KR20150101789A - Bacillus subtilis BK418 strain having complex enzyme productivity and antifungal activity and uses thereof - Google Patents

Abstract

The present invention relates to a Bacillus subtilis BK418 (KACC91903P) strain having composite enzyme productivity and antifungal activities, and a use of the same. More specifically, the present invention relates to a probiotic composition, an antibacterial composition, an environmentally purifying composition, a cosmetic ingredient, a fermented food, and a health functional food, comprising a Bacillus subtilis BK418 strain producing protein, starch, lipid, and cellulose decomposing enzyme and having antibacterial power in both staphylococcus which is a gram positive bacillus and colon bacillus which is a gram negative bacillus, or a culture medium thereof as an active ingredient. The Bacillus subtilis BK418 strain or the culture medium thereof of the present invention does not exhibit antibacterial activities to effective microorganisms such as lactobacillus and yeast to have excellent antibacterial activities and antagonistic effects specifically to pathogenetic bacteria, thereby being provided as a composite microorganism with an effective microorganism.

Description

(Bacillus subtilis BK418 strain having complex enzyme production and antifungal activity and uses thereof)

The present invention relates to a Bacillus subtilis BK418 strain having a complex enzyme production ability and an antimicrobial activity, and more particularly to a Bacillus subtilis BK418 strain producing protein, starch, lipid and fibrinolytic enzyme, The present invention relates to a biocide composition, an antimicrobial composition, an environmental purification composition, a cosmetic raw material, a fermented food, and a health functional food containing a Bacillus subtilis BK418 strain or a culture thereof having antibacterial activity against both Escherichia coli and Gram-

Chungkukjang has been traditionally used as a protein source for a long time in Korea as soybean fermented food. Other representative soybean fermented foods include soy sauce and soybean paste. The manufacturing process of Chungkukjang is to boil the beans in rice straw or silkworm, then ferment for 23 days in a warm room and use them for food. Chungkookjang is expected to have anticancer effects such as anticancer, antioxidant, blood sugar control, hypertension, immune enhancement, thrombolysis, diabetes prevention, heart disease, osteoporosis inhibition and antibacterial effect. Interest in food is on the rise. It is known that Bacillus, which is a fermenting microorganism of Chungkukjang which contributes to the generation of such physiological activity, weakens activity of intestinal microbes and has antimicrobial activity against pathogenic bacteria. In other words, it is a beneficial bacteria that suppresses the activity of the decaying bacteria and reduces the carcinogenic substances such as ammonia, indole, amine and the like, and adsorbs and excretes harmful substances.

On the other hand, Bacillus subtilis is a bacterium belonging to the genus Bacillus that lives in soil and is gram-positive apo-forming bacteria. It is aerobic and has heat resistance. It is an organic substance decomposing microorganism which is metabolized by various saccharides such as glucose and starch as an energy source and is also used for fermented foods such as meju and chungkukjang. Bacillus subtilis is known as a microorganism that breaks down pectin and polysaccharides in plant tissues. It is also a microorganism capable of producing enzymes capable of degrading various substances such as lipids, proteins and starches. In addition, Bacillus subtilis, which produces antimicrobial substances capable of inhibiting pathogenic bacteria, is also known as a biological agent because of its various biochemical and physical properties and its stability against bacteria.

As a conventional technique, Patent Document 1 relates to Bacillus subtilis isolated from meju and an antimicrobial composition comprising the same. The antimicrobial composition comprises Bacillus subtilis MJP1 having antimicrobial activity against Gram-positive bacteria and antifungal activity, However, Bacillus subtilis, which has antimicrobial activity against both Gram-positive bacteria and negative bacteria, has not been proposed while producing various degrading enzymes.

1. Korean Patent No. 10-0919892

The present invention has been made in view of the above needs, and it is an object of the present invention to provide a microorganism capable of producing a complex enzyme capable of degrading protein, lipid, starch and fibrin, and capable of producing Escherichia coli and Staphylococcus aureus , And Bacillus subtilis strain BK418 which is excellent in antimicrobial activity against pathogenic bacteria such as Bacillus subtilis strain BK418 and a culture thereof.

