KR20160120569A - - Bacillus amyloliquefaciens strain having protease activity and -glucosidase inhibition activity and uses thereof - Google Patents

Abstract

The present invention provides a Bacillus amyloliquefaciens strain having a protein decomposing activity and an -glucosidase inhibiting activity; a probiotic composition a composition for producing protease or a composition for inhibiting an alpha glucosidase activity containing the strain or a culture liquid thereof as an active ingredient; a food containing the composition for producing protease; a health functional food for preventing or alleviating diabetes, a pharmaceutical composition for preventing or treating diabetes, and a composition for dissolving blood clots containing the strain or a culturing liquid thereof as an active ingredient.

Description

Bacillus amyloliquefaciens strain having proteolytic activity and? -Glucosidase inhibitory activity and use thereof <br> Bacillus amyloliquefaciens strain having protease activity and α-glucosidase inhibition activity and uses thereof

The present invention relates to a Bacillus amyloliquefaciens strain having a proteolytic activity and an? -Glucosidase inhibitory activity and a use thereof, and more particularly to a Bacillus amyloliquefaciens strain having proteolytic activity and? -Glucosidase inhibitory activity Bacillus amyloliquefaciens strain having the above-mentioned strain or a culture solution thereof as a active ingredient, a composition for producing protease or a composition for inhibiting alpha-glucosidase activity, A food containing the composition for producing protease, a health functional food for preventing or ameliorating diabetes containing the strain or a culture thereof as an active ingredient, a pharmaceutical composition for preventing or treating diabetes, and a composition for thrombolysis.

In recent years, modern people have been increasing their interest in health as well as improving their living standards. Microbiological food supplements called probiotics have been widely used for the purpose of promoting health, forming new natural microflora in the body, or activating the immune system. In general, microorganisms used as probiotics are resistant to gastrointestinal stomach, gall bladder bile, and various digestive enzymes secreted from the small intestine. They must have the ability to grow and colonize the colon and rectum. At present, probiotics include Lactobacillus acidophilus), Bacillus poly flops mentee kusu (Bacillus polyfermenticus , and Bacillus subtilis . Among them, Bacillus spp. Is an industrially important species and has been used for many years in food, pharmaceuticals and various fermentation industries. Bacillus Genes in the genus can produce various digestive enzymes or peptides with physiological activity. Typical digestive enzymes are protease and xylanase. As a digestive enzyme, protease has characteristics of casein degradation ability, thrombolytic ability and gelatin degradation ability. In particular, thrombolytic enzyme can dissolve thrombosis composed of fibrin and use microorganism of Bacillus that secretes thrombolytic enzyme And thus can be said to be suitable as a functional food for preventing cardiovascular diseases. Recently, various studies have been conducted to apply microorganisms isolated from various domestic fermented foods such as kimchi, meju, soy sauce, soybean paste, etc. as probiotics.

Protease is an enzyme that hydrolyzes peptide bonds between amino acids in other proteins and accounts for 60% of the worldwide sales of industrial enzymes. In addition to basic research fields such as protein structure and function prediction, it is used in various industrial fields related to the production of seasonings, softening of food, prevention of liquor turbidity, cheese aging, baking and other food industry, pharmaceutical industry such as digestive agent, detergent, The demand is increasing. Proteases are produced in all organisms and are produced in large amounts by microbial cells such as bacteria, fungi and yeast, but Bacillus sp. Strains are used for industrial production. The protease produced by microbial cells can be stably produced through mass culture of cells and can be improved in productivity and economical efficiency by optimizing the process, so that it is very useful in industrial aspects and is constantly developed and optimized according to its use and function .

The α-glucosidase in the small intestine is an essential enzyme for carbohydrate digestion, which enables the absorption of carbohydrates contained in diets by converting them into monosaccharides. The α-glucosidase inhibitor inhibits the absorption of carbohydrates by ingesting and immediately discharges the resulting carbohydrate, leading to a decrease in postprandial blood glucose. Thus, the α-glucosidase inhibitor is interested in the potential for development of a metabolic disease such as type II insulin-independent diabetes mellitus, obesity and hyperlipemia. The production of inhibitors by microorganisms is considered to be the most effective strategy because of the difficulties of mass production of natural materials.

Korean Patent Laid-Open Publication No. 2014-0050412 discloses a soybean fermented product having enhanced antidiabetic effect including Bacillus amyloliquefaciens subspecific strains F2189 and a process for producing the same, No. 2013-0033026 discloses a novel strain of Bacillus amyloliquefaciens BY04 having improved protease production ability and a method for producing the protease using the same. However, as in the present invention, the 'proteolytic activity and α-glucosidase inhibitory activity Bacillus amyloliquefaciens strain and its use 'have not been disclosed at all.

