KR101873629B1 - Novle Clostridium butyricum against pathogenic bacteria and use thereof - Google Patents

Abstract

The present invention relates to a novel Clostridium butyricum KNU-L09 strain (Accession No. KCTC 12975BP) having antagonistic ability against a pathogenic microorganism, a microorganism preparation using the same, and a preparation method thereof. The novel strain of the present invention has been experimentally confirmed to have an antagonistic ability against a pathogenic microorganism and can be usefully used as an environmentally friendly microorganism preparation for a formal action. In addition, since the safety was confirmed through the whole gene sequence analysis of the strain, there is an advantage that it can be safely used as a microorganism preparation for a dressing as well as a livestock.

Description

A novel Clostridium butyricum strain having an antagonistic ability against a pathogenic microorganism and its use.

The present invention relates to a novel Clostridium butyricum KNU-L09 strain (Accession No. KCTC 12975BP) having an antagonistic ability against a pathogenic microorganism, a microorganism preparation using the same, and a preparation method thereof.

The use of antibiotics has improved productivity, such as prevention and treatment of livestock diseases such as diarrhea, promotion of growth, and improved feed efficiency in the livestock industry. However, the use of antibiotics has been limited due to the emergence of resistant strains, the identification of residual antibiotics in livestock products, and human safety issues due to the resistance of bacteria to humans in humans. Recently, the EU Commission (European Commission) Prohibited the use of antibiotics added to feed as an animal growth promoter. In Korea, the total use of antibiotics is continuously decreasing in the livestock industry, and since 2012, antibiotics excluding antiparasitic and antacidides have been prohibited from being used for animal feed.

With the ban on the use of antibiotics and the growing interest in environmentally friendly products, studies on useful microorganisms for livestock feed have been made steadily. The feed additive microorganisms which are currently being developed and used include microorganisms having antagonistic action against pathogenic microorganisms and microorganism preparations for environmentally friendly livestock using microorganisms having various suitability functions.

For example, probiotics have recently attracted the greatest attention. Probiotics are defined as live-food additives that are generally beneficial to health, but they have recently been defined as living microorganisms in foods, feeds, or dietary supplements designed to improve the health of humans and animals. Bacteria that are most commonly used as a probiotic is Lactobacillus and the like (Lactobacillus) as a lactic acid bacterium and Streptococcus (Streptococcus).

Probiotics should be safe, resistant to acid and bile, and adherent to the epithelium of the intestine. In addition, antagonistic activity against pathogenic microorganisms producing toxic substances should be exerted by replacing them, and even if they have resistant genes, they should not be transferred to antibiotic resistance.

However, lactic acid bacteria have many useful properties such as antimicrobial activity, immune activity and anticancer activity. However, most of the lactic acid bacteria currently used as probiotics are not correctly evaluated as probiotic bacteria, There has been no development of a system capable of suppressing the theft of the user. In addition, it is ideal to separate and develop livestock prod- ucts for livestock in the intestines of livestock, but such strains can not be used as many probiotics.

Therefore, the present inventors isolated and identified a novel strain having an antagonistic ability against a pathogenic microorganism in the feces of a healthy pig, and confirmed that it was safe for human as well as for livestock through gene analysis of the strain. Therefore, the strain may be usefully used as a microorganism preparation for environmentally friendly dressing.

As a result of intensive studies to develop an environmentally friendly microorganism preparation, the present inventors have found a novel Clostridium butyricum KNU-L09 strain having antagonistic ability against a pathogenic microorganism from a healthy swine feces, thereby completing the present invention.

Accordingly, an object of the present invention is to provide Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP) having an antagonistic ability against pathogenic microorganisms and microorganism preparations using the same.

In addition, another object of the present invention is to provide a method for the preparation of Clostridium butyricum strain KNU-L09.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides Clostridium butyricum KNU-L09 strain (Accession No. KCTC 12975BP) having antagonistic ability against pathogenic microorganisms.

The present invention also relates to the above-mentioned strain of Clostridium butyricum KNU-L09 (Accession No. KCTC 12975BP), a culture of the strain, a concentrate of the culture, a dried product of the culture, As an active ingredient, at least one selected from the group consisting of: < RTI ID = 0.0 >

The present invention also provides a dressing composition comprising the microbial agent.

The present invention also relates to a method for culturing Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP), adding skim milk to the culture to a final concentration of 5-10% Freeze-drying and pulverizing at -60 to -80 占 폚.