The present invention also provides a novel method for culturing Bacillus subtilis strain BK418 using sugar or molasses as a single nutrient source.

The present invention also provides a Bacillus subtilis BK418 strain and a culture thereof and a biocide composition, an antimicrobial composition, an environmental purification composition, a cosmetic raw material, a fermented food and a health functional food containing the same as an active ingredient.

The Bacillus subtilis strain BK418 (KACC91903P) according to the present invention for the above-mentioned problem to be solved is characterized by having a complex enzyme producing ability and antimicrobial activity.

In one embodiment of the present invention, it has a digestive enzyme production ability with respect to protein, starch, lipid and fibrin, the gram-positive staphylococci (Staphylococcus aureus) And antimicrobial activity against Escherichia coli which is gram-negative bacterium.

In an embodiment of the present invention, a culture of the Bacillus subtilis strain BK418 is provided.

In one embodiment of the present invention, the culture of the Bacillus subtilis strain BK418 is carried out by inoculating the Bacillus subtilis strain BK418 in a TSB medium sterilized at 121 DEG C for 15 minutes at a temperature of 30 DEG C and 180 rpm for 18 hours Preparing a culture medium by mixing 25 to 50 g / l of molasses or sugar with distilled water, and culturing the seed culture in the culture medium with 1% of the seedlings inoculated and culturing the cells at 30 DEG C and 180 rpm And culturing the cells for 2 weeks.

As an embodiment of the present invention, there is provided a prophylactic composition, an antimicrobial composition, an environmental purification composition, a fermented food or a health functional food containing the Bacillus subtilis BK418 strain or a culture thereof as an active ingredient.

The Bacillus subtilis strain of the present invention has the ability to produce complex enzymes capable of degrading protein, lipid, starch and fibrin, and is useful as an antimicrobial agent for Escherichia coli and Staphylococcus aureus , And the like.

In addition, the Bacillus subtilis BK418 strain or its culture has excellent antimicrobial activity and antagonistic effect against pathogenic bacterium because it does not show antibacterial activity on effective microorganisms such as lactic acid bacteria and yeast, Can be provided as a complex microorganism.

In addition, the Bacillus subtilis strain BK418 can be cultured in a simple and easy new culture method using sugar or molasses as a single nutrient source.

Also, it is possible to provide a Bacillus subtilis BK418 strain and a culture thereof and a biocide composition, an antimicrobial composition, an environmental purification composition, a cosmetic raw material, a fermented food and a health functional food containing the same as an active ingredient.

Brief Description of the Drawings Fig. 1 is a graph showing the results of a comparison between Escherichia coli ( Escherichia coli) and Escherichia coli lt; RTI ID = 0.0 > coli ) < / RTI >
FIG. 2 is a photograph showing the results of measurement of antimicrobial activity against Staphylococcus aureus in the supernatant of Bacillus subtilis strain BK418 according to the present invention. FIG.
FIG. 3 is a photograph showing activity results of protease, amylase and cellulase through supernatant of Bacillus subtilis strain BK418 according to the present invention. FIG.
4 is a nucleotide sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention.
5 is a phylogenetic diagram showing the phylogenetic relationship with other bacteria based on the base sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings illustrate only the essential features of the invention in order to facilitate clarity of the invention and are not to be construed in a limiting sense since they are not shown in the accompanying drawings.

The present invention provides a Bacillus subtilis BK418 (KACC91903P) (hereinafter referred to as 'BK418') strain having a complex enzyme production ability and having an antibacterial activity.

The Bacillus subtilis BK418 was deposited at the National Institute of Genetic Resources (KACC) under accession number KACC91903P.

The Bacillus subtilis BK418 morphologically corresponds to Gram-positive bacilli and has a homology of 99% with Bacillus subtilis.