The present invention has been made in view of the above-mentioned needs. In the present invention, in order to select a new probiotic, two kinds of Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens) amyloliquefaciens strains were isolated. The two strains showed resistance to artificial gastric juice and bile acid, and adhesion to intestinal epithelial cells. In particular, the strains showed excellent α-glucosidase inhibitory activity and proteolytic enzyme secretion ability Respectively.

The known Bacillus amyloliquefaciens strain is known to secrete proteases or have an? -Glucosidase inhibitory activity. However, as in the present invention, protease-releasing activity and? -Glucosidase inhibitory activity The present invention has been accomplished by confirming the potential usefulness as a probiotic industrial microorganism by virtue of the fact that no strain has been disclosed at all.

In order to solve the above problems, the present invention provides a Bacillus amyloliquefaciens strain having proteolytic activity and? -Glucosidase inhibitory activity.

The present invention also provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.

The present invention also provides a composition for producing a protease comprising the strain or a culture thereof as an active ingredient.

The present invention also provides a food containing the composition for producing the protease.

The present invention also provides a composition for inhibiting alpha-glucosidase activity comprising the strain or a culture solution thereof as an active ingredient.

The present invention also provides a health functional food for preventing or ameliorating diabetes containing the strain or a culture thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating diabetes comprising the strain or a culture thereof as an active ingredient.

Further, the present invention provides a composition for fibrinolysis comprising the strain or a culture solution thereof as an effective ingredient.

According to the present invention, two kinds of Bacillus amyloliquefaciens strains having proteolytic activity and? -Glucosidase inhibitory activity can be obtained, and the strain of the present invention and its culture medium A related composition including a probiotic composition, a composition for producing protease, a composition for inhibiting alpha glucosidase activity, a health functional food for preventing or improving diabetes, and a pharmaceutical composition for preventing or treating diabetes, and the like Can be very useful.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the results of analysis of the growth of Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 isolated in the present invention in a nutrient broth culture.
FIG. 2 shows the results of analysis of protease activity by incubation time after culture of Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 strains isolated in the present invention in a nutrient broth medium.
FIG. 3 shows the results of analysis of the degree of growth of the Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 strains isolated according to the present invention.
FIG. 4 shows the results of analysis of the growth of Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 isolated according to the present invention.
FIG. 5 shows the results of analysis of the growth of Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 isolated according to the present invention.
FIG. 6 is a result of in vitro analysis of the binding ability of Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 strains isolated from the present invention to human endothelial cells (HT-29).
FIG. 7 shows the results of analysis of α-glucosidase inhibitory activity of the Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 strains isolated in the present invention.
FIG. 8 shows the results of comparing the fibrinogen resolution of the Bacillus amyloliquefaciens CDD5, CPD4 and CGD3 strains isolated in the present invention.

In order to achieve the above object, the present invention has a proteolytic activity and α- glucosidase (α-glucosidase) inhibitory activity Bacillus amyl Lowry Quebec Pacific Enschede (Bacillus amyloliquefaciens ).

In the strain according to an embodiment of the present invention, the Bacillus amyloliquefaciens strain may be a strain having adhesion, acid resistance, and bile resistance to intestinal epithelial cells, but is not limited thereto.

According to the present invention it was isolated from a strain paste, of which were isolated and the proteolytic activity α- glucosidase (α-glucosidase) excellent inhibitory activity against the strain, this Bacillus amyl Lowry Quebec Pacific Enschede (Bacillus amyloliquefaciens ). The Bacillus amyloliquefaciens strain may be Bacillus amyloliquefaciens CGD3 strain (KACC92048P) or Bacillus amyloliquefaciens CPD4 strain (KACC92049P) and deposited on March 19, 2015 at the Institute of Agricultural Biotechnology . The known Bacillus amyloliquefaciens strain is known to secrete proteases or have an? -Glucosidase inhibitory activity. However, as in the present invention, protease-releasing activity and? -Glucosidase inhibitory activity It was the first time that a strain was found.

The present invention also provides a probiotic composition comprising the strain or a culture thereof as an active ingredient.

