JP6471210B2 - Bacillus subtilis LRCC1002 strain having enzyme-producing ability, bile resistance, and antibacterial and thrombolytic ability against Helicobacter pylori - Google Patents

Description

  The present invention relates to a Bacillus subtilis LRCC1002 strain, and more particularly, to a Bacillus subtilis LRCC1002 strain having enzyme-producing ability, bile resistance, and antibacterial and thrombolytic ability against Helicobacter pylori. It is.

  Bacillus subtilis is also called a Bacillus subtilis and is a bacterium belonging to the genus Bacillus that mainly inhabits the soil, and forms Gram-positive spores. Such Bacillus subtilis is aerobic, moves with periflagellate flagella, is a heat-resistant fungus, metabolizes various sugars such as glucose or starch as an energy source, It is a microorganism that can be used as a fermenting strain during the production of fermented foods such as Chang, and can decompose organic matter.

  On the other hand, Bacillus subtilis is known as a microorganism that degrades pectin and polysaccharides in plant tissue, and also produces enzymes that can degrade various substances such as proteins and starches. It is a microorganism that can Bacillus subtilis, which produces antibacterial substances that can control pathogenic bacteria, is also known by research and has various biochemical and physical properties, and has been proven to be safe against bacteria. In fact, it is widely used as a pharmaceutical preparation. In addition, Koreans are not only deeply related as fermented microorganisms of Chung Kuk Chang, but are also used as many microbial preparations.

  On the other hand, Korean Journal of Microbiology. 2012, Vol. 48, no. 3, pp. 220-224. Discloses a Bacillus subtilis strain having a biogenic amine resolution isolated from traditional soy sauces.

  In addition, since Bacillus subtilis is known to possess thrombolytic ability, it can be expected to have a preventive effect on adult diseases caused by thrombosis, and various useful physiologically active functions have been reported. Attention is increasing as a sexual health food.

  Thrombus is a polymer formed by converting fibrinogen into fibrin by thrombin, and thrombosis, a disease in which thrombus is formed in the cerebral blood vessel or cardiovascular, is fatal. Therefore, in order to dissolve the thrombus, urokinase, streptokinase, tissue type plasminogen activator (Tissue Type Plasminogen Activator tPA) and the like have been used. However, such a preparation has a problem that it has a short half-life in blood, has side effects such as bleeding, and is expensive. Therefore, research to find a thrombolytic substance that can solve such problems has been actively conducted.

  However, no strain has been found that has a high ability to regulate the intestine (enzyme secretion ability, bile resistance, intestinal mucosa adhesion) and thrombolytic ability, and has an antibacterial action against Helicobacter pylori, a pathogenic microorganism. Therefore, there has been a need for research on novel microorganisms that can be used in the production of high value-added foods having these simultaneously.

Kim Young-san et al., Korean Journal of Microbiology. 2012, Vol. 48, no. 3, pp. 220-224.

  An object of the present invention is to provide a Bacillus subtilis LRCC1002 strain having enzyme-producing ability, bile resistance, and antibacterial and thrombolytic ability against Helicobacter pylori.

Bacillus subtilis according to one aspect of the present invention (Bacillus subtilis) LRCC1002 strain (Accession Number: KCCM11880P) has excellent enzyme-producing capability, bile resistance, and have a superior thrombolytic capability and having an antibacterial ability against Helicobacter pylori The enzyme is an amylase or a protease.
A health food for preventing or ameliorating thrombosis according to another aspect of the present invention includes one or both of Bacillus subtilis LRCC1002 strain (Accession No .: KCCM11880P) or a culture solution of the strain as an active ingredient. be able to.
The health food for improving intestinal function according to still another aspect of the present invention may contain one or both of Bacillus subtilis LRCC1002 strain (Accession No .: KCCM11880P) and a culture solution of the strain as an active ingredient. it can.

