KR20030090243A - Bacillus subtilis DM185(KCCM-10372) which produces fibrinolytic enzymes - Google Patents

Abstract

PURPOSE: A microorganism Bacillus subtilis DM185(KCCM-10372) producing a fibrin lysis enzyme is provided. The microorganism can mass-produce the fibrin lysis enzyme which can prevent various circulatory diseases caused by excess fibrin. CONSTITUTION: A microorganism Bacillus subtilis DM185(KCCM-10372) producing a fibrin lysis enzyme is provided, wherein Bacillus subtilis DM185(KCCM-10372) is isolated by collecting samples from salted seafood, diluting the samples in 0.85% physiological saline solution, heating the diluted solution at 85 deg. C for 30 minutes, diluting the heat treated solution in 0.85% physiological saline solution by stages, spreading the diluted solution on a nutrient agar medium, culturing the medium at 37 deg. C for 48 hours, isolating colonies grown, dropping the isolated colony cultured medium on a fibrin plate, and selecting one strain showing the excellent fibrin lysis activity.

Description

Bacillus subtilis DM185 (KCCM-10372) which produces fibrinolytic enzymes}

The present invention relates to Bacillus subtilis microorganisms producing thrombolytic enzymes.

The thrombolytic enzyme is a kind of protease and, like Plasmin, is an alkaline protease of the serine family. These enzymes are produced by strains of the genus Bacillus during the manufacturing process of traditional fermented foods, and many studies are being conducted under the assumption that ingestion of such fermented foods can have an effect on the prevention and treatment of thrombosis. Thrombus is a product of metabolic processes that naturally produce and degrade equilibrium during reactions in vivo, and is related to blood coagulation. The production of thrombi begins when platelets adhere to damaged vascular endothelial cells to form fibrin. The cause of thrombus formation is not yet known.

Congenital genetic diseases such as disseminated intravascular coagulation, changes in physiological function according to aging of the body, and stress are cited as causes. Especially, stress caused by excessive work of modern people is involved in blood clotting. It is considered as one of the causes of abnormal thrombus by changing the amount of the factor. Thrombosis, a disease caused by excessive blood clots in the blood, can cause fatal damage to the human body.

Therapeutic agents used for the treatment of thrombosis are currently used as anti-thrombotic agents such as coumarin, coumarin, warfarin, and hirudin, which are produced from leeches. (urokinase), streptokinase, and tissue-type plasminogen activator are commonly used as treatments, but these treatments are inexpensive and have difficulty in oral administration except for urokinase. .

Accordingly, the present invention is to provide a microorganism that produces a highly active and stable enzyme by isolating and selecting microorganisms producing thrombolytic enzymes in salted fish, which is a Korean traditional food.

FIG. 1 is a diagram showing a clear ring formed when 50 microliters of Bacillus subtilis DM185 (KCCM-10372) culture solution was added to a fibrin plate containing 0.7% fibrinogen and reacted at 37 ° C. for 16 hours.

2 is a diagram showing the morphology of the fungi formed when the Bacillus subtilis DM185 (KCCM-10372) was cultured on TSA solid medium (Tryptic Soy Agar) and cultured for 48 hours at 37 degrees Celsius.

Sample used in the invention

The feed used in the present invention used kimchi and soy sauce dipped in homes in Seoul and suburbs of Korea, as well as various types of salted salted seafood.

Isolation of bacteria from the sample

In order to exclude pathogenic bacteria and to isolate heat-resistant spore-producing bacteria, approximately 1 gram of sample was taken and suspended in 100 milliliters of 0.85% saline, and then heat-treated at 85 degrees Celsius for 30 minutes. The heat treated suspension was serially diluted in 0.85% saline and plated on Nutrient agar. After incubating the plated nutrient agar for 48 hours at 37 degrees Celsius, the resulting bacterium was taken as platinum and streaked on a nutrient agar medium, followed by pure separation until pure colonies were observed.