Each of the Bacillus amyloliquefaciens CGD3 strain, Bacillus amyloliquefaciens CPD4 strain or the culture thereof of the present invention is excellent in adherent cell adhesion, acid resistance and biliary cholesterol, and has a proteolytic activity and α-glucosidase (α -glucosidase inhibitory activity, all of the above-mentioned conditions to be provided as probiotics are satisfactorily satisfied, and therefore, they can be effectively used as a probiotic composition.

The composition of the present invention may be prepared according to a conventional method for producing a prod- uct composition, and may be in the form of a lyophilized or encapsulated culture or a suspension or a dry powder. The effective amount of the above-mentioned Bacillus amyloliquefaciens CGD3 strain, Bacillus amyloliquefaciens CPD4 strain, or culture thereof as a main component may be added to an effective amount of one or more pharmaceutically acceptable conventional carriers or one or more kinds of additives May be selected to prepare a composition of a conventional formulation.

The carrier may be selected from one or more selected from among diluents, lubricants, binders, disintegrants, sweeteners, stabilizers and preservatives. Examples of the additives include one or more of flavorings, vitamins and antioxidants Can be used.

In the present invention, the carrier and the additive may be any pharmaceutically acceptable ones. Specific examples of the diluent include lactose monohydrate, trehalose, corn starch, soybean oil, Microcrystalline cellulose or mannitol is preferred and the lubricant is preferably magnesium stearate or talc and the binder may be polyvinylpyrrolidone (PVP) or polyvinylpyrrolidone And hydroxypropylcellulose (HPC). The disintegrant may be selected from carboxymethylcellulose calcium (Ca-CMC), sodium starch glycolate, polacrylin potassium, or cross-linked polyvinylpyrrolidone And the sweetener is selected from white sugar, fructose, sorbitol or aspartame. Examples of the stabilizer include sodium carboxymethylcellulose sodium (Na-CMC), beta-cyclodextrin (beta-cyclodextrin) white bee's wax or xanthan gum and the preservative is selected from the group consisting of methyl p-hydroxy benzoate, methlparaben, propyl p-hydroxybenzoate, propylparaben, or potassium sorbate potassium sorbate).

In addition, the strain culture obtained by inoculating and culturing the Bacillus amyloliquefaciens CGD3 strain and the Bacillus amyloliquefaciens strain CPD4 strain and further culturing the microorganism having the probiotic activity, The active ingredient may be administered to an animal as a probiotic having a tic activity or may be administered to an animal together with a food, a medicament, an animal drug or a lactic acid bacterium, and is preferably used as a feed additive or an auxiliary feed for livestock, The nutrients and useful physiologically active substances in the pigs can increase the growth rate of pigs and can prevent the occurrence of diarrhea in pigs.

The present invention also provides a composition for producing a protease comprising a strain or a culture solution thereof as an active ingredient. The method for culturing the strain of the present invention can be cultivated according to a method commonly used in the art. The protease produced by the strain of the present invention can be used for producing seasonings, softening of meat, prevention of main turbidity, , Food industry such as baking, pharmaceutical industry such as fire extinguishing agent, detergent, leather, and various industries related to the environment.

The present invention provides a composition for fibrinolysis comprising the strain or a culture thereof as an active ingredient. Since the proteolytic enzyme produced by the strain of the present invention is excellent in thrombolytic activity, it can be provided as a health food for fibrinolysis containing it as an effective ingredient. For example, such a fibrinolytic health food may be ingested for the prevention and treatment of circulatory diseases including thrombotic diseases such as coronary artery disease, pulmonary thromboembolism, cerebrovascular disease, hypertensive disease, ischemic heart disease and the like.

The present invention also provides a food containing the composition for producing the protease. In the present invention, the strain or the protease isolated from the culture can be used as it is or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, health or therapeutic treatment). In particular, the food composition of the present invention can be used in food processing stages such as milk, baking, and cheese.

The present invention also provides a composition for inhibiting alpha-glucosidase activity comprising the strain or a culture solution thereof as an active ingredient. α-glucosidase has a function of cleaving glycosidic bonds between a sugar and a sugar, a sugar and a lipid in a polysaccharide, or between a sugar and a protein in a cell, and when the glucosidase enzyme reaction is inhibited, the absorption of the sugar in the small intestine is delayed It is effective for diabetes treatment.

The present invention also provides a health functional food for preventing or ameliorating diabetes containing the strain or a culture thereof as an active ingredient.

In addition to the active ingredient, the functional food of the present invention may further contain various auxiliary ingredients as necessary. In the case of the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.

