KR101740545B1 - Bacillus subtilis strain YD-6 and uses thereof - Google Patents

Abstract

Bacillus subtilis, which has excellent enzymatic activity, thrombolytic ability and antimicrobial substance-releasing ability, has a high histamine and tyramine-resolving ability, Bacillus subtilis YD-6 (KACC91956P) and a fermented food containing the same. A microorganism preparation containing the strain or a culture thereof as an active ingredient, a method of producing a microorganism preparation comprising the step of culturing the strain, and a fermented food including the strain.

Description

Bacillus subtilis strain YD-6 and its use {Bacillus subtilis strain YD-6 and uses thereof}

The present invention relates to a Bacillus subtilis YD-6 strain having biosgenic amine-decomposing ability, enzyme activity and thrombolytic ability, and a use thereof. More particularly, the present invention relates to a Bacillus subtilis YD-6 strain having a biogenic amine- Bacillus subtilis YD-6 strain (KACC 91956P), which is excellent in thrombotic resolution and excellent in antimicrobial substance-releasing ability, a microorganism preparation containing the strain or a culture thereof as an active ingredient, and a step of culturing the microorganism preparation A production method thereof, and a fermented food comprising the strain.

Cheonggukjang is one of the traditional fermented foods of Korea, which is one of the traditional fermented foods that have been traditionally used to date with soy sauce, soybean paste, and kochujang, and it plays an important role as a source food of essential amino acids and fatty acids, . In addition to nutritional research on soybean fermented foods such as Chungkukjang, functional studies such as thrombolytic, antioxidant, hypotensive, and atherogenic inhibition have been actively conducted in soybean fermented foods from all over the world.

Chungkookjang has the advantages of short fermentation period compared to other chapters of Korea. It has fermentation process and it is able to ferment the proteins, oligosaccharides, isoflavones, lipids Etc. are decomposed into amino acids, free sugars, isoflavone aglycones, fatty acids and the like which are easily digestible, and secondary products having antioxidative and thrombolytic functions are produced. Cholangiocarcinol has been reported to lower cholesterol, antioxidant, lower blood pressure, antimicrobial and anticancer effects, and it has recently been reconsidered as a functional food by the action of Bacillus.

Soybean products mainly made from soybeans have been produced and used by self-produced before the 1960s (Agricultural, Shrimp and Newspaper Foods, 1995), but the mass-produced products can be easily found on the market due to the development of the food industry. Recently, however, the demand for Chungkookjang has increased as the traditional manufacturing method has become more and more convenient by purchasing goods through the Internet and using courier service rather than commercialized Chungkookjang. The number of products with homogeneous quality is small because the production methods and microorganisms used in the traditional manufacturing method are not the same in each province. In particular, quality varies depending on the type of strains used in fermentation of Cheonggukjang, so it is important to select and use high-performance microorganisms for production of high-quality Cheonggukjang.

Chungkookjang has excellent nutritional and physiological functions, but its consumption is limited due to the content of odor and biogenic amine produced during fermentation. The odor of Chungkookjang is caused by volatile amines produced by the action of microorganisms such as fermenting microorganisms in foods with high protein content, and the biogenic amines are basic nitrogen compounds, which are generally formed by decarboxylation with amino acids as precursors . The formation of biogenic amines in foods affects not only smell but also health. It also affects blood pressure and can cause an allergic reaction to a single person when there is a certain degree of abnormality in the food, causing problems on migraine and bowel. Recently, studies on biogenic amines produced by food microorganisms and studies on reduction of biogenic amines in traditional fields have been actively conducted.

Therefore, the present invention seeks to isolate and isolate strains from traditional fermented chongkukjang to select and characterize microorganisms having excellent functions. To confirm the safety of strains isolated from Chungkookjang prepared by traditional fermentation method and to confirm the secretion of substances capable of inhibiting contaminants by performing the discrimination of 7 kinds of B. cereus toxin genes, Enzyme activity, thrombolytic ability, biogenic amine degradation ability, sensory evaluation, and to provide the best selected strains for use as basic data for setting the quality index of Cheonggukjang when used in the food industry such as fermented starter of Chongkukjang .

A related art is Korean Patent No. 10-0779267, a method for producing chongkukjang strain using Bacillus subtilis strain BN-NUC1 (KCCM-10839P) and Korean patent No. 10-0864850. HA, a method for producing chungkukjang using the same, and chungkukjang produced by the method.

