KR101730063B1 - Bacillus methylotrophicus strain MH-2 and uses thereof - Google Patents

Abstract

Bacillus methylotrophicus ( Bacillus methylotrophicus) , which has excellent enzyme activity and low production and resolution of biogenic amines, low volatile ammonia and amine production during production of chungkukjang , good odor, good taste and quality, ) MH-2 (KACC92038P) 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 methylotrophicus MH-2 strain and its use {Bacillus methylotrophicus strain MH-2 and uses thereof}

The present invention relates to a Bacillus methylotrophicus MH-2 strain (KACC 92038P) having an enzyme activity and a biogenic decomposition ability, a microorganism preparation containing the strain or a culture thereof as an active ingredient, and a step of culturing the microorganism preparation And a fermented food containing 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.

Chungkukjang, which has a short fermentation period compared to other chapters in Korea, has a fermentation process that can ferment soybeans by the secretion of various enzymes such as amylase, cellulase, protease and lipase of microorganisms. A free amino acid, a free sugar, an isoflavone aglycon, a fatty acid and the like contained in the protein, oligo polysaccharide, isoflavone, and lipid contained in the protein, oligosaccharide, isoflavone and lipid are decomposed into a secondary product having antioxidative and thrombolytic functions. Antioxidants, antihypertensive effects, antimicrobial and anticancer effects have been reported as cholangiocarcinol effects, and they have recently been reexamined as functional foods due to 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. In order 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 carrying out discrimination of 7 kinds of B. cereus toxin genes, Enzyme activity, thrombolytic ability, biogenic amine degradation ability, sensory evaluation and volatile amine test were conducted. In order to use as a basic data for setting the quality index of Cheonggukjang when used in the food industry such as fermented starch fermented starch, ≪ / RTI >

Korean Priority Patent No. 10-1379230 (Bacillus licheniformis SCK B11 strain having antimicrobial activity against harmful microorganisms and biogenic amine degrading activity and its use) and Korea Patent No. 10-0779267 (Bacillus sub- Tillis NNT-0701 strain and Chungkookjang produced thereby) and Korea Patent No. 10-0831420 (Bacillus genus MK-15 having thrombolytic ability).

An object of the present invention is to provide a method for producing Bacillus methyltropicus MH, which has excellent enzyme activity and low production and resolution of biogenic amines, produces less volatile ammonia and amines when producing chungkukjang, has no odor, -2 strain, a microorganism preparation containing the same, and a fermented food.

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 methylotrophicus MH-2 containing the nucleotide sequence of SEQ ID NO: 1 and deposited with Accession No. KACC92038P.

The strain is characterized by an enzyme activity.

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

The strain is characterized in that it does not produce any one or more of histamine and tyramine in the biogenic amine.

The strain is characterized in that it has the ability to decompose at least one of tyramine, cadaverine, and putrescine in the biogenic amine.

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

The toxin genes include nheA, nheB, nheC , hemolytic enterotoxin genes hblA , hblC And cytok < RTI ID = 0.0 > Or more.

The strain is characterized by being capable of producing any one or more of cysteine and methionine which are the limited amino acids of soybean.

The present invention also provides a microbial agent comprising Bacillus methylotrophicus MH-2 strain (KACC92038P) 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 methylotrophicus MH-2 (KACC92038P).

The present invention also provides a fermented food comprising Bacillus methylotrophicus MH-2 strain (KACC92038P).

The fermented food is characterized by having one or more of the restricted amino acids cysteine, histidine and methionine of soybean.

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

The content of volatile ammonia and amine in the fermented food is lower than the content of volatile ammonia and amine in the fermented food not containing the strain.

In the present invention, the Bacillus methylotrophicus MH-2 strain isolated from a traditional herb has low production and resolution of biorenic amines, has excellent enzyme activity, produces less volatile ammonia and amines when producing chungkukjang , It was confirmed that the production capacity was excellent. Therefore, Bacillus methylotrophicus MH-2 of the present invention is a safe strain, so it can be used as a useful strain that can be used in the production of traditional soup with no germination and excellent in taste and nutrition .