In addition, among the functional foods of the present invention, health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau Martin and stevia extract, and synthetic sweetening agents such as saccharin and aspartame. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

The present invention also provides a pharmaceutical composition for preventing or treating diabetes comprising the strain or a culture thereof as an active ingredient. The pharmaceutical composition can prevent or treat a disease that is controlled by an? -Glucosidase inhibitory activity. Since the strain of the present invention exhibits? -Glucosidase inhibitory activity and exhibits an excellent therapeutic effect against diseases such as diabetes, the strain of the present invention or a culture thereof can be used as a pharmaceutical composition for the prevention or treatment of diabetes . The strain is preferably Bacillus amyloliquefaciens CGD3 strain (KACC92048P) or Bacillus amyloliquefaciens CPD4 strain (KACC92049P), more preferably a Bacillus amyloliquefaciens strain.

The pharmaceutical composition of the present invention can be safely absorbed into the body regardless of the intake amount. The dosage or amount may vary depending on factors such as the type and amount of the active ingredient and the other ingredients contained in the pharmaceutical composition, the type of the formulation and the age, body weight, general health condition, sex and diet, time of administration, Duration of administration, drug used concurrently, and the like. When the pharmaceutical composition of the present invention is administered to a human body, there is no fear of side effects as compared with chemically synthesized α-glucosidase inhibitor-containing foods and medicines. The food or pharmaceutical composition of the present invention may be formulated to contain one or more carriers acceptable for pharmaceutical or food addition to the above-mentioned effective ingredients. The pharmaceutical or food acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease, or according to each ingredient, using the method disclosed in the appropriate field in the art, or the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

Formulations of the pharmaceutical compositions of the present invention may be granules, powders, granules, coated tablets, tablets, capsules, sticks, extracts, suppositories, syrups, juices, suspensions, emulsions, have.

The capsules of the present invention are composed of a coating material and a core material to be coated. In the capsules of the present invention, the nuclear material preferably contains the strain of the present invention or? -Glucosidase separated from the culture thereof. In the capsules of the present invention, the coating material surrounding the nuclear material uses a water-soluble polysaccharide having excellent water absorption, dispersibility, and stickiness. The water-soluble polysaccharides usable in the present invention include starch, agar, carrageenan, alginic acid, sodium alginate, polymethylmethacrylate, wheat protein, soy protein, methylcellulose, hydroxypropylcellulose cellulose derivatives such as hydroxypropylcellulose and hydroxypropylmethylcellulose, xanthan gum, arabic gum, locust bean gum, guar gum, tamarind A gum such as tamarind gum, Tara gum, karaya gum, tragacanth gum, ghatti gum, and gellan, xanthan, pectin, (LM, HM type), dextran, glucan, glucomannan, arabino galactan, furcelleran, pullulan, glucosamine, Gelatin, casein (ca sein. &lt; / RTI &gt;

The amount of the coating material in the preparation of the capsules of the present invention may be appropriately adjusted depending on the application and purpose, and it is preferable to use the coating material in an amount ranging from 1 to 80% by weight, which is generally used on the basis of nuclei Do. The capsules of the present invention may further contain an emulsifying agent, a protecting agent and a plasticizer as necessary. The capsule preparation of the present invention may be prepared by using an appropriate one of the commonly used encapsulation methods. The composition containing the capsules of the present invention can be specifically used as dairy products (milk, soy milk, processed milk), fermented milk (liquid yogurt, yogurt yogurt), fermented foods (kimchi, .

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

Isolation of strain

Specimens used to isolate strains from traditional pastes were 16 varieties of soybean paste and 5 varieties of chongkukjang which were traditionally produced at home. 80 strains were isolated from the 21 species of strains collected in this study, and the isolates with protease releasing ability were isolated as follows. The diluted solution was diluted to 0. 100% in 0.85% physiological saline by decidual dilution. The diluted solution was plated on nutrient broth (Difco, Detroit, MI, USA) and incubated at 37 ° C for 24 hours. The colonies were inoculated on a plate medium The strains that form clear active rings due to proteolytic enzyme activity were isolated as protease producing strains. As a method to identify the active rings formed by the protease activity, the strain was inoculated into a nutrient agar medium containing 2% skim milk and cultured at 37 ° C for 24 hours. The protease-producing strains isolated in this study were used in the experiments while stored in a -70 ° C freezer.

Identification of isolated strains

In order to confirm that the strain isolated as a protease producing strain from the soybean was a microorganism applicable to the food, the strain was identified by isolating the 16S ribosomal RNA gene from the strain isolated from Solgent Co., Korea.

