KR20130095127A - BACILLUS AMYLOLIQUEFACIENS KC41 HAVING HIGH ACTIVITY OF γ-GLUTAMYLTRANSPEPTIDASE AND FIBRINOLYTIC ENZYME, AND CHUNGKOOKJANG FERMENTED BY USING THE SAME - Google Patents

Abstract

PURPOSE: Bacillus amyloliquefaciens KC41 is provided to have activities of gamma-glutamyltranspeptidase and fibrinolytic enzyme and to develop cheonggukjang (fermented bean paste) with excellent functionality and preference. CONSTITUTION: Bacillus amyloliquefaciens KC41 (KCCM 11249P) has a nucleic acid sequence of sequence number 1 as a 16S rDNA gene sequence. A method for making cheonggukjang comprises the step of fermenting soybeans using Bacillus amyloliquefaciens KC41 as a spawn. [Reference numerals] (AA) Absorbance; (BB) Isolated strain

Description

Bacillus amyloliquefaciens KC41 having high activity of γ-glutamyltranspeptidase and fibrinolytic enzyme, and Chungkookjang fermented by using the same }

The present invention provides Bacillus amyloliquefaciens KC41 ( Bacillus) having high activity of gamma glutamyltranspeptidase and fibrin degrading enzyme. amyloliquefaciens KC41) and Cheonggukjang prepared using the same.

In Korea, where rice is a staple food, Cheonggukjang is an important source of protein and has been used as a very important fermented food as a base of Korean food taste along with miso, red pepper paste, and soy sauce. In particular, Cheonggukjang has been widely used because it can be produced by fermentation in comparison with doenjang. Cheonggukjang of Korea produced by self-method is not uniform in taste and flavor in each province, which is reported to change the taste and flavor of Cheonggukjang according to the types of fermented microorganisms.

The quality of Cheonggukjang differs depending on the type of microorganisms involved in fermentation. The main microorganisms present in Cheonggukjang are Bacillus subtilis , Bacillus licheniformis , Bacillus pumilus , Bacillus megaterium , Bacillus amyloliquefaciens and the like are known. Various physiologically active substances produced during fermentation of Cheonggukjang have been shown to exhibit functional effects such as blood pressure increase suppression, immune enhancement, antimutation, antioxidant effect, and thrombolytic activity. Reported to be present. In addition, thrombolytic enzyme called nattokinase was found in Natto of Japan, which is similar to Korea's Cheonggukjang.

The viscosity and flavor component of Cheonggukjang is levan, a polymer of poly-γ-glutamic acid polymerized with D, L-glutamic acid, γ-polyglutamate (γ-PGA) and fructose. have. γ-Glutamyltranspeptidase (γ-GTP) is an enzyme that hydrolyzes γ-glutamylpeptide and transfers γ-glutamyl to other amino acids or peptides. In γ-PGA acid, it becomes α-helix structure, and in neutral, the molecule is changed into a random coil structure with long elongation. Although a kind of polypeptide, the γ-carboxyl group of glutamic acid and the α-amino group of glutamic acid are distinguished by a single amino acid polymer composed of amide bonds. It is a highly water-soluble and biodegradable anionic amino acid polymer material, and has a wide range of applications for high value-added medicines, cosmetics, functional foods, environmental use, and industrial use. Cheonggukjang is fermented by a large number of unidentified microorganisms, so it is necessary to study bioreaction control functions by various components of soybeans and microbial metabolites decomposed by various microbial enzymes.

The present invention seeks to provide a new microorganism with high activity of gamma glutamyltranspeptides and fibrin degrading enzymes, and to provide Cheonggukjang with excellent efficacy and flavor prepared using it as a seed.

As a means for solving the above problems, the present invention is Bacillus amyloliquefaciens KC41 ( Bacillus) which is a novel microorganism isolated from Cheonggukjang amyloliquefaciens KC41).

Bacillus amyloliquefaciens KC41 according to the present invention ( Bacillus amyloliquefaciens KC41) has the nucleic acid sequence of SEQ ID NO: 1 as a 16S rDNA gene sequence.

In one embodiment, the Bacillus amyloliquefaciens KC41 may be deposited with the accession number KCCM 11249P.