The object of the present invention is to provide a Bacillus subtilis YD-6 strain having excellent thrombolytic and enzyme activity and having a biogenic amine-decomposing ability and less volatile ammonia and amine production during the production of chungkukjang, and a microorganism preparation and a fermented food containing the same .

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

In order to achieve the above object, the present invention provides a strain of Bacillus subtilis YD-6 (KACC91956P) containing the nucleotide sequence of SEQ ID NO: 1 and deposited with Accession No. KACC91956P.

The strain is characterized by its thrombolytic ability and enzyme activity.

The enzyme is characterized by being at least one of amylase, cellulase, protease, and lipase.

The strain is characterized by having an antibacterial substance-releasing ability.

The antimicrobial substance is an antimicrobial substance against at least one of fungi, anthracnose, anthracnose, and invertebrates.

The strain is characterized in that it has the ability to decompose at least one of histamine or tyramine in the biogenic amine.

The strain is characterized in that it does not contain a toxin gene.

The toxin gene is the gene of the enterotoxin Non-hemolytic nheA, nheB, nheC, Hemolytic enterotoxin gene of hblA, hblC and Cytotoxin K gene cytK Or more.

The present invention also provides a microorganism preparation comprising Bacillus subtilis YD-6 strain (KACC91956P) or a culture thereof as an active ingredient.

The present invention also provides a method for producing a microorganism preparation comprising culturing a strain of Bacillus subtilis YD-6 (KACC91956P).

The present invention also provides a fermented food comprising a strain of Bacillus subtilis YD-6 (KACC91956P).

The fermented food is characterized in that it is at least one selected from the group consisting of meju or Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed soup, Korean soy sauce, brewed soy sauce and mixed soy sauce.

In the present invention, it was confirmed that the strain Bacillus subtilis YD-6 isolated from the traditional soybean was excellent in the enzyme activity and thrombolytic ability and the antimicrobial substance-releasing ability with biosgenic amine-decomposing ability. Therefore, Bacillus subtilis YD-6 of the present invention can be used as a useful strain capable of producing a product having excellent functionality and low odor by using it in combination with a seed culture in the production of a traditional product, since it is a safe strain .

Brief Description of the Drawings Figure 1 is a diagram showing the results of amylase, cellulase, lipase and protease activity of Bacillus subtilis YD-6 strain of the present invention.
(A) Amylase, (B) Cellulase, (C) Lipase, (D) Protease
FIG. 2 shows the results of thrombolysis of the strain Bacillus subtilis YD-6 of the present invention.
3 is a diagram showing the presence or absence of the B. cereus toxin gene of the strain Bacillus subtilis YD-6 of the present invention.
FIG. 4 is a graph showing the antimicrobial activity of the strain Bacillus subtilis YD-6.
(A) B, cinerea, (B) C. acutatum, (C) P. capsici, (D) P. palmivora.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail, and a detailed description of known functions and configurations that may unnecessarily obscure the gist of the present invention will be omitted.

The present invention provides a strain of Bacillus subtilis YD-6 (KACC 91956P), which has excellent biosynthetic amine-resolving ability, excellent enzyme activity and thrombolytic ability, and excellent antimicrobial substance-releasing ability.

The Bacillus subtilis strain YD-6 (KACC 91956P) was isolated from a conventional soybean, and has no toxin gene, and has a biogenic amine-degrading ability, thrombolytic activity, and enzyme activity. The Bacillus subtilis YD-6 strain was deposited at the National Institute of Agricultural Science and Technology on Jun. 10, 2014 (Accession No .: KACC 91956P)

In a strain according to an embodiment of the present invention, the biogenic amine may be histamine or tyramine, but is not limited thereto.

The biogenic amine of the present invention is formed from an amino acid by the action of an amino acid decarboxylase of a microorganism. When a biogenic amine is ingested from a food in excess of the decomposition limit of the human body, the biogenic amine may cause rash, local skin inflammation, Vomiting, nausea and diarrhea.

In a strain according to an embodiment of the present invention, the enzyme may be, but not limited to, amylase, cellulase, protease, and lipase.

In the strain according to an embodiment of the present invention, the antimicrobial substance may be, but not limited to, fungus germ, anthrax anthracis, anthracnose germs, and rot fungi.