Measuring the growth rate of cultured isolates

In order to confirm the growth of the strains according to the incubation time, the shaking culture was diluted with 0.85% physiological saline at 3, 6, 9, 12, 24, 48, 72, 96 hours intervals by decidual dilution method, (Difco, Detroit, MI, USA) and cultured at 37 ° C for 24 hours. Growth was measured by grown colonies.

Measurement of protease activity of isolated strains

To measure the protease activity of each isolate culture, 50 mM sodium phosphate After dissolving 0.6% casein in a buffer solution (pH 7.0), 0.5 mL of the culture supernatant was added to the culture supernatant, and the mixture was reacted at 37 ° C for 30 minutes. Then, 0.44 M trichloroacetic acid (TCA) The reaction was terminated by adding 0.55 M Na ₂ CO ₃ and phenol reagent. The absorbance at 660 nm was measured and converted to the amount of tyrosine from the standard curve. In this study, 1 unit of enzyme activity ) Was defined as the amount of enzyme producing 1 μmol of tyrosine per minute.

Identification of growth conditions of isolates

In order to identify the growth conditions of the identified microorganisms, the degree of growth of the microorganism was measured by absorbance at 600 nm with different initial pH, NaCl and glucose conditions. After adjusting the pH of the nutrient broth (Difco, Detroit, USA) to 3 to 11, 1% (v / v) of the pre-cultured strain was inoculated into each medium and cultured for 24 hours. To investigate the effects of NaCl and glucose concentrations on bacterial growth, NB medium was prepared with NaCl at different concentrations of 3, 6, 9, 12 and 15%, and glucose was also added at 5, 10, 15, 20, 25, 30, 35, and 40% of the culture medium was prepared, and 1% (v / v) of the pre-cultured strain was inoculated into each culture medium and cultured for 24 hours.

The artificial gastric juice of the isolate and In bile acid  Evaluation of resistance

For the evaluation of resistance to artificial gastric juice, the strain was preincubated in nutrient agar medium (Difco, Detroit, USA) and centrifuged to collect only the cells. 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 artificial gastric juice was measured by adding 0.3% (w / v) pepsin to 0.5% NaCl (pH 3.0) and incubating the artificial gastric juice with 10% each of the suspensions for 3 hours. And the viable cell count was measured. As a control group, 10% each of the suspension of the strain was inoculated into artificial gastric juice prepared by adding 0.3% (w / v) pepsin to 0.5% NaCl (pH 3.0) and then plated on agar medium. The strains were inoculated in 0.5% NaCl (pH 3.0) at 10% each, and the plate was plated on agar medium for 3 hours. The resistance of the isolate to artificial gastric juice was expressed as% compared with the number of the live cells counted for the control group and the number of live cells counted for the normal group after the reaction for 3 hours.

For the evaluation of resistance to bile acids, the isolates were preincubated in NB medium, centrifuged to collect cells, washed with sterilized water, and dispersed in sterilized water at the end of washing to prepare a suspension. The bile acid resistance was measured by adding 0.5% of the suspension to NB medium supplemented with 0.3% bile salt, incubating for 24 hours, and measuring absorbance at 600 nm using a UV-vis spectrophotometer (UVIKON XS, SECOMAN) Were measured. As a control, the group treated with the suspension of NB alone medium was used, and the resistance to bile acid was expressed as a percentage of the control group relative to the absorbance after culturing for 24 hours.

Evaluation of intestinal adhesiveness of isolated strains

The HT-29 cells used in this experiment were intestinal epithelial cells derived from human colon cancer and were used in Korean cell line bank (KCLB). HT-29 cells were treated with 10% FBS (Fetal bovine serum, Welgnen co., Daegu, Korea), 1% penicillin / streptomycin (Gibco, NY, USA), 25 mM NaHCO ₃ and 25 mM HEPES (N-2-Hydroxyethylpiperazine (Welgene co., Daegu, Korea) supplemented with 5% CO ₂ at 37 ° C. When the cells form a monolayer, the cells were subcultured and the medium was exchanged every other day. For intestinal adhesiveness experiments, HT-29 cells were seeded in 24-well plates at a concentration of 1 × 10 ⁵ cells / mL per well, cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours, Experiments were carried out with pure RPMI 1640 medium without FBS and penicillin / streptomycin. The isolated strain was inoculated on nutrient broth and preincubated for 24 hours at 37 ° C., and the strain was recovered and washed three times with 0.1 M PBS (phosphate buffered saline) to remove FBS and penicillin / streptomycin And dispersed in RPMI1640 medium. The isolate was inoculated with 500 μL of the HT-29 cells in a well plate, incubated at 37 ° C under 5% CO ₂ for 2 hours, and then washed with 0.1 M PBS to remove unattached strains. For the measurement of viable cell count, 1 mL of 0.1% triton X-100 was added to each well, and the mixture was reacted at 37 ° C. for 30 minutes, and then recovered and diluted. The plate was plated on agar medium and cultured at 37 ° C. for 24 hours Respectively. Intestinal adhesiveness (%) was calculated by counting the number of viable cell counts after treatment for the initial number of inoculated cells.