In one embodiment of the present invention, the strain is characterized in that it comprises the 16S rRNA base sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the strain comprises at least one pathogenic microorganism antagonist gene selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

In another embodiment of the present invention, the pathogenic microorganism is selected from the group consisting of Bacillus cereus , Salmonella choleraesuis , Salmonella entrica , Salmonella typhimurium , Streptococcus agalactiae ). < / RTI >

Clostridium butyricum strain KNU-L09 of the present invention has been experimentally confirmed to have an antagonistic ability against a pathogenic microorganism, and thus it can be effectively used as an environmentally friendly microorganism preparation for a formal action. In addition, since the safety of the strain was confirmed through whole gene sequence analysis and harmfulness test, it could be safely used as a microorganism formulation for humans as well as for livestock.

FIG. 1 is a photograph showing that an inhibitory ring is formed by treating the strain of the present invention with Clostridium butyricum strain KNU-L09 on a plate on which a pathogenic microorganism has been stained.
Fig. 2 is a nucleotide sequence of a gene (rDNA) encoding 16S rRNA of Clostridium butyricum strain KNU-L09 of the present invention.
3 is a phylogenetic diagram showing the nucleotide sequence (1,497 bp) of a gene (rDNA) encoding 16S rRNA of Clostridium butyricum strain KNU-L09 of the present invention in comparison with a known strain.
4 is a photograph showing a powder type microorganism preparation obtained by lyophilizing Clostridium butyricum strain KNU-L09 of the present invention.
FIG. 5 is a graph showing that the survival rate of the strain was remarkably improved by adding 7% skim milk as a preservative in the preparation of a microorganism preparation containing Clostridium butyricum strain KNU-L09 of the present invention.
Fig. 6 shows the results of the whole gene sequence analysis of the strain KNU-L09 of Clostridium butyricum of the present invention.
FIG. 7 is a graph showing the results of analysis of the genes (Aminodeoxychorismate lyase (EC 4.1.3.38)) involved in the production of natural antibiotics related to the antagonistic ability to pathogenic microorganisms in the Clostridium butyricum strain KNU-L09 of the present invention. , Anthranilate synthase, amidotransferase component (EC 4.1.3.27), anthranilate synthase, aminase component (EC 4.1.3.27), para-aminobenzoate synthase, amidotransferase component (EC 2.6.1.85)).
FIG. 8 shows the result of performing a toxicity test on the whole gene of the strain KNU-L09 of Clostridium butyricum of the present invention.

The present invention provides Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP) having antagonistic ability against pathogenic microorganisms.

In the present invention, the Clostridium butyricum KNU-L09 strain comprises the 16S rRNA gene of SEQ ID NO: 1 and the 16S rRNA nucleotide sequence has 99% homology with Clostridium butyricum DSM 10702T And 1% mutagenicity was recognized between the two strains, which proved to be a novel strain.

Thus, the present inventors named the novel strain obtained as Clostridium butyricum KNU-L09 and deposited it with KCTC 12975BP on Feb. 11, 2016 at Korea Research Institute of Bioscience and Biotechnology.

In one embodiment of the present invention, it was confirmed that when the novel strain cultured from a healthy swine feces was treated with a pathogenic microorganism-treated plate, a pronounced inhibitory effect was produced, thereby confirming antagonistic ability against pathogenic microorganisms 1). The pathogens in the present invention is Bacillus cereus (Bacillus cereus, KACC 10097), Salmonella choleraesuis could device (Salmonella choleraesuis, KCTC 2929), Salmonella Entebbe Rica (Salmonella entrica, KACC 10763), Salmonella typhimurium (Salmonella typhimurium, KCTC1926 ), And Streptococcus agalactiae (ATCC 13813), but the present invention is not limited thereto.

In another embodiment of the present invention, in order to analyze the sugar metabolism characteristics of the novel strain of the present invention, the strains were cultured using various active ingredients and the growth of the strains was observed. As a result, cellobiose, esculin Aesculin, Lactose, Maltose, Ribose, Salicin, and Sucrose were used as energy sources (see Example 2).