The Bacillus subtilis BK418 has the ability to produce digestive enzymes against proteins, starch, lipids and fibrin, and is resistant to Gram-positive bacteria such as Staphylococcus aureus , And Escherichia coli which is a Gram-negative bacterium.

The present invention provides a culture of the Bacillus subtilis strain BK418. The culture means a culture medium or a culture medium in which the BK418 strain has been cultured, and the resultant cultured in the culture medium or culture, and the culture may or may not include the BK418 strain. The culture is not particularly limited in its formulation, and may be a formulation conventionally used in the art. For example, the culture may be liquid or solid, and may be in the form of a round, or a concentrated form, or a dried form of the culture.

More specifically, the method for culturing the culture of the Bacillus subtilis strain BK418 may include culturing the seed culture, preparing the culture medium, and culturing the seed culture inoculating the culture medium.

The Bacillus subtilis strain BK418 was inoculated on a TSB medium sterilized for 15 minutes at 121 ° C. and cultured at 30 ° C. and 180 rpm for 18 hours to prepare a seed culture. 25 to 50 g / l of molasses or sugar was added to distilled water To prepare a culture medium, and 1% of the seeds can be inoculated in the culture medium and cultured at 30 DEG C and 180 rpm for 2 weeks.

The present invention also provides a probiotic microbial composition, an antibacterial composition, an environmental purification composition, a cosmetic raw material, a fermented food, and a health functional food, which contain the Bacillus subtilis BK418 strain or a culture of the strain as an active ingredient .

The above-mentioned probiotic composition means a microorganism or a component thereof which shows a beneficial effect on the health of a host such as a human or an animal. The prophylactic composition fixes on the wall of digestive tract in the intestine of the host and functions to inhibit the establishment of other harmful microorganisms, The beneficial digestive enzymes produced by the composition can facilitate absorption and utilization of nutrients.

In addition, the Bacillus subtilis BK418 strain or the culture thereof has excellent antimicrobial activity and antagonistic effect against pathogenic bacteria because it does not show antibacterial activity on effective microorganisms such as lactic acid bacteria and yeast, A biocide composition, an antimicrobial composition, a composition for environmental purification, a cosmetic raw material, a fermented food, and a health functional food.

In addition, the Bacillus subtilis strain BK418 can be easily cultured by a simple and easy new culture method using sugar or molasses as a single nutrient source, and can be conveniently used as an inexpensive medium for the production of a biocide composition, an antimicrobial composition, Cosmetics raw materials, fermented foods, and health functional foods.

Hereinafter, the present invention will be described in more detail by describing experimental examples.

Experimental Example 1

Bacillus subtilis ( Bacillus subtilis ) Isolation and Identification of BK418 Strain

(1) Isolation of strain

In order to isolate Bacillus subtilis strain BK418, 1 g of chungkukjang was added to 9 ml of sterilized distilled water and mixed for 10 minutes. The supernatant was diluted with sterilized distilled water and applied to TSA (tryptic soy agar, BD, USA) And cultured at 30 DEG C for 18 hours, and then each colony was separated. The selected strains were purified by TSA solid medium and purified. The selected strains were subjected to enzymatic degradation and antimicrobial tests.

(2) Identification of the strain

(2-1) Investigation of the use range of sugar with API 50 CHB kit

Biochemical identification was performed using API 50 CHB kit (Biomerieux, France) for carbohydrate availability. The strain to be identified was plated on a TSA solid medium and cultured at 30 ° C for 18 hours. The colonies were suspended in 2 ml of physiological saline, and the suspension was suspended in 5 ml of suspension medium. The turbidity was dispersed in 2 Mcalpalts McFarland). Turmeric solution was inoculated into 10 ml of API 50 CHB medium, and 150 μl of the solution was inoculated on an API 50 CH strip, followed by culture at 30 ° C for 24 hours and 48 hours. The identified results were analyzed by biochemical results through API identification system (apiweb ^TM ).