As can be seen in the following examples, Bacillus amyloliquefaciens KC41 according to the present invention is of the gamma glutamyl transpeptidase (γ-glutamyltranspeptidase) involved in the production of gamma glutamate (γ-glutamate), the main component of the cheonggukjang The activity and fibrinolytic enzyme (fibrinolytic enzyme) that dissolves the blood clots that cause thrombosis or heart attack is very high. In addition, Cheonggukjang prepared using Bacillus amyloliquefaciens KC41 showed higher taste and aroma than other Cheonggukjang prepared using other seeds, which makes it very useful as a seed for the production of Cheonggukjang. Found to be high.

Accordingly, the present invention also provides a method for preparing Cheonggukjang, which comprises fermenting soybeans using Bacillus amylocufaciens KC41 as a seed, and Cheonggukjang prepared accordingly.

Cheonggukjang according to the present invention can be prepared according to the conventional production method of Cheonggukjang using Bacillus amylocufaciens KC41 as a seed. For example, immerse the soybeans in water for 10-20 hours, boil for 4-5 hours, transfer the boiled soybeans, cool them to 50-60 ℃, and inoculate Bacillus amylocufaciens KC41 with spawn 42 It can be prepared by fermentation at ~ 45 ℃ for 36-48 hours.

The present invention also provides a food composition comprising Cheonggukjang prepared according to the above method. The food composition according to the present invention may be formulated in a conventional manner to be used as a functional food or added to various foods.

The present invention also provides a health functional food for improving thrombotic disease, comprising Cheonggukjang prepared according to the above method. Health functional food is a food prepared by adding active ingredients to food materials such as beverages, teas, spices, gums, confectionery, or by encapsulating, powdering, and suspensions. Meaning, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a food raw material. The health functional food of the present invention thus obtained is very useful because it can be consumed on a daily basis.

To date, various anticoagulants, antiplatelets, thrombolytics, etc. have been used for the prevention and treatment of thrombotic diseases, but the price is very high, and there are problems of high risk of side effects such as hemorrhagic side effects and gastrointestinal disorders and hypersensitivity reactions. On the other hand, when the intake of the cheongukjang according to the present invention as food, thrombotic diseases such as thrombosis, arteriosclerosis, embolism, ischemic heart disease, stroke, angina pectoris, cerebral infarction, intracranial bleeding, aneurysm, atherothrombosis, neurosis, myocardial infarction It provides functionality that can be prevented or improved.

The amount of Cheonggukjang added according to the present invention in such a health food cannot be uniformly defined depending on the kind of the health food to be treated, but may be added within a range that does not impair the original taste of the food. 0.01 to 50% by weight, preferably 0.1 to 20% by weight. In the case of food in the form of granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of Cheonggukjang hwan.

Bacillus amyloliquefaciens KC41 according to the present invention ( Bacillus amyloliquefaciens KC41, Accession No. KCCM 11249P), dissolves the activity of gamma glutamyltranspeptidase, which is involved in the production of gamma glutamate, the main component of cheonggukjang slime, and dissolves thrombus that causes cerebral thrombosis or heart attack. Fibrinolytic enzyme activity is very high, and it has excellent value as a spawn for the development of Cheonggukjang with excellent functionality and palatability.

1 is a graph showing the results of measuring the γ-GTP synthase activity of the microorganisms isolated from Chunggukjang.
Figure 2 is a photograph showing the thrombolytic activity on the fibrin plate of the isolate strain KC41.
Figure 3 is a photograph showing the results of the morphological investigation of KC41 isolate strains.
Figure 4 shows a systematic diagram showing the molecular biological correlation of the nucleotide sequence of the identified KC41 strain with the data of BLASTN.
Figure 5 is a graph showing the results of measuring the microbial viable count of Cheonggukjang prepared using the selected microorganism as the seed.
Figure 6 is a graph showing the results of measuring the viscous physical properties of Cheonggukjang prepared using the selection microorganism as a seed.
Figure 7 is a graph showing the palatability results of Cheonggukjang using the selection microorganism as a seed.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims.