In another embodiment of the present invention, in order to identify a related gene sequence exhibiting an antagonistic ability against a pathogenic microorganism in the novel strain of the present invention, DNA was extracted from the strain and the whole gene sequence was analyzed. As a result, genes that may be involved in the generation of natural antibiotics related to the antagonistic ability to pathogenic microorganisms, such as Aminodeoxychorismate lyase (EC 4.1.3.38), Anthranilate synthase; amidotransferase component (EC 4.1.3.27), Anthranilate synthase; aminase component (EC 4.1.3.27), Para-aminobenzoate synthase; amidotransferase component (EC 2.6.1.85)) was confirmed, and the safety of the whole gene was confirmed through the hazard test (see Example 4).

Thus, the novel Clostridium butyric rikum (Clostridium butyricum) KNU-L09 strain, at least one selected from the culture, the culture of a concentrate with water and a dry product of the strain, and the group consisting of an active ingredient of the present invention , It can be usefully used as a microorganism preparation for a primate, a livestock, and a variety of animals which are human or non-human.

In the present invention, the cultured product may be cultured in a medium such as a normal agar medium (Nutrient agar medium), a tryptic soy agar medium (TSA medium), a Standard Methods Agar (Plate Count Agar), a lactose medium Broth, BGlB medium, BGlB medium, Endo agar medium, Eosin methylene blue agar medium, Nutrient broth, But are not limited to, those obtained by separating from desoxycholate lactose agar or EC broth.

The microbial formulation according to the present invention can be formulated into a formulation according to a conventional method, and can be prepared in the form of a dry powder or a liquid, but is not limited thereto.

In the present invention, the microorganism preparation can be prepared by adding an additive such as a surfactant, a weight agent, and a nutrient to a strain or a culture thereof. The surfactant may be selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene Polyoxyethylene alkylarylpolyether, polyoxyethylene nonylphenyl ether, sodium sulphonate naphthalene formaldehyde, Triton 100 and Tween 80. The composition of claim 1, And one or more selected from the group consisting of soybean flour, rice, wheat, loess, diatomaceous earth, dextrin, glucose and starch is used as the extender and nutrient, and the disintegrant is bentonite ), Talc, dialite, kaolin, And calcium carbonate may be used.

The present invention also provides a dressing composition comprising the microbial agent.

The present invention also relates to a method for culturing Clostridium butyricum strain KNU-L09, which comprises adding skim milk to a culture solution to a final concentration of 5-10% Freeze-drying and pulverizing the microorganism preparation.

In the present invention, when skim milk was added as a preservative in the freeze-drying, it was confirmed that the skim milk maintained a survival rate of 50% or more as compared with the preservative-free sample. As a result, the microbial agent was excellent in survival activity, There will be.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example  One. Clostridium Buty Richem ( Clostridium butyricum ) KNU Isolation and Identification of -L09 Strain

1-1. Strain isolation and pathogenic microorganisms Antagonistic ability  Verification

1 g of fecal samples were diluted in saline (0.85% NaCl), and then treated with agar containing Reinforced clostridial medium, TSB (Tryptic Soy Broth) and YPD (Yeast extract Peptone Dextrose) agar) medium and cultured at 30 DEG C for 48 hours. Subsequently, an experiment was carried out to select strains which can be used as a formative agent for strains cultured from fecal samples according to the above method.

More specifically, the cultivated strains are selected from the group consisting of pathogenic microorganisms Bacillus cereus (KACC 10097), Salmonella choleraesuis ( KCTC 2929), Salmonella entrica (KACC 10763), Salmonella typhimurium ( Salmonella typhimurium , KCTC1926), and Streptococcus agalactiae (ATCC 13813) were transferred to the stained plate to observe whether they formed inhibitory rings.

As a result, as shown in Fig. 1, a strain having a marked inhibitory effect on pathogenic microorganisms was found, and as shown in Table 1, it was confirmed that antagonistic action was exhibited on all five pathogenic microorganisms.

Test microorganisms Inhibition B. cereus , KACC 10097 ○ S. choleraesuis , KCTC 2929 ○ S. enterica , KACC 10763 ○ S. typhimurium , KCTC 1928 ○ S. agalactiae , ATCC 13813 ○

1-2. Identification of isolated strains

In order to identify the strains having excellent antagonistic ability against the pathogenic microorganism through Example 1-1, genomic DNA was extracted from the cells, and PCR was performed using a universal primer The 16S rRNA fragment was amplified by polymerase chain reaction (PCR). The multipurpose primers used were 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), respectively. The amplified DNA products were purified using a PCR purification system (Solgent, Daejon, Korea) and then submitted to Solgent (Daejon, Korea) for sequencing. The resulting 16S rRNA nucleotide sequence (SEQ ID NO: 1) of FIG. 2 was compared using BLASTN of National Center for Biotechnology Information (NCBI). The homology and phylogenetic tree of the nucleotide sequence were Bioedit and Mega5 And analyzed using the neighbor-joining methods through the program.