As a result of the analysis, the availability of sugars to 49 kinds of carbohydrates was confirmed. As a result, the isolates produced various acids using various saccharides and their characteristics are shown in Table 1.

The results of the API 50 CHB kit for pure isolates were confirmed by apiweb ^TM for the API identification system, confirming that Bacillus subtilis was highly homologous with 99.8%.

(2-2) Analysis of strains using 16s rRNA nucleotide sequence

Genomic identification was performed by extracting genomic DNA from microorganisms and amplifying 16s rRNA which was used for identification by PCR method. After purification, PCR fragments were subjected to sequencing by SolGent Co., Ltd., a gene analysis center .

The nucleotide sequence of the strain obtained from the nucleotide sequence analysis can be found in the GeneBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC==) blasthome) to BLASTN and compared the similarity of 16s rRNA of previously reported microorganisms.

FIG. 4 is a nucleotide sequence of 16s rRNA of Bacillus subtilis BK418 according to the present invention, and FIG. 5 is a sequence listing of 16s rRNA of Bacillus subtilis BK418 according to the present invention It is a systematic diagram showing the genetic relationship.

As shown in FIG. 4 and the sequence listing, analysis of the 16s rRNA base sequence confirmed 1,433 bases.

As shown in FIG. 5, a phylogenetic analysis using the 16S rRNA nucleotide sequence showed a similarity rate of 99% (1430/1431) with the 16s rRNA gene of Bacillus subtilis strain D13 (accession No: KC441757) The isolated strain was named Bacillus subtilis BK418.

Experimental Example 2

Bacillus subtilis ( Bacillus subtilis ) Culture method of BK418 strain

Bacillus subtilis strain BK418 was inoculated on a TSB liquid medium sterilized for 15 minutes at 121 ° C and cultured at 30 ° C and 180 rpm for 18 hours.

25 g / l of molasses and 50 g / l of distilled water were added to the distilled water to determine the growth of Bacillus subtilis strain BK418 according to the amount of molasses. The medium was sterilized at 121 占 폚 for 15 minutes. In addition, 25 g / l of sugar was added to distilled water to confirm the growth of Bacillus subtilis BK418 strain.

1% of seeds were inoculated in a medium containing molasses or sugar and cultured for 2 weeks at 30 ° C and 180 rpm. The number of viable cells during the culture was diluted by a 10-fold dilution method and 100 μl was plated on a TSB solid medium to confirm the number of colonies.

As a result, the viable cell count of the Bacillus subtilis strain BK418 cultured in the molasses medium is shown in Table 2.

As shown in Table 2, even when 25 g / l of molasses was added at the time of preparing the medium, the number of viable cells similar to 50 g / l was confirmed. After 14 days, the number of viable cells was maintained at 10 ⁸ cfu / 1 was confirmed to be 10 ⁶ cfu / ml or more, it was confirmed that the composition could be used for the production of a composition containing Bacillus subtilis strain BK418 or a culture thereof as an active ingredient.

Experimental Example 3

Bacillus subtilis ( Bacillus subtilis ) Antibacterial activity of strain BK418

(1) Antimicrobial activity test for Gram negative pathogenic bacteria

Escherichia coli used in this experiment is a Gram-negative pathogenic bacterium which is present in human and animal intestines and causes abdominal pain, diarrhea and enteritis. It is inoculated into LB (Luria Bertani) liquid medium and is cultured under conditions of 30 ° C. and 180 rpm After culturing for 21 hours, the cells were adjusted to 1 x 10 < ⁷ > cfu / ml and 100 [mu] l was plated on LB solid medium. A paper disk (ADVANTEC, Japan) was placed on top of the stained LB solid medium to adsorb 50 μl of the antimicrobial substance and cultured at 30 ° C. for 24 hours. The diameter of the clear zone (mm) Respectively. The antimicrobial substance was obtained by inoculating Bacillus subtilis strain BK418 in a TSB liquid culture medium and culturing at 30 ° C. and 180 rpm for 18 hours. The supernatant obtained after centrifugation at 8,000 × g was filtered with a 0.2 μm membrane filter to measure antimicrobial activity And used as a sample.