  The Bacillus subtilis LRCC1002 strain according to the present invention has excellent enzyme-producing ability, excellent bile resistance, intestinal mucosal adhesion, and excellent antibacterial activity against Helicobacter pylori. It is applicable to highly functional foods such as food.

  In addition, the Bacillus subtilis LRCC1002 strain according to the present invention has an excellent thrombolytic ability, and thus can be applied to health foods for prevention or improvement of thrombosis. Furthermore, such health foods are caused by blood clots. It can be expected to have a preventive effect on lifestyle-related diseases.

It is a figure which shows the enzyme secretion ability confirmation result of Bacillus subtilis LRCC1002 strain by this invention. It is a figure which shows the intestinal adhesion confirmation result of Bacillus subtilis LRCC1002 strain by this invention. It is a figure which shows the antibacterial activity measurement result of Bacillus subtilis LRCC1002 strain by this invention. It is a figure which shows the thrombolytic ability measurement result of Bacillus subtilis LRCC1002 strain by this invention.

  While the invention is susceptible to various modifications, and may involve various embodiments, specific embodiments are illustrated in the drawings and will be described in detail below. However, this should not be construed as limiting the invention to the specific embodiments, but should be construed as including all transformations, equivalents and alternatives falling within the spirit and scope of the invention. In the description of the present invention, when it is determined that there is a possibility that a specific description of a related known technique may obscure the gist of the present invention, the detailed description thereof is omitted.

  According to one aspect of the present invention, Bacillus subtilis LRCC1002 strain (Accession No .: KCCM11880P, hereinafter, description of Accession No. KCCM11880P of LRCC1002 strain is omitted) has an enzyme production ability, bile resistance, and antibacterial activity against Helicobacter pylori And has thrombolytic ability.

  According to the prior art, there is no strain that has high anti-intestinal function (enzyme secretion ability, bile resistance, intestinal mucosal adhesion) and thrombolytic ability, and has antibacterial activity against Helicobacter pylori, a pathogenic microorganism. There was a point.

  Therefore, the present inventors have studied and identified strains having excellent enzyme production ability, excellent bile resistance, intestinal mucosal adhesion, and excellent antibacterial activity against Helicobacter pylori, and completed the present invention. It was.

  Hereinafter, the Bacillus subtilis LRCC1002 strain which is a specific example of the invention will be described in more detail.

  The Bacillus subtilis LRCC1002 strain was deposited with the Korean Culture Center of Microorganisms on August 12, 2016 (Bacillus subtilis LRCC1002 strain).

  The strain can simultaneously have enzyme-producing ability, bile resistance, and antibacterial ability and thrombolytic ability against Helicobacter pylori.

  The enzyme is a digestive enzyme, and examples thereof include amylase, cellulase, protease, and lipase, and amylase or protease is preferable.

  According to one aspect of the present invention, it is possible to provide a health food for improving intestinal function comprising the Bacillus subtilis LRCC1002 strain according to the present invention or a culture solution of the strain as an active ingredient.

  Meanwhile, it is possible to provide a health food for preventing or ameliorating thrombosis comprising the Bacillus subtilis LRCC1002 strain according to the present invention or a culture solution of the strain as an active ingredient.

  Specifically, for the purpose of improving bowel function or preventing or improving thrombosis, the strain dry powder or strain culture solution of the present invention can 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 can be added in an amount of 0.01 to 1.0% by weight of the total weight of the food, and the health drink composition is 0.1% based on 1000 ml. It can be added at a rate of ˜10.0 g.

  As long as the health functional beverage composition of the present invention includes the strain dry powder or strain culture solution as an essential component, the other components are not particularly limited, and various sweeteners or natural carbohydrates as in a normal beverage Etc. can be blended as additional components. Examples of natural carbohydrates described above are monosaccharides such as glucose, fructose, etc .; disaccharides such as maltose, sucrose, etc .; and polysaccharides such as common sugars such as dextrin, cyclodextrin, and xylitol, sorbitol, erythritol, etc. It is a sugar alcohol. In addition to those described above, natural sweeteners (such as thaumatin, stevia extract, rebaudioside A, and glycyrrhizin) and synthetic sweeteners (such as saccharin and aspartame) can be used as sweeteners.