[ Example ]

Example  1: Exploration of Microorganisms for Fermentation

Cheonggukjang for microbial separation used in this study was purchased and used in Yeoju-gun, Gyeonggi-do, Wonju-si, Gangwon-do, Sunchang-gun, Jeollabuk-do, and Anseong-si, Gyeonggi-do. Separation of microorganisms from Chungkookjang was carried out in the following manner. Cheonggukjang samples were taken and mixed in sterile physiological saline in a 1: 9 ratio, homogenized using a homogenizer (Stomacher 400, Seward, England) for 10 minutes, and then a suspension was prepared. Decimal dilution of this suspension with sterile saline solution, 100 μl of tryptic soy agar (TSA; Difco, Detroit, MI, USA) plate and incubated at 37 ° C. for 24 hours, followed by different microorganisms Was separated.

(One) Gamma - glutamyltranspeptidase  (? GTP Activity measurement of

Γ-glutamyl transpeptidase (γ-GTP) was measured using γ-glutamyl-ρ-nitroanilide as a substrate to select microorganisms with high γ-polyglutamate production in 1,399 strains isolated from Cheonggukjang.

Separation microorganism was incubated in soy medium for 24 hours at 37 ℃ and centrifuged (10,000 × g / 10 min), the supernatant was used as coenzyme solution. Soy medium production for the separation microorganism to produce the enzyme is heat-treated with soybean (Geosan-gun, Chungbuk 2010) and distilled water (1: 4) at 121 ℃ for 15 minutes, soybeans are removed using a filter network and the filtrate is 1N The pH was adjusted to 3.5 using HCl, and then allowed to stand at 4 ° C. for 6 hours to remove precipitated protein with filter paper (Whatman, England). Prepared by readjusting the pH to 6.5 using 1N NaOH. γ-GTP enzyme activity was measured by adding 0.25 mL of coenzyme solution and 0.25 mL of 20 mM glycylglycine to 0.5 mL of substrate solution (1.0 mM γ-glutamyl-ρ-nitroanilide, 50 mM Tris-HCl buffer, pH 8.0) for 10 minutes at 37 ° C. After the reaction, 0.5 mL of 3.5 M acetic acid was added to inhibit the enzymatic activity, and the absorbance was measured at 410 nm. As a control group, a substrate enzyme reaction solution without 20mM glycylglycine was used.

After γ-GTP enzyme activity, the absorbance (OD) was measured at 410 nm and 50 strains with the highest activity of γ-GTP synthase were first selected (data not shown). As a result of measuring activity, a total of 4 weeks were selected secondarily (FIG. 1). A total of 4 microorganisms were selected as HC188, HH28, MC108, and KC41, respectively, and among them, KC41 isolates having the highest γ-GTP enzyme activity were finally selected.

(2) Determination of activity of thrombolytic enzymes

The thrombolytic activity of the final selected KC41 isolate was examined. The activity of the thrombolytic enzymes of the isolated microorganisms was dissolved in 10 mL of 0.1 M sodium phosphate buffer (pH 7.8) so that fibrinogen was 0.8% and 100 μl of thrombin (100 NIH / ml) was added. 10 mL of 1% agarose solution dissolved in the same buffer solution was added to the mixture, and then sufficiently mixed, immediately poured into a square dish, left at room temperature for 5-10 min, and solidified to prepare a fibrin plate. After incubating the isolated microorganisms in soy medium for 24 hours at 37 ℃ and centrifugation (10,000 × g / 10 min), put the paper disk (8mm, Toyo Roshi, Japan) on the fibrin plate prepared, 10 ㎕ of the culture supernatant After the reaction was added dropwise at 37 ℃ for 24 hours, the size of the resulting transparent degradation ring was compared with the control plasmin (plasmin, 1 unit / ml).

As a result, when compared to the control unit concentration of plasmin 1 unit / ml (100%), KC41 isolates showed thrombolytic activity of about 86% (Fig. 2).

Example  2: KC41 Isolate  Sympathy

(1) Morphological characterization

The microorganisms were isolated and identified by Gram staining and apoptotic staining to examine their morphological characteristics. Morphological observations of KC41 strains with the highest γ-GTP enzyme activity showed microscopic colonies on irregular TSA solid media with irregular wrinkled morphologies. As a result, the gram-positive bacilli that form heat-resistant spores showed the characteristics of a typical Bacillus strain (FIG. 3).