FIG. 1 is a photograph showing the antimicrobial activity of Escherichia coli of the supernatant of Bacillus subtilis strain BK418 according to the present invention. As shown in FIG. 1, And Bacillus subtilis strain BK418 or culture thereof showed strong antibacterial activity against Gram negative pathogenic bacteria.

(2) Antimicrobial activity against gram-positive pathogenic bacteria

Staphylococcus aureus used in this experiment is a typical Gram-positive pathogen causing food poisoning and skin disease. It is inoculated into TSB liquid medium and cultured for 21 hours at 30 ° C and 180 rpm. Cells were treated with 1 x 10 ⁷ cfu / Ml < / RTI > and plated on TSA solid medium. The paper disk (ADVANTEC, Japan) was placed on a solidified TSA solid medium, and 50 μl of the antimicrobial substance was adsorbed on the solidified TSA solid medium and cultured at 30 ° C. for 24 hours. Then, the diameter of a clear zone Respectively. The antimicrobial substance was obtained by inoculating Bacillus subtilis strain BK418 in a TSB liquid culture medium and culturing at 30 ° C. and 180 rpm for 18 hours. The supernatant obtained after centrifugation at 8,000 × g was filtered with a 0.2 μm membrane filter to measure antimicrobial activity And used as a sample.

FIG. 2 is a photograph showing the antimicrobial activity of Staphylococcus aureus in the supernatant of Bacillus subtilis strain BK418 according to the present invention. As shown in FIG. 2, the antimicrobial activity of Staphylococcus aureus zone), the Bacillus subtilis BK418 strain or culture thereof showed strong antimicrobial activity against gram-positive pathogenic bacteria.

As a result of this paper disk antimicrobial test, both Gram-negative and Gram-positive pathogenic bacteria have antimicrobial activity. Therefore, Bacillus subtilis strain BK418 or a culture thereof is used as an active ingredient in various fields such as antimicrobial composition, . It can also be used as a natural preservative to replace fermented food fermentation process quickly and safely and to act as a chemical preservative for long-term preservation.

(3) Antibacterial activity against non-pathogenic bacteria

The lactic acid bacteria and yeast used in this experiment are known as useful microorganisms. They are inoculated into MRS and SDA liquid medium and cultured at 37 ° C and 180 rpm for 36 hours. The strains were cultured in 1 × 10 ⁷ cfu / Ml < / RTI > and plated on MRS and SDA solid media. A paper disc (ADVANTEC, Japan) was placed on the stained MRS and SDA solid medium, and 50 μl of the antimicrobial material was adsorbed thereon. After incubation at 30 ° C. for 24 hours, the diameter (mm) of the clear zone indicating the growth inhibition was measured Paper disk method. The antimicrobial substance was obtained by inoculating Bacillus subtilis strain BK418 into a TSB liquid medium and culturing the mixture at 8,000 xg for 18 hours at 30 ° C and 180 rpm. The supernatant obtained was filtered with a 0.2 μm membrane filter to measure the antimicrobial activity And used as a sample. As a result, the useful microorganisms such as lactic acid bacteria and yeast did not show antibacterial activity because no transparent ring was formed.

In addition, when lactic acid bacteria and Bacillus subtilis strain BK418 were inoculated to the TSA liquid medium and cultured at 30 ° C. and 180 rpm for 36 hours, the viable cell counts of 10 ⁷ cfu / ㎖ or more were measured in both strains. Thus, the Bacillus subtilis strain BK418 And non - pathogenic strains were found to maintain a mutually complementary symbiotic relationship.

Bacillus obtained by separation in this study subtilis BK418 strain or its culture is beneficial microorganism is specific to antibiotic activity and antagonistic against pathogenic bacteria such as but not antibacterial activity and E. coli (Escherichia coli) and Staphylococcus aureus (Staphylococcus aureus) And it was confirmed that it was effective as a complex microorganism with effective microorganism.