In the case of the strain according to an embodiment of the present invention, the toxin gene may be a toxin gene produced by Bacillus cereus, which is a harmful microorganism occurring in a soybean, but may be nheA , nheB, nheC , hblA, hblC , cytK, plcRpapR Do not.

The present invention also provides a microorganism preparation comprising the Bacillus subtilis YD-6 strain or a culture thereof as an active ingredient.

The microbial formulation according to one embodiment of the present invention may be prepared in the form of a liquid, and may be added to the granule formulation in the form of a powdered powder or may be granulated by formulating it. However, the formulation is not particularly limited.

The present invention also provides a method for producing a microorganism preparation comprising culturing the Bacillus subtilis strain YD-6. The Bacillus subtilis YD-6 strain of the present invention can be cultured by a conventional method known in the art.

The present invention also provides a food comprising the strain Bacillus subtilis YD-6.

The food according to one embodiment of the present invention is a fermented food, and the fermented food may be a soup, but is not limited thereto.

The fermented food according to an embodiment of the present invention may be a soup made of meju or Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed soup, Korean soy sauce, brewed soy sauce and mixed soy sauce Do not.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, and it is to be understood by those skilled in the art that the present invention is not limited thereto It will be obvious.

Example 1 Preparation of Bacillus subtilis of the present invention ( Bacillus subtilis) Isolation and identification of YD-6 (KACC 91956P) strain, selection

A variety of strains isolated by R2A agar and Raka-Ray agar were streaked on R2A agar (R) agar, which was diluted to 0.85% in saline buffer. And cultured on a plate medium at 28 ° C for 1 to 2 days.

The YD-6 strain was isolated from pure strains by amylase, cellulase, protease and lipase experiments, enzymatic resolution, presence of toxic genes, biosynthetic amine decomposition, and production of volatile ammonia and amines during fermentation of chungkukjang. Were selected.

In order to classify the strain YD-6 of the present invention, the base sequence (SEQ ID NO: 1) was determined by amplifying 16S rRNA using PCR as follows.

In order to classify isolated strains, 16S rRNA was amplified using PCR as follows to determine the base sequence. Genomic DNA was extracted (DNA purification kit, Promega, USA) from Geno Tech Corp. (Genotech, Deajeon, Korea) to amplify the 16S rRNA nucleotide sequence, and a universal PCR primer 27F (5 ' -AGAGTTTGATCCTGGCTCAG-3 '; SEQ ID NO: 2) and 1492R (5'-GGTTACCTTGTTACGACTT ?? 3; SEQ ID NO: 3). Using the 16S rRNA seqeunce obtained from the above experiment, the homology between the isolates and the registered strains was compared using the Blast Search program of Ezbiocloud (http://www.ezbiocloud.net/eztaxon) provided by Chunlab, Inc. . In order to determine the phylogenetic location of the isolates, we used the Advenced Blast Search program of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) The nucleotide sequences were analyzed using the MegAlign program (DNA STAR Lagergene, version 8) and the Mega 6.0 program neighbor-joining method.

The homology of the 16S rRNA sequence of the YD-6 strain was compared with that of Bacillus subtilis . The strain of the present invention was named Bacillus subtilis YD-6 and deposited under the accession number KACC 91956P on June 10, 2014 at the National Institute of Agricultural Science (National Institute of Agricultural Science and Technology, KACC).

≪ Example 2 > Analysis of enzyme activity

In order to investigate the enzyme activity of the strain Bacillus subtilis YD-6 of the present invention, enzymatic activity and thrombolytic activity of amylase, cellulase, protease and lipase were examined in a selection medium.

- amylase activity

R2A (Difco) was added with 0.5% soluble starch, which is an amylase production inducer. Two strains were inoculated into the plate culture medium and incubated at 28 ° C for 24 hours. The culture medium was stained with Gram's iodine solution (1.0 g of iodine crystal, 0.2 g of potassium iodine, and 30 ml of distilled water) Was confirmed through a clear zone.

- cellulase activity

The R2A medium supplemented with 0.4% CMC (carboxymethyl cellulose) was inoculated in a confluent culture method, cultured at 28 ° C for 24 hours, stained with 0.1% congo red solution, washed with 1M NaCl, Respectively.

- protease activity

2% skim milk and 3% agar were sterilized to prepare a culture medium. Then, the strain was inoculated with two strains in a confluent culture method and cultured at 28 ° C for 24 hours to confirm a clear zone.