The α- Glucosidase (? glucosidase ) Inhibition activity measurement

α-glucosidase inhibitory activity was obtained by mixing 50 μL of 0.2 unit / mL α-glucosidase enzyme solution and 50 μL of 0.1 M potassium phosphate buffer (pH 6.8) in 60 μL of each sample, pre-incubating the mixture at 37 ° C. for 20 minutes, mM ρNPG (ρ-nitrophenyl α-D-glucopyranoside). The reaction was carried out at 37 ° C for 20 minutes, and 0.1 M sodium carbonate was stopped. The absorbance at 405 nm was measured and the enzyme inhibitory activity was calculated based on the control group containing the same amount of buffer solution as the sample solution. The enzyme inhibitory activity was expressed as a percentage of the absorbance of the sample by subtracting the absorbance of the sample from the absorbance of the control and divided by the absorbance of the control.

Isolate Thrombolysis ability  Measure

To measure the fibrinolytic activity of the isolated strain, fibrinogen was dissolved in PBS buffer (10 mM, pH 7.25) to a final concentration of 0.3% (w / v) and poured into a plate, followed by adding 20 units of thrombin Thick fibrin clots were formed and left at room temperature for 30 minutes to be used for the measurement of thrombolytic enzyme. Activity measurements were made by placing 50 μL of the culture broth in a paper disc (8 mm, ToyoRoshi, Tokyo, Japan) on the fibrin plate and measuring the diameter of the transparent region produced after 6 hours at 37 ° C. Respectively.

Thrombolytic activity (%) = dissolution area (mm) by sample / dissolution area by plasmin (mm) × 100

Example  1. Isolation and Identification of Protease Producing Strain

As a result of isolating the protease producing strains from the soybean, three strains exhibiting excellent active rings in the solid medium supplemented with skim milk were selected as protease producing strains (Table 1). The protease activity of casein was examined using the culture supernatant obtained by liquid culture of the isolated strains, and enzyme activity of the isolated strains was confirmed. Finally, BLAST analysis using the 16S ribosomal RNA gene sequence revealed that all three strains isolated were identified as Bacillus amyloliquefaciens and 98-99% similarity in all strains there was.

Protease activity of strains isolated from soybean Regions Isolated strains Species Identity (%) protease activity (U / mL) Daegu CDD5 Bacillus
amyloliquefaciens 99 7.00 + 0.06 Gyeongsang buk-do Pohang-si CPD4 Bacillus
amyloliquefaciens 99 7.12 + - 0.35 Gyeongsang buk-do
Gumi-si CGD3 Bacillus
amyloliquefaciens 98 7.55 0.30

All values were mean ± SD (n = 3)

Example  2. Depending on incubation time Growth support  Protease activity

Isolated Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens In order to confirm the growth time and protease activity of CGD3 at the time of culture, the growth characteristics of the strains were examined while culturing the strains in a nutrient medium for 96 hours (FIG. 1). When the strain was grown from 0 to 3 hours and cultured for 6 hours, the Bacillus amyloliquefaciens strain CDD5 and Bacillus amyloliquefaciens CPD4 showed maximum growth of 6.91 log CFU / mL, respectively, and Bacillus amyloliquefaciens CGD3 showed maximum growth at 6.67 log CFU / mL for 12 hours. The protease activity after incubation for 0 to 3 hours showed no protease activity, and the protease activity was observed after incubation for 6 hours. The protease activity of Bacillus amyloliquefaciens strain CGD3 and Bacillus amyloliquefaciens CDD5 showed the highest activity at 9.8 and 7.0 Unit / mL when cultured for 72 hours, and Bacillus amyloliquefaciens CPD4 exhibited a maximum activity of 5.4 Unit / mL when cultured for 48 hours (Fig. 2).