As a result, as shown in Fig. 3, the isolated strain was found to have 99% homology with the previously reported published strain, Clostridium butyricum DSM10702T, and 1% different from the published strain And it was recognized that it was a novel strain belonging to Clostridium butyricum species. Thus, the isolate was named Clostridium butyricum KNU-L09, and the strain was deposited at the Korea Research Institute of Bioscience and Biotechnology (Accession No. KCTC 12975BP).

Example  2. Analysis of glucose metabolism

To investigate the sugar metabolism of the novel Clostridium butyricum KNU-L09 strain isolated in Example 1, the strains shown in the following Table 2 were cultured in a 48 ° C incubator at 30 ° C for 48 After incubation for a period of time, the growth of the strain was analyzed.

As a result, as shown in the following Table 2, the novel strain of the present invention can be used as a strain of cellobiose, Aesculin, Lactose, Maltose, Ribose, Salicin, And sucrose as an energy source.

Active ingredient result Cellobiose + Aesculin + Lactose + Maltose + Ribose + Salicin + Starch - Sucrose + Trehalose -

Example  3. Preparation of Microbial Agents Using New Strain

The microorganism preparation containing the novel strain of Clostridium butyricum KNU-L09 isolated through Example 1 was prepared. To this end, the strain was incubated at 30 ° C for 48 hours, centrifuged at 25 ° C and 7,000 rpm for 15 minutes to recover only the cells, and once with sterile physiological saline, the same saline was added to 12.5 And the cells were suspended. Skim milk was added as a preservative in order to increase the viability of the cells during lyophilization, and the mixture was added to the suspension to a final concentration of 7%, and the mixture was homogeneously mixed. The mixed bacterial solution was completely frozen at -70 ° C and then freeze-dried for 3 days using a freeze dryer. The sample after the freeze-drying was uniformly pulverized as shown in Fig. 4, and the survival rate of the cells was examined.

As a result, as shown in FIG. 5, when skim milk was not added, there was almost no viable cell in comparison with that before freeze drying, but when the skim milk was added, the survival rate was 50% or more even after lyophilization. In addition, it was confirmed that when the preparation was refrigerated, viability was maintained for 30 days. The results indicate that the microbial agent of the present invention can be used as an environmentally friendly animal microbial agent.

Example  4. Identification of genes involved in the action of the new strain and harmfulness of the gene sequence

In order to identify a related gene sequence exhibiting an antagonistic ability against a pathogenic microorganism in the novel Clostridium butyricum KNU-L09 strain isolated through Example 1, a genomic DNA was extracted from the strain The entire gene sequence of the strain was analyzed using the PacBio SMRT sequencing kit. The results are shown in FIG. Aminodeoxychorismate lyase (EC 4.1.3.38), anthranilate synthase, which may be involved in antagonistic action against pathogenic microorganisms; amidotransferase component (EC 4.1.3.27), Anthranilate synthase; aminase component (EC 4.1.3.27), Para-aminobenzoate synthase; The nucleotide sequences of the amidotransferase component (EC 2.6.1.85) (SEQ ID NOS: 2 to 5) were confirmed, and the results are shown in FIG.

Furthermore, as shown in FIG. 8, two toxin-like genes were found in 4152 genes. However, hypothetical genes were found to be harmless to humans and livestock Proved.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC12975BP