  In addition, the strain dry powder or strain culture solution of the present invention includes various nutrients, vitamins, minerals (electrolytes), fragrances such as synthetic fragrances and natural fragrances, colorants and fillers (cheese, chocolate, etc.), pectinic acid And a salt thereof, alginic acid and a salt thereof, an organic acid, a protective colloid thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used in carbonated beverages, and the like. In addition, the dried strain or strain culture solution of the present invention can be blended with fruit juice for producing natural fruit juice, fruit juice drink, and vegetable drink. Such components can be used alone or in combination.

  In the health food for improving bowel function or preventing or improving thrombosis according to one aspect of the present invention, the food is a dairy product, fermented milk, drink, meat, sausage, bread, chocolate, candy, snacks, confectionery, It is selected from the group consisting of pizza, ramen, gums, ice creams, soups, beverages, alcoholic beverages and vitamin complexes, but is not limited thereto.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, these examples are only for illustrating the present invention, and the scope of the present invention should not be construed as being limited by these examples.

Test Example 1: Identification of isolated strains and isolation of strains excellent in enzyme secretion ability After separating strains from traditional fermented foods, nine strains having good growth and vivid colony shapes were selected. In order to confirm whether the selected strain is a microorganism that can be used as a food raw material, an analysis was conducted by requesting Biofact Co., Ltd. (BIOFACT Co. Ltd., Korea) to identify the strain based on the 16s ribosomal DNA base sequence. The result was obtained. As a result of the identification, all were microorganisms belonging to the genus Bacillus, and among these, four edible microorganisms were all Bacillus subtilis. As a result of confirming the extracellular enzyme secretion ability, amylase was detected in Bacillus subtilis LRCC1002 strain. (Amylase) and protease (Protease) enzyme activities were the best (FIG. 1). As a control group for the experiment, Bacillus subtilis KCCM12512 distributed from Korea Cell Line Bank was used.

Test Example 2: Evaluation of Resistance to Artificial Gastric Fluid and Bile Acid In order to evaluate the resistance of the isolated strain to artificial gastric juice, the strain was pre-cultured in Tryptic Soy Broth (Difco, Detroit, USA) and then centrifuged. Only the cells were collected after separation. The collected cells were washed with sterilized 0.5% NaCl and dispersed in 0.5% NaCl at the final stage of washing to prepare a strain suspension. The resistance of the artificial gastric juice was measured by inoculating the artificial gastric juice prepared by adding 0.04% (w / v) pepsin to 0.5% NaCl (pH 2.5) and reacting for 3 hours. The plate was smeared on an agar medium and the viable cell count was measured. The number of viable bacteria in the control group was measured by inoculating a plate on an agar medium immediately after inoculating the artificial gastric juice. The resistance of the isolated strain to artificial gastric juice was calculated by the ratio of the initial viable cell count and the viable cell count after the reaction for 3 hours and expressed in Log%.

  In order to evaluate resistance to bile acids, isolates should be pre-cultured in a liquid medium, centrifuged to collect the cells, washed with sterile water, and dispersed in sterile water at the final stage of washing. A strain suspension was prepared. The bile acid resistance was measured by inoculating the strain suspension in a liquid medium in which 0.3% bile salt (Bile Ox Gall salt) was added to 0.5% NaCl (pH 7.0), and allowed to react for 3 hours. Plated agar (Agar) medium and counted the number of viable bacteria. The number of viable bacteria in the control group was measured by inoculating a bile acid solution and immediately smearing it on an agar medium. The resistance of the isolated strain to bile acids was calculated by the ratio of the initial viable cell count and the viable cell count after the reaction for 3 hours and expressed in Log%.