(2) On carbon  Usability survey

The API 50 CHB kit (bioMerieux Co., France) was used to investigate the availability of 49 carbon sources of the KC41 strain, and the results were tentatively analyzed using the API 50 CHB database V3.0 (http://apiweb.biomerieux.com). I identified it. As a result, KC41 strain showed 87% homology with Bacillus amyloliquefaciens , but it was confirmed that there was a difference in 5 sugar utilizations of B. amyloliquefaciens KCCM12090 standard strains such as glycerol, D-xylose, lactose, Trehalose, and β-Gentiobiose. Table 1).

Carbohydrate Result Carbohydrate Result KC41 Bacillus  Amy Lorikyu facience KCCM12090 KC41 Bacillus  Amy Lorikyu facience KCCM12090 Control - - Esculine + + Glycerol - + Salicine + + Erythritol - - Cellobiose + + D-Arabinose - - Maltose + + L-Arabinose + + Lactose + - Ribose + + Melibiose - - D- Xylose + - Saccharose + + L-Xylose - - Trehalose + - Adonitol - - Inulin - - β Methyl-xyloside - - Melezitose - - Galactose - - D-Raffinose + + D-Glucose + + Amidon + + D-Fructose + + Glycogen + + D-Mannose + + Xylitol - - L-Sorbose - - β Gentiobiose - + Rhamnose - - D-Turanose - - Dulcitol - - D-Lyxose - - Inositol - - D-Tagatose - - Mannitol + + D- Fucose - - Sorbitol + + L- Fucose - - αMethyl-D-mannoside - - D-arabitol - - αMethyl-D-glucosamine + + L- Arabitol - - N Acetyl glucosamine + + Gluconate - - Amygdaline + + 2 ceto - gluconate - - Arbutine + + 5 ceto - gluconate - -

* +, positive or slow positive; -, negative or weakly positive

(3) 16S ribosomal DNA gene sequencing  analysis

For more accurate identification, KC41 strain was identified by 16S ribosomal DNA gene sequencing analysis. After taking the cells of the isolated microorganisms and washing the cells twice in sterile physiological saline, DNA was extracted using a DNeasy tissue kit (Qiagen, Valecia, CA, USA).

Universal primer for amplifying the 16S ribosomal DNA gene of the extracted DNA; 27F (5'-AGAGTTTGATCATGGCTCAG-3 ') and 1492R (5'-GGATACCTTGTTACGACTT-3') primers were used. PCR reaction when Takara Perfect Premix (0.4mM dNTP, 0.5units Taq polymerase, 4mM Mg 2 + containing the PCR buffer) DNA template (20㎍ / ml) in 10㎕ 1㎕, each of the forward and reverse primer (1.0μM) 1 μl each was added and the rest was added to distilled water to prepare a total volume of 20 μl. PCR amplification was performed with a Mastercycler gradient (Eppendorf, Hamburg, Germany). PCR reactions were performed 30 cycles for 5 minutes (initial denaturation) at 95 ° C, 45 seconds at 94 ° C (denaturation), 45 seconds at 52 ° C (annealing) and 1 minute at 72 ° C, and 5 minutes at 72 ° C. Final extension was performed. The amplified fragment of about 1400 bp was transformed after binding to a T vector (Invitrogen, Carlsbad, CA, USA). Sequence determination was performed using a T vector sequencing primer. The 16S rDNA gene sequence of KC41 strain was shown as the nucleic acid sequence of SEQ ID NO: 1.

The nucleic acid sequence of SEQ ID NO: 1 was identified by comparison with ribosomal DNA gene sequencing of GenBank (NCBI, Bethesda, MD, USA) using the BLAST search (http://www.ncbi.nlm.nih.gov) program. The phylogenetic tree showing the molecular biological associations obtained as a result of the identification is shown (FIG. 4). According to these results, strain KC41 is Bacillus The closest flexible relationship with amyloliquefaciens was seen. Thus Bacillus strain KC41 Identified as amyloliquefaciens and Bacillus amyloliquefaciens It was named KC41, and it was deposited with Korea Microorganism Conservation Center on Jan. 11, 2012 and was given accession number KCCM 11249P.