Measurement of thrombolytic activity against isolated strain

As a screening method for selecting strains having excellent thrombolytic activity, a predetermined amount of the culture solution of the strains was added and reacted on a fibrin plate, and then the presence or absence of the formation of a transparent ring was confirmed. When the reaction solution is added with a culture solution of a strain that produces a thrombolytic enzyme on the fibrin plate, the fibrin is dissolved by the enzyme and a clear ring is formed around the culture solution. Therefore, for each of the isolated strains, it was determined that strains that produce thrombolytic enzymes can be selected by adding cultures of the strains to the fibrin plates and reacting them, and then observing the production of clear rings.

Example 1

As an experiment for selecting strains that produce thrombolytic enzymes, a method using fibrin plates was used. Purely isolated strains were inoculated in 10 milliliters of Nutrient broth, stirred and incubated for 24 hours at 37 degrees Celsius, and cultured to remove the cells by centrifugation. Used as. Take 0.7 grams of fibrinogen, dissolve in 100 milliliters of phosphate buffer (10 mM, pH 7.4), filter with filter paper, and dispense 10 milliliters into Petri dishes. Add 100 microliters of thrombin (100 NIH units). Mix and leave at room temperature to solidify. A paper disk was placed on the coagulated fibrin plate, and the coenzyme solution was inoculated at 50 microliters to 100 microliters and reacted for 16 hours at 37 degrees Celsius, and then the diameter of the dissolved transparent ring was measured.

Strains with large diameters of the resulting transparent rings were first selected as strains with excellent thrombolytic activity, and their forms were as shown in FIG. 1.

Example 2

The tubing method was used to determine the more accurate activity of thrombolytic enzymes for the primary selected strains. 1.4 ml of 0.05 mol Borate buffer (pH 8.5, NaCl) and 0.4 ml of fibrinogen solution (0.72%) were added to the test tube and allowed to stand for 5 minutes in a 37 ° C constant temperature water bath, followed by a thrombin solution (1000 unit / ml). 100 microliters of the mixture was mixed well and then reacted for 10 minutes in a constant temperature water bath at 37 degrees Celsius. 100 microliters of the crude enzyme solution to be measured was added to the coagulated reaction solution, followed by reaction for 1 hour in a constant temperature water bath at 37 degrees Celsius. After 1 hour of reaction, 2 ml of a 0.2 mol trichloroacetic acid solution was added to stop the reaction. After adding trichloroacetic acid solution, the reaction mixture was further reacted in a constant temperature water bath at 37 degrees Celsius for 20 minutes, and then 1 milliliter of the reaction solution was centrifuged at 15,000 rpm for 5 minutes. After centrifugation, the supernatant was carefully taken and the absorbance was measured at 275 nm. Enzyme activity was calculated by comparing the obtained absorbance value with the control. The final strain was selected as the best enzyme activity was named DM185, the enzyme activity is shown in Table 1.

Table 1. Enzyme activity of DM185 by incubation time

DM185, the final selected strain, was cultured on a TSA solid agar medium (Tryptic Soy Agar) and cultured for 48 hours at 37 degrees Celsius, and the resulting colonies were observed.

Identification and Biochemistry Testing of Selected Bacteria

DM185 (KCCM-10372), the final strain selected as the highest enzyme activity, was identified using biochemical properties, sugar availability, and membrane fatty acid composition ratio.

Example 3

Morphological, physiological and biochemical experiments on DM185 (KCCM-10372) are shown in Table 2.

Table 2. Morphological, Physiological and Biochemical Properties of DM185 (KCCM-10372)

Based on the results shown in Table 2, the selected strain was considered as Bacillus sp.

Example 4

The sugar fermentation test of 49 strains considered to be of the genus Bacillus using API 50CHB kit (API bioMerieux, France) was performed as shown in Table 3 below.

Table 3. Sugar Fermentation Test Results of DM185 (KCCM-10372)

Table 3. Then

Table 3. Then

Example 5

In order to analyze the cell membrane fatty acid component of the strain, an analysis method of Microbial Identification System (MIDI; Microbial ID, Inc., Newark, Delaware, U.S.A) was used. For cell membrane fatty acid analysis of the strain, gas chromatography (GC) pretreatment was first performed. The isolates were incubated for 24 hours at 28 degrees Celsius on a BHI agar medium, and then the resulting colonies were subjected to a saponification process with sodium hydroxide solution suspended in methanol in the resulting pellets. This was again methylated with 6M hydrochloric acid solution suspended in methanol, extracted with hexane and methyl-tert-butyl ether, and washed with sodium hydroxide solution. After the washing process, the organic solvent layer was taken, transferred to GC vial, and the burned carbon in methyl ester fatty acid of microbial sample prepared by gas chromatography was used as data for microorganism identification. The strain was identified by comparing the fatty acid content in the experimental strain and the identification library stored in the database. The results of analysis of the cell membrane fatty acid component of the starter strain are shown in Table 4.

