, 3 g of xylitol, 5 g of polymentisin at 60-70 degreeC The mixture was put in a preheated mixer and mixed, and 7 g of the flavor was added while maintaining the mixer internal temperature at 55 ° C. and mixed for about 20 to 30 minutes. Then, the formed gum was rolled, aged for 24 hours, cut and packaged to prepare a gum containing polymentincin SCD.

Example 5 Preparation of Polyfermentin SCD-Containing Beverages

After dissolving 0.5 g of polypermentin SCD in 20 g of purified water, 1 g of glucose, 2 g of fructose, 2 g of sugar, 0.05 g of citric acid, 0.02 g of malic acid, 0.01 g of sodium citrate, 0.02 g of vitamin C, 30 g of apple juice, lemon After adding 1 g of juice and 0.5 g of honey, stirring it to completely dissolve it, passing it through a 1 / ㎛ filtration filter, adding purified water to final 100 ml, and filling into a can or bottle and sterilizing at 90 ° C. for 5 to 10 minutes. Polyfermentin SCD-containing beverages were prepared.

Example 6: Bacillus polyfermentus ( B. polyfermenticus ) Preparation of Processed Dried Yeast Food Containing SCD

B. polyfermenticus SCD 14 g supplied in apo form was added to 80 g of beer yeast, 5 g of sorbitol, and 1 g of seaweed calcium to prepare a dried yeast processed food.

Example 7: Bacillus polyfermentus ( B. polyfermenticus Manufacture of Wires Including SCD

15 g of glutinous rice, 20 g of barley rice, 15 g of brown rice, 10 g of brown rice, 5 g of white beans, 1 g of crude, 1 g of wheat, wheat 1 g, buckwheat 1 g, corn 5 g, chestnut 1 g, pine nuts 1 g, walnut 1 g, carrot 1 g, spinach 1 g, hemp 1 g, lotus root 1 g, safflower seed 1 g, roundle 1 g, kelp 2 g, Haitai 1 g, mulberry leaf 3 g, silkworm 3 g, cordyceps 3 g Wire was prepared by adding 5 g of B. polyfermenticus SCD supplied in apo form.

As can be seen in the detailed description of the invention, the composition containing the probiotic Bacillus polyfermenticus SCD bacteria (KCCM 10104) or polypermancincin SCD produced therefrom is a Helicobacter pylori ( Helicobacter pylori ) is a very useful invention that can be used for foods, medicines, etc. because it has an excellent antimicrobial activity against bacteria.

Claims (2)

A composition for preventing and treating gastrointestinal diseases containing Bacillus polyfermenticus SCD strain (KCCM 10104) having an antimicrobial effect against Helicobacter pylori strains as an active ingredient. A bacteriocin produced by Bacillus polyfermenticus SCD strain (KCCM 10104), which has an antimicrobial effect against Helicobacter pylori strains, for the prevention and treatment of gastrointestinal diseases containing polyfermentin SCD as an active ingredient. Composition.