In addition, the secondary screening microorganisms HC188, HH28, and MC108 were identified as Bacillus amyloliquefaciens subsp.plantarum, Bacillus licheniformis, Bacillus subtilis, respectively.

Experimental Example : Characteristics of Cheonggukjang prepared using the microorganism according to the present invention

(1) Manufacture of Cheonggukjang

B. amyloliquefaciens with the highest γ-GTP enzyme activity Cheonggukjang was prepared using Baektae, Goesan-gun, Chungbuk, which was harvested in 2010, using KC41 selection strains and MC108, HC188, and HH28 isolates as fermenters. The selected microorganisms were incubated in soy medium liquid medium at 37 ° C. for 24 hours (10 ⁷ CFU / mL) and used as a seed for fermentation of Chungkookjang. Raw soybeans were soaked at 10 ° C. for 24 hours and then drained to increase the temperature at 121 ° C. for 60 minutes. After the increase, the mixture was cooled to 50 ° C. and cultured in a soy medium liquid medium (37 ° C., 24 hr) to inoculate 1% (v / w) of soybean with spawn seed. Cheonggukjang fermentation conditions were fermented for 48 hours at 45 ℃ while the humidity inside the thermostat was maintained at 70%.

(2) Investigation of Physicochemical Properties

The physicochemical properties of Cheonggukjang prototypes prepared by the method were compared.

The microbial test was serially diluted 10 times with sterile distilled water and plated on tryptic soy agar (TSA; Difco, Detroit, MI, USA) plate and incubated for 14 hours at 37 ℃ to count the number of colony produced. The number of viable cells inoculated with boiled beans was fermented at 4.60 to 4.95 Log CFU / g for 48 hours, and 8.81 to 9.68 Log CFU / g.

The moisture content of Chunggukjang was measured using atmospheric pressure drying method. pH was measured by using a pH meter after adding 5 mL of distilled water to 5 g of Cheonggukjang. Amino nitrogen content was measured by Formol titration and crude protein content was measured by Kjeldahl method. For ammonia nitrogen, take 0.1 mL of the same sample liquid as amino nitrogen, and then add 10 g of phenol, 0.05 g of sodium nitroprusside dihydrate, and 1 L of distilled water, 0.9 g of Na ₂ HPO · 12H ₂ O, 6 g of NaOH, 10 mL of NaOCl. 2 mL of 1 L distilled water was added to each solution for 20 minutes at 37 ° C., and then the absorbance was measured at 630 nm using a spectrophotometer. Calibration curves were prepared using ammonia sulfate to calculate ammonia nitrogen. Cheonggukjang viscosity was measured by adding distilled water to Cheonggukjang and suspending (180 rpm, 15 min) to separate the filtrate using a filter network. The filtrate was centrifuged (10,000 × g / 30 min) to take pellet. After washing three times with distilled water, suspended in 50 mL of distilled water, stirred three times by adding cold ethanol of three volumes, centrifuged (14,000 × g / 15 min), and then measured the weight of the precipitate and the maximum length of Cheonggukjang viscous It was. The control group was compared with physicochemical properties using boiled soybean without seed spawn.

The physicochemical characteristics of the prepared Cheonggukjang prototype are shown in Table 2.

Cheonggukjang Prototype Moisture content
(%) pH Amino nitrogen content
( mg %) Ammonia nitrogen content
( mM ) Crude Protein Content
(%) KC41 49.2 7.4 450.0 25.6 57.5 MC108 53.2 7.5 411.7 34.0 55.6 HC188 49.1 7.4 305.8 28.7 55.8 HH28 51.6 7.2 291.6 20.5 57.5 Control ^* 61.0 6.3 26.1 2.0 53.1

As can be seen in Table 2, the water content was maintained in 49.1 ~ 53.2% of all Cheonggukjang, pH was about 7.2 ~ 7.5.