- lipase activity

To 450 ml of R2A agar, 50 ml of mixture [5 ml of glycerol tributyrate, 1 ml of 0.5 M CaCl ₂ , 2 ml of 5 M NaCl and 2.5 g of gum arabic (from acacia tree) were mixed using a mixer After sterilization with a high pressure steam sterilizer at 121 ° C for 15 minutes, a solid medium was prepared by mixing, and the clear zone was confirmed by inoculation of the bacteria with the substitution culture method.

- thrombotic resolution

Fibrin plate method. 0.5% fibrinogen (containing 0.067M sodium phosphaste buffer) was prepared, and the mixture was completely dissolved at 37 ° C for 30 minutes. Thrombin was added at 1% and the mixture was divided. After 8 hours of inoculation with the strain on the hard medium, the solubility of fibrin in the medium was confirmed.

Brief Description of the Drawings Fig. 1 is a schematic view showing the structure of Amylase (Fig. 1A), cellulase (Fig. 1B), lipase (Fig. 1C), Bacillus subtilis YD- ), And protease (Fig. 1 (D)). FIG. 2 is a photograph showing the result of thrombolysis of Bacillus subtilis strain YD-6 of the present invention by the fibrin plate method. It was confirmed that the strain of the present invention had high thrombolytic ability I could.

As shown in FIG. 1 and Table 1, the Bacillus subtilis YD-6 strain of the present invention showed high activity in both amylase, cellulase, protease and lipase, which are fermentation-related enzymes, Which is higher than that of Bacillus subtilis KACC16479 and Bacillus subtilis A.

Enzyme activity of YD-6 Strain Blast Enzyme activity Amy. Cell. Pro. Lip. Fibrin. YD-6 B. subtilis subsp . subtilis + + + + (+) KACC16749 B. subtilis subsp . subtilis + + + + - A B. subtilis subsp . subtilis + + + + (+)

Symbols: -; negative activity, (+); weak activity, +; positive activity, Amy .; amylase, Cell; cellulase, Pro .; protease, Lip .; lipase, Fibrin .; fibrinolysis KACC 16749, A: comparative strain

≪ Example 2 > Bacillus cereus ( B.cereus ) Check for toxin gene

In order to ensure the safety of the strain Bacillus subtilis YD-6 of the present invention, Bacillus cereus , 6 genes for diarrhea toxin and 1 gene for diarrhea toxin expression transcriptional regulation were identified by PCR. Control strains were purchased from KACC Bacillus cereus Standard strain (KACC 11204) was used.

The toxin genes of Bacillus cereus used in this experiment are as shown in Table 2, and include 3 genes ( nheA ; nheB ; nheC ) of non-hemolytic enterotoxin, 2 types of hemolytic enterotoxin genes ( hblA ; hblC ) Cytotoxin K gene (cytK), and diarrhea toxin expression transcriptional regulatory protein, phospholipase C regulatory gene (plcRpapR) of the protein, conditions for PCR, after 2 minutes at 94 ℃ initial denaturation, 94 ℃ 1 bungan denaturation, 56 ℃, 58 ℃, The amplification was carried out at 72 ° C for 5 minutes. The amplification was carried out at 72 ° C for 30 minutes (72 ° C for 1 minute, 72 ° C for 1 minute).

Bacillus cereus toxic gene sequence and binding temperature. Primer pair Sequence Target sequence
(GeneBank accession no.) Size
(bp) Annealing temp. nheAF ATATGCGCAAAATGTAATTGCTCCA
(SEQ ID NO: 4) Non-hemolytic enterotoxin,
nheA (AY835995) 935
56 ℃
nheAR TGCCTTCTTCAACATTTGTTTGAATTT
(SEQ ID NO: 5) nheBF GACCAGCAGGATTCCCAGATGTAAT
(SEQ ID NO: 6) Non-hemolytic enterotoxin,
nheB (AY835995) 972
62 ° C
nheBR CCACGCCTTCATGTAATTTTTCTGT
(SEQ ID NO: 7) nheCF ACCAGTAGCGACTTTTGCAAGTGAA
(SEQ ID NO: 8) Non-hemolytic enterotoxin,
nheC (AY835995)
930
62 ° C
nheCR TTTTGAGCTGCATTCTCAATATGCC
(SEQ ID NO: 9) HBlAF ACCAGTAGCGACTTTTGCAAGTGAA
(SEQ ID NO: 10) Hemolytic enterotoxin,
HBla (AY822584)
909
56 ℃
HBlAF TTTTGAGCTGCATTCTCAATATGCC
(SEQ ID NO: 11) hblCF ATCAATACTCTCGCAACACCAACTG
(SEQ ID NO: 12) Hemolytic enterotoxin,
hblC (AY822584)
957
62 ° C
hblCR ATGTGCTCGTTGCTCTGCTGTTAAT
(SEQ ID NO: 13) cytKF CCGCTGTTTTTGCTAGTAGTGCTGT
(SEQ ID NO: 14) Cytotoxin K,
cytK (DQ019311)