  In order to apply probiotic microorganisms to products, they must survive in low pH conditions of the gastrointestinal tract and in environments where there is bile rich in digestive enzymes. Therefore, the selected Bacillus subtilis was searched for a strain having high resistance to artificial gastric juice and bile acids, and the results are shown in Table 1 below. As a control group for the experiment, Bacillus subtilis KCCM12512, Bacillus subtilis KCCM12027, and Bacillus subtilis KCCM12511 were used as described above. As a result of evaluating the resistance to artificial gastric juice, Bacillus subtilis LRCC1002 showed the highest survival rate of Log 67.6%, and the resistance to bile acids produced using 0.3% bile salts is all four strains. The log was 99% or more, and there was no significant difference.

Test Example 3: Intestinal Adhesion Evaluation Caco2 Cell used in this experiment was an intestinal epithelial cell derived from human colorectal cancer, and was distributed from ATCC (American Type Culture Collection). Caco2 Cell (HTB-37, ATCC) is Eagle's supplemented with 10% fetal bovine serum (FBS; Fetal bovine serum) (Welgene co., Daegu, Korea), 1% penicillin / streptomycin (Gibco, NY, USA). Using a medium of Medium Essential Medium (EMEM, Welgene co., Daegu, Korea), the cells were cultured at 37 ° C. in an incubator supplied with 5% CO ₂ . Cells in which the cells formed a monolayer were subcultured and used for experiments with medium changes every other day. For the intestinal adhesion experiment, Caco2 Cell was collected in a 24 Well Plate at a concentration of 1 × 10 ⁵ cells / mL per well and cultured at 37 ° C. in a 5% CO ₂ incubator for 24 hours. The isolated strain was inoculated into Trypsin Soy Medium and pre-cultured at 37 ° C. for 24 hours, and then the strain was recovered and washed with 0.1 M phosphate buffered saline (PBS). After washing twice, FBS and penicillin / streptomycin were dispersed in EMEM medium without addition. After inoculating 500 μl of the strain culture solution separated into 24 Well Plate in which Caco2 Cell was cultured, and culturing at 37 ° C. under 5% CO ₂ condition for 2 hours, 0.1M phosphorus was removed to remove the non-adhered strain. Washed with acid buffered saline. This was collected and diluted, and then plated on a Tryptic Soy Agar medium and cultured at 37 ° C. for 24 hours to confirm the viable cell count. Intestinal adherence (Log%) was calculated by the ratio of the number of bacteria that were treated and adhered to Caco2 Cell to the initial number of inoculated bacteria.

  In order to evaluate whether it can normally adhere to the intestinal epithelial cells and survive when it passes through the digestive organs and reaches the intestine which is the final target site, KCCM12512, KCCM12027, The results of evaluating the intestinal adhesion of KCCM12511 are shown in FIG. Bacillus subtilis LRCC1002 and Bacillus subtilis KCCM12511 showed high intestinal adherence with Log 80.3% and Log 78.2%, respectively.

Test Example 4: Evaluation of antibacterial activity against Helicobacter pylori bacteria Helicobacter pylori ATCC 40449 was transferred from the Korean Microbiology Center of Microorganisms (KCCM). Helicobacter pylori was inoculated into a blood agar plate (BAP; Blood Agar Plate) containing 10% sheep blood (Hanil Comed, South Korea) and enriched with a 10% CO ₂ incubator. Helicobacter pylori cultured for 3 to 4 days was suspended in 0.85% physiological saline and prepared. The experimental medium was prepared by dividing into Bottom Agar (containing Agar 1.2%) and Top Agar (containing Agar 0.3%). BBL ^™ Brucella Broth (Becton, Dickinson and Company, USA) was sterilized, cooled to 40 ° C., and fetal bovine serum (FBS; Fetal Bovine Serum) was added in a total volume of 10%. Bottom Agar (10 mL) was first collected in a Petri dish (Petri Dish) and solidified at room temperature for about 2 hours. Sterilized Top Agar was cooled to 40 ° C., inoculated with fetal bovine serum (FBS) and the prepared Helicobacter pylori in a volume of 10% of the total, and 3 mL was stocked on solidified Bottom Agar and solidified for 30 minutes. A 10 mm paper disk (Toyo Roshi Kaisha, Ltd, Japan) was then placed on the plate. The culture solution of LRCC 1002 strain was centrifuged (8,000 rpm, 10 minutes) to separate the supernatant and the cell fluid, and 30 μL of each sample was aliquoted on a paper disk and cultured at 37 ° C. and 10% CO _2. The cells were cultured in a vessel for 3 to 4 days, and the presence or absence of inhibition circle formation was observed (FIG. 3). The Bacillus subtilis LRCC 1002 strain isolated from traditional fermented foods is judged to have the property of inhibiting harmful bacteria applicable as probiotics.