Brief Description of the Drawings Fig. 1 is a diagram showing the presence or absence of B. cereus toxin gene of Bacillus methylotrophicus MH-2 strain (KACC 92038P), which is a strain of the present invention.

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 Bacillus methylotrophicus MH-2 strain (KACC 92038P), which is excellent in enzyme activity and has little volatile ammonia and amine production during fermentation of chongkukjang .

The Bacillus methylotrophicus MH-2 strain (KACC 92038P) was isolated from a traditional herb, has no toxin gene, does not produce a biogenic amine, has a resolution, and has an enzyme activity. The strain Bacillus methylotrophicus MH-2 was deposited at National Academy of Sciences on Feb. 11, 2015 (Accession No .: KACC 92038P)

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

The biogenic amines having a resolution in the strain according to an embodiment of the present invention may be, but not limited to, tyramine, cadaverine or putrescine.

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 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 methylotrophicus MH-2 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.

In addition, the present invention provides a method for producing a microbial agent comprising culturing the Bacillus methylotrophicus MH-2 strain. The Bacillus methylotrophicus MH-2 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 methylotrophicus MH-2.

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.

In the fermented food according to an embodiment of the present invention, the soup is selected from the group consisting of meju, Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed soup, Korean soy sauce, brewed soy sauce and mixed soy sauce But the present invention is not limited thereto.

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 methylotrophicus ( Bacillus methylotrophicus) Isolation and Identification of MH-2 (KACC 92038P) 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.

In the case of pure isolates, the enzyme activity (amylase, cellulase, protease, lipase), the presence of toxic genes, the production and resolution of biogenic amines, the production of volatile ammonia and amines during fermentation of chongkukjang, The strains were selected.

To classify the MH-2 strain of the present invention, 16S rRNA was amplified using PCR as follows to determine the nucleotide sequence (SEQ ID NO: 1).

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 MH-2 strain was compared and showed similarity to Bacillus methylotrophicus . The strain of the present invention was designated as Bacillus methylotrophicus MH-2 and deposited with the National Academy of Agricultural Sciences (KACC) on Feb. 11, 2015 to receive the accession number KACC 92038P.

≪ Example 2 > Analysis of enzyme activity

The enzymatic activity of amylase, cellulase, protease and lipase was confirmed in the selection medium to investigate the enzyme activity of the Bacillus methylotrophicus MH-2 strain of the present invention. Bacillus subtilis KACC16749 and Bacillus subtilis A were used as control strains.

- 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 the R2A agar, 50 ml of the mixture [5 ml of glycerol tributyrate, 1 ml of 0.5 M CaCl ₂ , 2 ml of 5 M NaCl, 2.5 g of gum arabic (from acacia tree)] was mixed using a mixer After sterilization using a high pressure steam sterilizer at 121 ° C for 15 minutes, a solid medium was prepared and the bacteria were inoculated in a confluent culture method to confirm a clear zone.

As shown in Table 1, the Bacillus methylotrophicus MH-2 strain of the present invention is similar to Bacillus subtilis , which is a control strain, and contains amylase, cellulase, protease , And lipase, respectively.

Enzyme activity of MH-2 Strain Blast Enzyme activity Amy. Cell. Pro. Lip. MH-2 Bacillus methylotrophicus + + + + 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 .; KACC 16749, A: control strain

≪ Example 3 > Bacillus cereus ( B.cereus ) Check for toxin gene

In order to ensure the safety of the Bacillus methylotrophicus MH-2 strain 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 methylotrophicus MH-2 is not only a phylogenetically close to Bacillus cereus but also a strain isolated from chungkukjang prepared by a conventional fermentation method, not by the method of inoculating the strain, by confirming presence or absence of the Bacillus cereus toxin gene And to secure the safety of the test strains. As shown in FIG. 1, the Bacillus methylotrophicus MH-2 strain had no genes for six diarrheal toxin genes of Bacillus cereus and one gene for regulating diarrhea toxin expression transcription. As a control strain, Bacillus cereus strain KACC 11204, which was distributed in KACC, was used.

<Example 4> Biogenic amines production ability and resolution analysis

The biosynic amine production and resolution of Bacillus methylotrophicus MH-2 of the present invention were analyzed.