Example  3. Growth characteristics according to culture conditions

Isolated Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens In order to confirm the optimal growth characteristics of CGD3, the growth characteristics of the present strain were examined while culturing the strain at a different initial pH (FIG. 3). As a result, all of the strains were uniformly grown in a medium having an initial pH of 5 to 10, and in particular, Bacillus amyloliquefaciens CGD3 was found to survive the most stable at pH 5. It was confirmed that the growth of all strains isolated under pH 5 or lower and pH 10 or higher was remarkably decreased, and no bacterial growth was observed at pH 3, 4 and 11.

Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens In order to confirm the salt tolerance of CGD3, the growth characteristics of the bacteria in the medium supplemented with NaCl were examined (FIG. 4). As a result, Bacillus amyloliquefaciens CGD3 showed the highest growth with an absorbance value of 0.542 ± 0.05 at the concentration of 6%, and at the same concentration, Bacillus amyloliquefaciens strain CDD5, Bacillus amyloliquefaciens strain The absorbance of CPD4 was 0.482 ± 0.03 and 0.430 ± 0.01, respectively, and the highest growth was observed at 9% concentration. The absorbance of each isolate was 0.606 ± 0.01 and 0.617 ± 0.03, respectively. And did not proliferate at 15% concentration. Taken together, these results indicate that Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4 is a Bacillus amyloliquefaciens It can be seen that the salt resistance is somewhat stronger than that of CGD3.

Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens In order to confirm the endurance of CGD3, bacterial growth characteristics in a medium supplemented with glucose at different concentrations were examined (FIG. 5). As a result, Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens CGD3 all showed a growth of up to a concentration of 5%, but the growth was remarkably lowered at a concentration of 10% or more. In particular, Bacillus amyloliquefaciens CPD4 was found to inhibit Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CGD3 strains were lower than those of CGD3 strains.

Example  4. Artificial gastric juice and In bile acid  Resistance to

In order to function as a probiotic, it must survive in the conditions of the digestive tract. The pH of the pure gastric juice is maintained at about 1.4 to 2.0, and most of the microorganisms are killed in the gastric juice or the original activity is lowered. The probiotics taken through the oral cavity can pass through stomachs and stomachs where various enzymes are present and reach the target area of the duodenum through which the bile is present. Among them, acid resistance to artificial gastric juice should be shown in order to survive at low pH such as gastric acid. Therefore, Bacillus amyloliquefaciens strain, which is selected as an edible strain isolated from domestic traditional fermented food, CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens The resistance of artificial gastric juice and bile acid to 3 kinds of CGD3 were confirmed. As a result of evaluating the resistance to artificial gastric juice prepared by adding 0.3% (w / v) pepsin to pH 3.0 adjusted with NaCl, Bacillus amyloliquefaciens CDD5 showed the highest survival rate (67.23%), and Bacillus amyloliquefaciens CPD4 , Bacillus amyloliquefaciens And 42.34% and 47.63%, respectively, in CGD3 (Table 2). As a result of evaluating the tolerance to bile acid prepared using 0.3% bile salt, the Bacillus amyloliquefaciens CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens And 78.47%, 74.84%, and 77.58% in CGD3, respectively. All three strains showed high resistance to bile acid. Therefore, among three strains isolated from miso, Bacillus amyloliquefaciens strain CDD5 was relatively superior to artificial gastric juice tolerance in comparison to other strains, whereas Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens CGD3 also showed high resistance and was confirmed to pass through the upper gastrointestinal tract and meet the requirements of the probiotic microorganism.

Digestive Juice Bacillus amyloliquefaciens Viability (%) Artificial gastric Juice CDD5 67.23 ± 3.28 CPD4 42.34 + - 2.32 CGD3 47.63 + - 4.25 Even Artificial CDD5 78.47 ± 3.63 CPD4 74.84 + - 6.59 CGD3 77.58 + - 4.27

All values were mean ± SD (n = 3)

Example  5. Intestines Attachment

To be characterized as probiotics, it must pass through the stomach and duodenum and reach the target area, the target site, and attach normally to the epithelial cells of the intestine to proliferate and function. Caco-2 cells were generally used to confirm intestinal adherence. However, since the mucous layer is exposed in the small intestine and large intestine, HT-29 cells, which secrete mucus during the differentiation process, are used to treat Bacillus amylolique Fassien CDD5, Bacillus amyloliquefaciens CPD4, Bacillus amyloliquefaciens The intestinal adhesiveness of CGD3 was confirmed and shown in Fig. As a result of experiment, it was confirmed that all three strains were adhered to each other. Among them, Bacillus amyloliquefaciens The highest adherence was found to be 0.19% in CPD4, and Bacillus amyloliquefaciens CDD5 and Bacillus amyloliquefaciens And CGD3 were 0.16% and 0.03%, respectively. These results indicate that even Bacillus sp. ( Bacillus sp.) Of the same species exhibits different adhesion to HT-29 cells because of the concentration of protein components, polysaccharide, lipoteichoic acid, Of the physiological activity.