901
58 ℃
cytKR ACGTCTTTTACGTTGTTTCCAACCC
(SEQ ID NO: 15) plcRF CRGGYGCRGTATACCCAAGT
(SEQ ID NO: 16) Phospholipase C regulatory protein, ( plcRpapR (58 ) 888
58 ℃
plcRR TGAAATACCCCATGYCATYG
(SEQ ID NO: 17)

Since Bacillus subtilis YD-6 is not only a phylogenetically close to Bacillus cereus, but also a strain isolated from a fermentation broth prepared by a conventional fermentation method, not a method of inoculating a strain, the Bacillus subtilis YD- And to confirm the safety of the test strains. As shown in FIG. 3, Bacillus subtilis YD-6 did not have six diarrhea toxin genes of Bacillus cereus and one gene of diarrheal toxin expression transcriptional regulatory gene.

≪ Example 3 > Quantitative analysis of resolving ability of biogenic amine

The histamine and tyramine resolving ability of Bacillus subtilis YD-6 of the present invention was quantitatively analyzed by HPLC in order to examine the biosgenic amine degradability.

The strain was inoculated with nutrient broth containing 1% histamine and tyramine and then cultured at 38 ° C for 20 days. After incubation, centrifugation was carried out at 1300 rpm for 5 minutes. To 200 상 of the supernatant, 2 내부 of internal standard solution 2M Diaminoctane was added.

After adding 300 μl Na ₂ CO ₃ solution (100 mg / ml in water) and 800 μl dansyl chloride solution (7.5 mg / ml in acetone), the mixture was dansilated at 60 ° C for 20 minutes.

After adding 100 μl of proline solution (50 mg / ml in water), the mixture was allowed to stand at 60 ° C for 10 minutes and then cooled at 5 ° C. Finally, 100 μl of toluene was added and shaken, followed by centrifugation for 5 minutes, followed by HPLC analysis using the supernatant.

The analysis conditions of the HPLC for the above experiment are as follows.

Amines were extracted using an Alliance e2695 HPLC system (Waters Co., Milford, MA, USA) and a 474 fluorescence detector (Waters Co., Milford, Mass., USA).

The flow rate was 1 ml / min using a C18 column (Waters, X Bridge C18, 4.6 X 250 mm 3.5 μm), and the concentration gradient of the mobile phase is shown in Table 3 below.

The concentration gradient of the HPLC mobile phase used in the present invention min A (Water)% B (Acetonitrile)% 0 60 40 5 60 40 35 15 85 40 15 85 54 0 100 55 60 40 60 60 40

 After 20 days, the resolving ability of 1% histamine, tyramine was confirmed. As shown in Table 4, YD-6 showed 88.3% histamine and 40.1% tyramine resolution, and showed higher resolution than KACC16749 and A, the control group.

Biogenic amine resolution of YD-6 Strain Blast Histamine Tyramine before after 20days before after 20days YD-6 B. subtilis subsp . subtilis 62832189 7323201 82912994 49610797 KACC16749 B. subtilis subsp . subtilis 54895888 10869051 74636877 48827886 A B. subtilis subsp . subtilis 58014297 12590688 77478043 49389937

≪ Example 4 >

Five strains of phytopathogenic fungi were purchased from KACC. The strains that were sold were Asymptomatic fungi (KACC40574, Botrytis cinerea); Red pepper anthracis (KACC40042, Colletotrichum acutatum); Anthracnose fungi (KACC40003, C. gloeosporioides); Pepper sprout (KACC40157, Phytophthora capsici); (KACC40176, P. palmivora). ≪ / RTI > The R2A agar was inoculated with the fungus in the center and 4 different strains were used to inoculate the chungkukjang isolate. Bacillus subtilis KACC16479 and Bacillus subtilis Mpul-4 were used as control strains.