Checked on: 20160211

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Novle Clostridium butyricum against pathogenic bacteria and use          the <130> MP15-192 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 1497 <212> DNA <213> Clostridium butyricum KNU-L09 16S rRNA <400> 1 ctaagattaa actttttatt gagagtttga tcctggctca ggacgaacgc tggcggcgtg 60 cttaacacat gcaagtcgag cgatgaaact ccttcgggag tggattagcg gcggacgggt 120 gagtaacacg tgggtaacct gcctcataga ggggaatagc ctttcgaaag gaagattaat 180 accgcataag attgtagtac cgcatggtac agcaattaaa ggagtaatcc gctatgagat 240 ggacccgcgt cgcattagct agttggtgag gtaacggctc accaaggcga cgatgcgtag 300 ccgacctgag agggtgatcg gccacattgg gactgagaca cggcccagac tcctacggga 360 ggcagcagtg gggaatattg cacaatgggg gaaaccctga tgcagcaacg ccgcgtgagt 420 gatgacggtc ttcggattgt aaagctctgt ctttagggac gataatgacg gtacctaagg 480 aggaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggatttac tgggcgtaaa gggagcgtag gtggatgttt aagtgggatg tgaaataccc 600 gggcttaacc tgggtgctgc attccaaact ggatatctag agtgcaggag aggaaaggag 660 aattcctagt gtagcggtga aatgcgtaga gattaggaag aataccagtg gcgaaggcgc 720 ctttctggac tgtaactgac actgaggctc gaaagcgtgg ggagcaaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgaat actaggtgta ggggttgtca tgacctctgt 840 gccgccgcta acgcattaag tattccgcct ggggagtacg gtcgcaagat taaaactcaa 900 aggaattgac gggggcccgc acaagcagcg gagcatgtgg tttaattcga agcaacgcga 960 agaaccttac ctagacttga catctcctga attactctgt aatggaggaa gctacttcgg 1020 tggcaggaag acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttattgtta gttgctacca tttagttgag cactctagcg 1140 agactgcccg ggttaaccgg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200 gtctagggct acacacgtgc tacaatggtc ggtacaatga gatgcaacct cgcgagagtg 1260 agcaaaacta taaaaccgat ctcagttcgg attgtaggct gaaactcgcc tacatgaagc 1320 tggagttgct agtaatcgcg aatcagaatg tcgcggtgaa tacgttcccg ggccttgtac 1380 acaccgcccg tcacaccatg agagttggca atacccaaag ttcgtgagct aaccgcaagg 1440 aggcagcgac ctaaggtagg gtcagcgatt ggggtgaagt cgtaacaagg tagccgt 1497 <210> 2 <211> 726 <212> DNA <213> Aminodeoxychorismate lyase <400> 2 atggacatta tattcgatga aggatattca tttggattag gaatatttga aacaatatca 60 gttgtagaca atcatcttgt tctattggat tatcacttag atagacttag tgaaggttca 120 aattttttgg gaataaattt aaatgttaca aaagaaaaaa tacaggatta tataacaaat 180 aatccaatgg aaaatggtgt gctaaaaata atagcttcag aaaaaaatac aatatttcaa 240 tgcagagaaa ataattatac tgatgataag tataaaaaag ggtttacaat aaaaataagt 300 tctgttgtga gaaatgaaag ttctccgttt acttatataa aatctttgaa ttatggtgat 360 aatatcatag aaaaaagaaa ggcagcaaaa aaaggttatg atgaacccgt ctttttaaac 420 atgaaaggtc agttgactga aggagcaaca actaatatat tttttgttaa taacaatcaa 480 attgttacac ctaaattatc ctgtgggctt cttaatggaa caataagaaa atatattctt 540 gataagtatg atgttgtaga aaaatatatt tatccatacg aagtaagtac tttcgatgag 600 atgatttgtaa caaattcgct tatgggtata atgccggttt ataaatttga agagcatatt 660 tttaaatcga gaaaaaaatc tgattatatt ttaagtgaat atttaaaaac aaagacttgc 720 ttataa 726 <210> 3 <211> 585 <212> DNA <213> Anthranilate synthase_amidotransferase component   <400> 3 atgatacttt taattgataa ttatgattca ttcacatata acctttatca gtatattggg 60 gaatttgcag aagttaaggt tgtgagaaat gatgaaataa ccattgatga aataatagaa 120 ttaaatccaa aaggaatagt gatttcacca ggtccaggta ctccagatga tgctggaatt 180 tcaatagatg tagtgcagaa gcttggagct agatatccta tacttggaat atgccttgga 240 catcagagta ttgcacaagc