- Production of biogenic amines

 PH 5.3 with 0.5% histidine, 0.5% tyrosine and 0.006% cresol red Inoculated with Nutrient agar medium [Nutrient Broth (Difco, USA), Bacto agar, destiled water] And the change in the color of the synthetic medium was confirmed after 24 hours at 28 ° C.

- Biogenic amines Resolution

The strains were inoculated into the minimal nutrient medium (0.1% tyramine, 0.05% cadaverine, 0.05% putrecine, 0.2% KH ₂ PO ₄ , 0.1% NaCl, 0.01% MgSO ₄ , 1.2% agar, pH 7.0) Colonies were observed after 24 hours.

In order to examine the histamine and tyramine production ability and tyramine, cadaverine and putrecine resolving ability, strains were inoculated into the synthetic medium and colonies were observed after 24 hours at 28 ° C Results: Among the control strains, KACC16749 produced histamine and tyramine and showed no resolution, and strain A showed histamine and tyramine production and resolution. However, the MH-2 strain produced less histamine and tyramine, and showed resolution of tyramine, cadaverine, and putrecine.

Amine production and resolution of MH-2 Strain Blast Biogenic amine Production Degradation MH-2 B. methylotrophicus (+) + KACC16749 B. subtilis subsp . subtilis + - A B. subtilis subsp . subtilis + +

Symbols: -; negative activity, +; positive activity, (+); week activity

&Lt; Example 5 > Production of Cheonggukjang and Analysis of Quality of Cheonggukjang

- Chongkukjang Manufacturing

1.5 times water was added to the washed soybeans and the mixture was heated at 121 ° C for 20 minutes. 10 g of boiled soybeans maintained at a temperature of 40 ° C. were mixed with 250 μl of Bacillus methylotrophicus MH-2 culture medium (1 × ^{10 9} CFU / ml) and cultured at 40 ° C. for 72 hours to prepare chungkukjang.

- Determination of volatile amines and ammonia

In order to measure volatile ammonia and amines of fermented Chungkookjang using Bacillus methylotrophicus MH-2, chungkukjang was first prepared by the above-mentioned method of producing chungkukjang. Bacillus subtilis KACC16749 and Bacillus subtilis A were used as control strains. Chungkukjang was prepared by the same method. The content of ammonia and amine in 50 ml of gas inside the silicon container was measured by using a detector tube type gas detector (GASTEC, GV-100S, Japan) after shaking for 1 minute and then waiting for 5 minutes. At this time, an ammonia detection tube (Ammonia; NH3, No.3L, Japan) and an amine detection tube (Amines (Methylamine); R? NH2 No.180, Japan) were used.

The measurement results are shown in Table 4. In 50 ml of gas, Bacillus methylotrophicus MH-2 showed 7 ppm ammonia and 14 ppm amine, which is similar to that of Bacillus subtilis KACC16749 among the control strains and lower volatile ammonia Amines were identified.

Strain Blast ppm / 50 ml Ammonia Amines MH-2 B. methylotrophicus 7 14 KACC16749 B. subtilis subsp . subtilis 6.5 13 A B. subtilis subsp . subtilis > 35 > 120

Symbols:>; excess

- ^One H-NMR analysis

0.1 g of chrysanthemum lyophilized powder was dissolved in 600 μl of deuterium oxide (D2O, 99.9%) and 5 mM sodium-2,2-dimethyl-2silapentane-5-sulfonate (DDS, 97%). After centrifugation, the supernatant was transferred to an NMR tube and measured using a 600 MHz NMR spectrometer (Avance 600, Bruker, Germany). Identification and concentration of amino acid metabolites were analyzed using the Chenomx NMR suite program (ver. 7.7, Chenomx, Canada).

As a result, the MH-2 was able to produce the important amino acids present in the natural environment including the 20 standard amino acids by using the polymer of the soybean, as well as the limited amino acid cysteine, histidine ), The production of methionine and the production of nine essential amino acids were also excellent (Table 5).