Experimental Example 4

Bacillus subtilis ( Bacillus subtilis ) Experiment with BK418 strain

Bacillus subtilis strain BK418 was cultured in SM medium consisting of 1% sodium chloride, 0.5% yeast extract, 1% peptone, and 2% skim milk and 2% soluble starch instead of skim milk. And CMC medium containing 1% carboxy methyl cellulase were prepared. Bacillus subtilis strain BK418 was inoculated into each solid medium and incubated at 30 ° C for 48 hours. The resulting clear zone was treated with protease, amylase and fibrinolytic enzyme cellulase) were investigated.

FIG. 3 is a photograph showing the results of activity of protease, amylase and cellulase through the supernatant of Bacillus subtilis strain BK418 according to the present invention. As a result, Starch and fibrin were detected through the formation of a clear zone. The quantitative analysis was performed to investigate the exact enzyme activity.

Experimental Example 5

Bacillus subtilis ( Bacillus subtilis ) Enzyme activity measurement of BK418 strain

To determine the enzyme activity of Bacillus subtilis strain BK418, the strain was inoculated into 50 ml of TSB liquid medium at a concentration of 10 ² cfu / ml. 1 ml of the cultured medium cultured at 30 rpm at 180 rpm for 24 hours was centrifuged at 10,000 g for 10 minutes, and the supernatant was sterilized with a membrane syringe filter having a pore diameter of 0.45 m.

The activity of the protease was determined by dissolving casein in 1 mM Tris-HCl buffer (pH 7.5) to 1%, and using 0.5 ml of substrate solution and 0.5 ml of substrate solution at 30 ℃ After reacting for 1 hour, 0.5 ml of 40% (w / v) trichloroacetic acid (TCA) solution was added to stop the reaction and left at room temperature for 15 minutes. After centrifugation at 12,000 g for 15 minutes, the supernatant was taken and the amount of soluble peptide was measured by absorbance at 280 nm. For the standard curve, L-tyrosine solution was used and 1 unit of enzyme activity unit was defined as the amount of enzyme producing tyrosine equivalent to 1 g per minute.

The activity of starch degrading enzyme (amylase) was 0.5 ml of 1% (w / v) soluble starch and 0.5 ml of coenzyme was added and reacted at 30 ℃ for 1 hour. The reaction was stopped by adding 1 ml of cold 1N HCl, and then 0.1 ml of Lugol's iodine solution was added to develop color. The solution was diluted with distilled water to 25 ml, and the absorbance was measured at 620 nm. The standard curve used maltose solution. The enzyme activity unit was defined as 1 unit of enzyme amount equivalent to 0.01% soluble starch reduction for 1 minute.

The activity of cellulase was determined by quantitative determination of reducing sugar liberated after enzyme reaction with 3,5-dinitrosalicylic acid (DNS) using Carboxymethyl Cellulose (CMC) as a substrate. 0.5 ml of 1.0% (w / v) CMC solution suspended in distilled water, 0.25 ml of 100 mM Tris-HCl buffer (pH 7.5) and 0.25 ml of enzyme solution were mixed and reacted at 55 ° C for 15 minutes. The reaction was stopped by adding 3 ml of the DNS reagent. The reaction solution was allowed to stand in boiling water for 5 minutes. After color development, the absorbance was measured at 540 nm and glucose was used as a standard sample. The amount of reducing sugar was determined. The enzyme activity of 1.0 unit was defined as the amount of enzyme that produces reducing sugars corresponding to 1 mol of glucose from CMC for 1 minute under the above conditions.