Test Example 5: Evaluation of thrombolytic activity Astrup, T .; and Mullertz method [The Fibrin Plate Method for Estimating Fibrinolytic Activity, Archivesbiochem. Biophysics, 1952] was modified to dispense a fibrinogen solution (0.17M borate buffer solution, 0.3% fibrinogen 10 mL) into a Petri dish, and this was added to a thrombin solution (0.17M borate buffer solution). , 500 U / mL thrombin) was added, and the mixture was stirred for 1 minute and solidified for 1 hour or more. A paper disk was placed on the solidified fibrin plate, 20 μL of a measurement sample was separated, and allowed to stand at room temperature for 3 hours, and the diameter of a transparent portion formed around the paper disk was measured. In the control group, plasmin (Plasmin, 1.0 units / mL), which is a purified thrombolytic enzyme, was used, and the thrombolytic ability was calculated by the following formula. The results of measuring the thrombolytic enzyme activity of the isolated strain LRCC1002 and KCCM12512, KCCM12027, and KCCM12511 as a comparison group are as shown in FIG.

* Fibrinolytic activity (%) = (Sample Clear Zone / Natkinase Clear Zone) × 100
As described above, the Bacillus subtilis LRCC1002 strain according to the present invention has an excellent enzyme-producing ability, an excellent bile resistance, intestinal mucoadhesiveness, and an excellent antibacterial ability against Helicobacter pylori. It was found that the present invention can be applied to highly functional foods such as health foods for improving intestinal function. Furthermore, since the Bacillus subtilis LRCC1002 strain according to the present invention has an excellent thrombolytic ability, it can be applied to a health food for preventing or ameliorating thrombosis. As a result, such a health food is generated by thrombus. It can be expected to have a preventive effect on lifestyle-related diseases.

  Although specific portions of the present invention have been described in detail above, such specific descriptions are merely preferred embodiments for those having ordinary knowledge in the art, which limit the scope of the present invention. It will be clear that this is not done. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Depositary institution: Korea Microbial Preservation Center Accession number: KCCM11880P
Date of deposit: 2016.08.12.

Claims (3)

Ability to produce an enzyme, bile resistance and antibacterial ability against Helicobacter pylori and thrombolytic ability possess,
The enzyme is Bacillus subtilis LRCC1002 strain (Accession No. KCCM11880P) which is amylase or protease. Ability to produce an enzyme, bile resistance, and have antibacterial ability and thrombolytic ability against Helicobacter pylori, wherein the enzyme is Bacillus subtilis amylase or protease (Bacillus subtilis) LRCC1002 strain (Accession No. KCCM11880P) or the strain A health food for preventing or ameliorating thrombosis comprising any one or both of the above culture solutions as an active ingredient. Ability to produce an enzyme, bile resistance, and have antibacterial ability and thrombolytic ability against Helicobacter pylori, wherein the enzyme is Bacillus subtilis amylase or protease (Bacillus subtilis) LRCC1002 strain (Accession No. KCCM11880P) or the strain A health food for improving intestinal function, comprising any one or both of the above culture solutions as an active ingredient.