The reason for the high pH during fermentation process is due to ammonia produced. The amino nitrogen content, a measure of Cheonggukjang's delicious taste, is a substance produced by the decomposition of protein during fermentation of Cheonggukjang, and the quality standard of Cheonggukjang products is regulated to more than 280 mg% in the Food Code. All of the produced Cheonggukjang contain amino nitrogen (291.6 ~ 450.0 mg%) above the standard prescribed in the food process, and amino nitrogen content is known to be mainly affected by protease activity. The ammonia nitrogen content of Chunggukjang was 20.5-34.0 mM. Ammonia nitrogen is known to form ammonia nitrogen as the soy protein is hydrolyzed in amino nitrogen form and fermentation proceeds simultaneously. In case of high ammonia nitrogen content in Cheonggukjang, the quality may be interpreted as poor result.

As a result of measuring the length of viscous peculiar to the Cheonggukjang and the amount of sediment deposit (FIG. 6), B. amyloliquefaciens having high γ-GTP enzyme activity The length of the viscous material of the Cheonggukjang prepared with KC41 as a spawn was 95 cm, and the weight of the precipitate was 1.7 g / 100 g, which was the highest. It was judged that there was a correlation between the viscous substance of chungkukjang and γ-GTP enzyme activity.

As a control, boiled soybean was measured with the lowest amino nitrogen and ammonia nitrogen content, and no viscous substance was found, so it was confirmed that the unique viscous substance was produced during fermentation by microorganisms.

B. amyloliquefaciens among fermented seedlings The KC41 strain produces the most viscous material and produces the highest amino amino nitrogen, which determines the taste of Cheonggukjang, and the small amount of ammonia nitrogen, which is a kind of unpleasant ingredient, produces less Chungcheongjang when fermented with KC41. It could be manufactured.

(3) palatability survey

The palatability of Cheonggukjang's prototypes (A, B, C, D samples) was investigated through the questionnaire to find out the tastes of the consumers of the majority and used for quality improvement. The palatability survey was performed to evaluate the appearance, taste, aroma, and comprehensive palatability of Cheonggukjang in 5 stages (very poor, poor, normal, good, very good). Respondents participated in the palatability questionnaire, and they surveyed males and females in their 20s and 60s.The respondents' genders were 54% and 46% for males and females, respectively. 43% for 20s, 9% for 30s, and 15s for 40s. % Were in their 50s, 22% and 11% in their 60s.

As a result, it was found that Cheonggukjang prototype KC41 was excellent in appearance, taste, fragrance, and overall palatability as a whole (Fig. 7). Cheonggukjang's prototype MC108 was too strong in odor and was evaluated for bitter taste. It is judged that consumers prefer the production of viscous substance and the delicious flavor and light taste compared to Cheonggukjang, and it can be judged to be excellent when manufacturing Cheonggukjang by using the fermented seed of fermentation of Cheonggukjang with excellent γ-GTP enzyme activity. .

Korea Microorganism Conservation Center (overseas) KCCM11249P 20120111

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Bacillus amyloliquefaciens KC41 having high activity of          gamma-glutamyltranspeptidase and fibrinolytic enzyme, and          Chungkookjang fermented by using the same <130> P120119 <160> 1 <170> Kopatentin 1.71 <210> 1 <211> 1048 <212> DNA <213> Bacillus amyloliquefaciens KC41 <400> 1 tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60 acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120 atgnttgttt gaaccgcatg gttcagacat aaaaggtggc ttcggctacc acttacagat 180 ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 240 ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300 ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360 gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtgc cgttcaaata 420 gggcggcacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480 ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540 tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600 aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660 tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720 gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780 agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840 gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900 catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960 tcctagagat aggacgtccc cttcgggggc agagtgacag gtggngcatg ggttgtcgtc 1020 agctcgtgtc gtgagatgtt gggttaag 1048

Claims (6)

Bacillus amylocufaciens KC41 ( Bacillus) having the nucleic acid sequence of SEQ ID NO: 1 as a 16S rDNA gene sequence amyloliquefaciens KC41).
The method of claim 1,
Bacillus amylocufaciens KC41 deposited with accession number KCCM 11249P.
A method for producing Chungkookjang comprising fermenting soybeans using Bacillus amylocufaciens KC41 of claim 1 or 2 as a seed.
Cheonggukjang prepared according to the method of paragraph 3.
Food composition comprising the Chunggukjang of claim 4.
Health functional foods for improving thrombotic disease, including the Chunggukjang of claim 4.

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