The lipase activity was determined by measuring the amount of p- nitrophenol produced by enzyme action from p- nitrophenyl palmitate used as substrate. 10 μl of coenzyme was added to 10 μl of 10 mM p- nitrophenyl palmitate dissolved in acetonitrile and to 350 μl of 100 mM Tris-HCl buffer (pH 7.5) buffer. The mixture was immediately mixed and reacted at 45 ° C. for 30 minutes. The increase in p-nitrophenol production was measured at 405 nm absorbance. Enzyme activity (unit) was defined as the amount of enzyme required to produce 1 μ mol of p- nitrophenol produced over 1 min.

As a result, the activity of each enzyme is shown in Table 3.

It was confirmed that protease, amylase, cellulase and lipase were all produced through the coenzyme obtained from the culture broth. Since the activity of the enzyme was good, Bacillus subtilis BK418 strain or a culture thereof can be used as an active ingredient to be used as a fermented food or a health functional food.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined in the appended claims. It will be possible.

<110> SEO, BUM-GU <120> Bacillus subtilis BK418 strain having complex digestive          productivity and antifungal activity and uses thereof <130> 0001 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1433 <212> DNA <213> Bacillus subtilis <400> 1 gctataatgc agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg 60 tgagtaacac gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa 120 taccggatgg ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt 180 acagatggac ccgcggcgca ttagctagtt ggtgaggtaa yggctcacca aggcaacgat 240 gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct 300 acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc 360 gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt 420 cgaatagggc ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc 480 agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc 540 aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa 600 ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta 660 gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga 720 gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga 780 gtgctaagtg ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg 840 cctggggagt acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg 900 gtggagcatg tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc 960 tgacaatcct agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg 1020 tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct 1080 tagttgccag cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg 1140 tggggatgac gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg 1200 acagaacaaa gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt 1260 cggatcgcag tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc 1320 atgccgcggt gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt 1380 gtaacacccg aagtcggtga ggtaaccttt aggagccagc cgccgaagtg aca 1433

Claims (10)

Bacillus subtilis BK418 (KACC91903P) strain having antibiotic activity with complex enzyme production ability. The method according to claim 1,
Staphylococcus aureus , a gram-positive bacterium, has digestive enzyme-producing ability against proteins, starch, lipids and fibrin. And Bacillus subtilis BK418 (KACC91903P) strain having an antimicrobial activity against Escherichia coli which is gram-negative bacterium. A culture of the strain of Bacillus subtilis BK418 (KACC91903P) according to claim 1. A method for culturing a culture of the strain of Bacillus subtilis BK418 (KACC91903P) according to claim 3,
Culturing the strain of Bacillus subtilis BK418 (KACC91903P) in a TSB medium sterilized at 121 ° C for 15 minutes at 30 ° C and 180 rpm for 18 hours;
Mixing distilled water with 25 to 50 g / L of molasses or sugar to prepare a culture medium; And
Culturing the above-mentioned culture medium with 1% of said seeds inoculated, and culturing at 30 DEG C and 180 rpm for 2 weeks; and culturing the culture of the strain of Bacillus subtilis BK418 (KACC91903P). A prophylactic or antimicrobial composition comprising the above-mentioned Bacillus subtilis BK418 (KACC91903P) strain or the culture of the strain described in claim 3 as an active ingredient. The antimicrobial agent composition according to claim 1, which comprises the above-mentioned Bacillus subtilis BK418 (KACC91903P) strain or a culture of the strain described in claim 3 as an active ingredient. An environmental cleansing composition comprising the Bacillus subtilis BK418 (KACC91903P) strain according to claim 1 or a culture of the strain described in claim 3 as an active ingredient. A cosmetic raw material characterized by containing the above-mentioned Bacillus subtilis BK418 (KACC91903P) strain or the culture of the above-mentioned strain according to claim 3 as an active ingredient. A fermented food comprising the above-mentioned strain of Bacillus subtilis BK418 (KACC91903P) according to claim 1 or a culture of the strain described in claim 3 as an active ingredient. A health functional food characterized by comprising the above-mentioned Bacillus subtilis BK418 (KACC91903P) strain or the culture of the strain described in claim 3 as an active ingredient.

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