As shown in FIG. 4, YD-6 was found to be more effective than yeast fungus (Fig. 4A), red pepper anthracis 4B), pepper root rot (FIG. 4C), and reverse rot (FIG. 4D). Table 5 shows the antimicrobial activity of Bacillus subtilis YD-6, Bacillus subtilis KACC16479 and Bacillus subtilis Mpul-4 of the present invention.

Antimicrobial substance releasing ability Strain Blast Antifungal test B C. a. C. g. P. c. P. p. YD-6 B. subtilis subsp . subtilis + + - + + KACC16749 B. subtilis subsp . subtilis (+) - (+) (+) (+) Mpul-4 B. subtilis subsp . subtilis - - (+) - -

Symbols: -; negative activity, (+); weak activity, +; positive activity, B .; B. cinerea , C. a ; C. acutatum , C. g; C. gloeosporioides , P. capsici , P. p; P. palmivora ..

Based on the above results, the present invention is based on the above findings, and it is an object of the present invention to provide a method for producing chrysanthemum pumice, which has excellent thrombolytic and enzymatic activity and antimicrobial activity and ability to degrade histamine and tyramine, Bacillus subtilis YD-6 strain and food containing the same can be provided.

Korea Agricultural Microbiology Resource Center KACC91956P 20140721

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Bacillus subtilis strain JP-5 and uses thereof <130> P2015-0040 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1492 <212> RNA <213> Bacillus subtilis YD-6 <400> 1 tggctcagga cgaacgctgg cggcgtgcct aatacatgca agtcgagcgg acagatggga 60 gcttgctccc tgatgttagc ggcggacggg tgagtaacac gtgggtaacc tgcctgtaag 120 actgggataa ctccgggaaa ccggggctaa taccggatgg ttgtttgaac cgcatggttc 180 aaacataaaa ggtggcttcg gctaccactt acagatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcgacgat gcgtagccga cctgagaggg tgatcggcca 300 cactgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca 360 atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag 420 ctctgttgtt agggaagaac aagtaccgtt cgaatagggc ggtaccttga cggtacctaa 480 ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt ggcaagcgtt 540 gtccggaatt attgggcgta aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc 600 cccggctcaa ccggggaggg tcattggaaa ctggggaact tgagtgcaga agaggagagt 660 ggaattccac cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg 720 cgactctctg gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag 780 ataccctggt agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct 840 tagtgctgca gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac 900 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac 960 gcgaagaacc ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt 1020 cgggggcaga gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt 1080 taagtcccgc aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta 1140 aggtgactgc cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct 1200 tatgacctgg gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg 1260 ttaagccaat cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg 1320 aagctggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt 1380 gtacacaccg cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt 1440 ttaggagcca gccgccgaag gtgggacaga tgattggggt gaagtcgtaa ca 1492

Claims (12)

(SEQ ID NO: 1) and has an enzymatic activity of at least one of thrombolytic ability and amylase, cellulase, protease, and lipase, histamine and histamine in biogenic amines (tyramine) of any one or more of having a resolution, Non-hemolytic gene nheA, nheB, nheC, Hemolytic enterotoxin gene of the enterotoxin hblA, hblC and Bacterium Bacillus subtilis YD-6 deposited with Accession No. KACC91956P, characterized in that it does not contain any one or more toxin genes of cytok , cytotoxin K gene.
delete delete The method according to claim 1,
Bacillus subtilis YD-6 strain capable of releasing antimicrobial substances against any one or more of fungus, pepper anthracnose, pepper spp., And invertebrate.
delete delete delete delete A microbial preparation comprising the Bacillus subtilis strain YD-6 (KACC91956P) or a culture thereof as an active ingredient according to any one of claims 1 to 4.
A method for producing a microbial agent, comprising culturing the Bacillus subtilis strain YD-6 (KACC91956P) of claim 1 or 4.
A fermented food comprising the strain Bacillus subtilis YD-6 (KACC91956P) of claim 1 or 4.
12. The method of claim 11,
Wherein the fermented food is at least one selected from the group consisting of meju or Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed jang, Korean traditional soy sauce, brewed soy sauce and mixed soy sauce.

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