Analysis of Amino Acid Components of Chungkukjang Fermented by Bacillus methylotrophicus MH-2 Strain. Compound μmol / g ^-1 dry C heonggukjang Control MH-2 common
amino acid field 224.7 54.68 arginine 597.7 80.3 asparagine 89.7 44.22 aspartate 320.6 110.31 cysteine - 71.08 glutamate 670.7 348.26 glutamine 121.3 112.19 glycine 995.1 55.71 histidine - 24.3 iso- leucine 43.8 152.27 leucine 123.4 443.85 lysine 150.9 129.59 메티오 로 - 54.8 피 phenylalanine 41.4 183.98 proline - 37.72 serine 1648.7 371.99 threonine 181.3 99.34 tryptophan 123.4 19.37 tyrosine 46.4 97.87 valine 59.5 275.48 betaine 9 28.01 cystine 235 12.17 pyroglutamate - 110.76 saccharopine 375.9 38.79 β-domain - 31.04

Symbols: -; undetected, Control is boiled soybeans. Control: Boiled beans Bold worlds: Essential amino acids

Based on the above results, the present invention has been made based on the above findings. It is an object of the present invention to provide a biosynthetic pharmaceutical composition which is excellent in enzyme activity, has low production and resolution of biogenic amines and produces less volatile ammonia and amines when producing chungkukjang, It is possible to provide a strain of Rottrophic MH-2 and a fermented food containing the same.

Korea Agricultural Microbiology Resource Center KACC92038P 20150311

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Bacillus methylotrophicus strain MH-2 and uses thereof <130> P2015-0043 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1409 <212> RNA <213> Bacillus methylotrophicus MH-2 <400> 1 gcagtcgagc ggacagatgg gagcttgctc cctgatgtta gcggcggacg ggtgagtaac 60 acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct aataccggat 120 ggttgtttga accgcatggt tcagacataa aaggtggctt cggctaccac ttacagatgg 180 acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcracg atgcgtagcc 240 gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300 cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360 tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg 420 gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480 taatacgtag gtggcaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt 540 tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 600 cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 660 gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc 720 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780 tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga 840 gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900 tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc 960 ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat cttagttgcc 1080 agcattcagt tgggcactct aaggtgactg ccggtgacaa accggaggaa ggtggggatg 1140 acgtcaaatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggacagaaca 1200 aagggcagcg aaaccgcgag gttaagccaa tcccacaaat ctgttctcag ttcggatcgc 1260 agtctgcaac tcgactgcgt gaagctggaa tcgctagtaa tcgcggatca gcatgccgcg 1320 gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca ccacgagagt ttgtaacacc 1380 cgaagtcggt gaggtaacct ttaggagcc 1409

Claims (12)

Bacillus methylotrophicus MH-2 strain containing the nucleotide sequence of SEQ ID NO: 1 and deposited with Accession No. KACC92038P.
The method according to claim 1,
Bacillus methylotrophicus MH-2 strain having an enzyme activity of at least one of amylase, cellulase, protease, and lipase.
The method of claim 1,
Bacillus methylotrophicus MH-2 strain having the ability to decompose at least one of tyramine, cadaverine, and putrescine among biogenic amines .
delete delete The method according to claim 1,
Bacillus methylotrophicus MH-2 strain having the ability to produce at least one of cysteine, histidine and methionine which are the limited amino acids of soybean.
A microbial agent comprising the Bacillus methylotrophicus MH-2 strain (KACC92038P) or a culture thereof as an active ingredient according to any one of claims 1 to 3 or 6.
A method for producing a microbial agent comprising culturing the Bacillus methylotrophicus MH-2 strain (KACC92038P) according to any one of claims 1 to 6.
A fermented food comprising Bacillus methylotrophicus MH-2 strain (KACC92038P) according to any one of claims 1 to 6.
10. The method of claim 9,
A fermented food having at least one of the restricted amino acids cysteine, histidine and methionine of soybean.
10. The method of claim 9,
Wherein the fermented food is selected from the group consisting of meju, Korean miso, miso, seasoned miso, kochujang, seasoned kochujang, chunjang, chungkukjang, mixed jang, Korean soy sauce, brewed soy sauce and mixed soy sauce.
10. The method of claim 9,
Wherein the content of volatile ammonia and amine in the fermented food is lower than the content of volatile ammonia and amine in the fermented food not containing the strain.

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