Example  6. α- Glucosidase (? glucosidase ) Changes in inhibitory activity

As a result of measuring the inhibitory activity of the α-glucosidase enzyme activity produced by the isolated three strains, it was confirmed that the two kinds of strains showed an excellent inhibitory effect as acarbose, which is a positive control group (FIG. 7). Bacillus Amiloriquepassien CDD5 did not exhibit the? -Glucosidase inhibitory activity, but Bacillus amyloliquefaciens CPD4 and Bacillus amyloliquefaciens CGD3 showed 96% and 97% inhibition activity, respectively. α-glucosidase is a disaccharide-degrading enzyme distributed in the small intestine, which is known to be induced specifically by dietary saccharides in diabetic patients. For diabetics, it is recommended to avoid simple sugars such as sugars. Simple sugars are easily absorbed in the intestine compared to polysaccharides and easily cause hyperglycemia. In particular, it is particularly important to control postprandial hyperglycemia in patients with non-insulin-dependent diabetes mellitus. In this experiment, the isolated Bacillus amyloliquefaciens CPD4 and Bacillus amyloliquefaciens CGD3 has potent α-glucosidase inhibitory activity, which is considered to be useful for improving symptoms of carbohydrate metabolism disorder.

Example  7. Thrombolytic enzyme activity

The isolated strains with three proteolytic activities isolated from the present invention were cultured in a nutrient broth to produce coenzyme. Then, in the fibrin plate, the relative clearance of each coenzyme to the plasmin (1.0 U / mL) And the fibrinolytic activity was measured (Table 3, Fig. 8). Bacillus Amiloriquepassien In the CDD5, no transparent ring was observed, but Bacillus amyloliquefaciens CPD4 and Bacillus amyloliquefaciens In CGD3, a transparent ring was observed, among which Bacillus amyloliquefaciens CPD4 showed the largest transparency, indicating that thrombolytic activity was the strongest. Bacillus Amiloriquepassien The fibrinolytic activity (%) of CGD3 showed a fibrinolytic activity equivalent to 81% of plasmin 1.0 U / mL, whereas the fibrinolytic activity of Bacillus amyloliquefaciens CPD4 showed almost the same activity as plasmin in 104%. The fibrinolytic activity of the isolates identified in this experiment was obtained by using the undifferentiated culture as it is, and it is expected that the fibrinolytic activity of the isolated strains will be higher when the separated and purified enzyme is used. Proteases with thrombolytic activity are serine enzymes, which have a serine residue at the active site and a triad structure consisting of Ser221, His64 and Asp32 at the active site. It is known that fibrinolytic enzymes such as Nattokinase differ in that fibrin is preferred to other proteins such as casein in preference to the substrate and there is no significant difference in other amino acid sequences . Thus, several differences in the amino acid sequence in the primary structure may result in fibrin specificity.

Bacillus amyloliquefaciens Relative fibrinolytic activity (%) CDD5 ND CPD4 104.51 + 1.81 CGD3 81.22 ± 3.66

ND: Not detected

All values were mean ± SD (n = 3)

Agricultural Biotechnology Research Institute KACC92048P 20150319 Agricultural Biotechnology Research Institute KACC92049P 20150319

Claims (10)

Bacillus amyloliquefaciens having proteolytic activity and? -Glucosidase inhibitory activity ( Bacillus sp. amyloliquefaciens ) strain. The strain according to claim 1, wherein the Bacillus amyloliquefaciens strain has adhesive activity, acid resistance and biliary cholesterol to intestinal epithelial cells. The strain according to claim 1, wherein the Bacillus amyloliquefaciens strain is Bacillus amyloliquefaciens CGD3 strain (KACC92048P) or Bacillus amyloliquefaciens CPD4 strain (KACC92049P). A probiotic composition comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A composition for producing a protease comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A food comprising a composition for producing a protease according to claim 5. A composition for inhibiting alpha-glucosidase activity comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A health functional food for preventing or ameliorating diabetes comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A pharmaceutical composition for preventing or treating diabetes comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A fibrinolytic composition comprising the strain of any one of claims 1 to 3 or a culture thereof as an effective ingredient.

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