Table 4. Cell Membrane Fatty Acid Components of DM185 (KCCM-10372)

Based on the morphological, physiological, biochemical, glycofermentative properties and cell membrane fatty acid component analysis, DM185 (KCCM-10372) was selected as Bacillus subtilis.

Enzyme Activity According to Culture Conditions of Selected Strains

In order to investigate the enzyme activity according to the culture conditions of Bacillus subtilis DM185 (KCCM-10372) enzyme activity was measured by varying the culture temperature, pH, culture medium and the like.

Example 6

An experiment was conducted to investigate the enzyme activity according to the culture medium of Bacillus subtilis DM185 (KCCM-10372). Bacillus subtilis DM185 (KCCM-10372) was inoculated in each of five media, followed by incubation, and each culture was taken, and centrifuged to remove cells. Culture conditions were shaken for 36 hours at 30 degrees Celsius, culture medium composition and enzyme activity for each is shown in Table 5. At this time, the enzyme activity was measured using the fibrin plate method, and the enzyme activity was determined as a relative ratio of the diameter of the transparent ring generated by reacting a known standard enzyme (Plasmin, 100 unit / ml) under the same conditions.

Table 5. Culture medium composition and enzyme activity for Bacillus subtilis DM185

As shown in Table 5 above, it showed the highest enzyme activity when cultured in LB liquid medium.

Example 7

An experiment was conducted to investigate the enzyme activity according to the culture temperature of Bacillus subtilis DM185 (KCCM-10372). Bacillus subtilis DM185 (KCCM-10372) was passaged twice in LB liquid medium, and then cultured at different incubation temperatures. The culture medium was taken, centrifuged to remove cells, and the enzyme activity of the supernatant was measured. Enzyme activity was measured using the fibrin plate method, and was determined by the relative ratio of the diameter of the transparent ring formed by reacting a standard enzyme of known titer (Plasmin, 100 unit / ml) under the same conditions. Enzyme activity for each culture temperature is shown in Table 6.

Table 6. Enzymatic Activities of Bacillus Subtilis DM185 (KCCM-10372) According to Incubation Temperature

As shown in Table 6 above, the highest enzymatic activity was obtained when cultured at around 35 degrees Celsius.

Example 7

An experiment was conducted to investigate the enzyme activity according to the culture pH of Bacillus subtilis DM185 (KCCM-10372). Bacillus subtilis DM185 (KCCM-10372) was inoculated into each LB liquid medium prepared at different pHs, and stirred cultured at 30 ° C. for 48 hours. After culturing, the culture solution was taken, cells were removed by centrifugation, and the enzyme activity of the supernatant was measured. Enzyme activity was measured using the fibrin plate method, and standard enzymes of known titers

(Plasmin, 100 unit / ml) was determined by the relative ratio of the diameter of the transparent ring formed by the reaction under the same conditions. Enzyme activity for each culture pH is shown in Table 7.

Table 7. Enzyme Activity by Culture pH of Bacillus subtilis DM185 (KCCM-10372)

As shown in Table 7, the highest enzyme activity was shown when the initial pH was around 8.0.

Bacillus subtilis DM185 (KCCM-10372), isolated from Korean traditional fermented food, has been shown to produce higher titers of thrombolytic enzymes than previously reported strains.

The thrombolytic enzyme produced by this microorganism can be used as a raw material for enzyme preparations or other dietary supplements to produce industrially valuable high-quality products suitable for Koreans, and have a comparative advantage in product quality and international competitiveness.

Claims (1)

Bacillus subtilis DM185 (KCCM-10372) microorganism producing enzymes with activity to dissolve blood clots