ttatggtgga aagataatac gtgcagacga aatataccat 300 ggaaagactt ccaaaatatc tgtaaagggt aaattaatat ttgaaggaat tccaaggaag 360 atggatatta tgagatatca ttctttaata gtagataata tgtcacttcc agaatgtctt 420 gatgttatag gatctacagt agatgacaat attataatgg cagtgaaaca taaagaacat 480 gatgtttatg gacttcaatt tcatccagaa tcaatttata cacctaaagg aaagcatata 540 ataagcaatt ttgttattaa tatctgtaaa gaagaacaga agtaa 585 <210> 4 <211> 1392 <212> DNA <213> Anthranilate synthase_aminase component <400> 4 atgataaata tttctaaaga tcagtttaat gaaaaaagaa aagaaaataa ggttttttca 60 ttaataacag aatatagagg tgatgaagtt acacccataa gaatttttaa tggatttaag 120 ggaagaagaa aattcatttt tgaaagtgga agtacagaaa attattttgg acgatactca 180 tacttaggtg aaaatccata taaggaaatt ttgggagaaa gtatagatga tattgatgaa 240 ctaaaaaaat caataagatt agattttgac gaaagtacaa atgatttttc ttttaaaggc 300 ggagctatag gatatatggg atatgaaaca atttgtttgt atgaaaaaag acttaaattt 360 aataatccag atatacttga cttaccttta ataagattta atttatacag cagatatata 420 tgctatgacc actttactca caaggtattt gtcattgata acatattaaa tgatgaccct 480 agggaatatg aaagcatagt tgaaaatcaa agagaatata tattttcact gttaagcaga 540 cctactaata tagaagaatt tgaagaaaag aaagatgtcc attttgaatt gtgcacctca 600 aaagaaaaat atgaagaaaa tgtacgtata ggaaaagaac atattcttgc aggtgatatt 660 tttcagtttg ttccatctct aaggatgaaa tgtataacac aaaaatcctt cgttgaaatt 720 tatagaagac ttagagaagt taatccatcc ccatacatgt atttaattga ttatgatgat 780 tatcaagtta taggagcatc accagaaagt gttgtaagtg ttaaaaatgg gagagcatct 840 acaaagccta ttgcaggtac aagaaaaaga ggcgaaactc aggaggaaga ttcagctctt 900 gaaaaagaac ttcttcagga taaaaaagaa cttgcagaac atgttatgct tgttgatctt 960 gcaagaaatg atatagggcg aataagtgaa attggaactg ttgaagtaaa agactttatg 1020 aaaattgaaa agttttctca cgtaatgcat attacaagta cagttacagg aaagacatta 1080 caaaatatag acggttttga agcattaagt acatgccttc cagcaggaac tttatcagga 1140 gctcctaaaa taagagcaat ggaaataatt gaagaattag aagaatatag acgtgatatt 1200 tatggagggt cagttgggta tttctcctat ggaggagata cagatatggc aatagcaata 1260 agaacaatag taatgaaagg caatactgca tatcttcaag caggtgcagg tgttgttttt 1320 gattctgtcc cagaaaagga atttgaagag gttcagaata aattaatggc attaaaggag 1380 gctttaagat ga 1392 <210> 5 <211> 735 <212> DNA <213> Para-aminobenzoate synthase_amidotransferase component <400> 5 atgaagccgg taatagggat tacatcatat ataagaaaag acgtatttag aaactattca 60 caagttggat atgagtatat agaaaaaatc gaaaaagcaa atggtatacc tatgattttg 120 ccagtgcttc agaaatatga tgatcaagaa cttaataaac ttatagattg tgtaaatgga 180 attatattta ctggaggatg taacgtagaa tcacaatggt atggagaaaa acctttagga 240 gaggaaacaa aagaagatgt tttacgtaat ggatttgaaa gggatttatt tttagcagca 300 aagaagaaga aaaaacctat acttggtgta tgcagagggt gtcagctcat taatgtaatg 360 cagggaggaa ctctagtaca gaatattgat agtcagctta atactgctgt atatcataaa 420 ggaactggtt gcagaataag tgagaaactt catagagttg tattagcaga agattcacgt 480 cttaaaaatg catataatga acaggaagtt ccagtcaatt cttttcatga gcaatgtatt 540 aaacaaattg gaacagatct tagaattaca gctaaatgtt gtgaggatgg tgtaattgaa 600 ggtgttgaat atgaagggga cttttatatg gcaggagttc agtggcatcc agaaggaatg 660 gaagaccaac ttaagctttt tgaagagttt gtggatatat gcttaaaaaa tagtgatgaa 720 ttggcattga aataa 735

Claims (7)

Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP), which has an antagonistic ability against pathogenic microorganisms,
The pathogenic microorganism may be selected from the group consisting of Bacillus cereus, Salmonella choleraesuis, Salmonella entrica, Salmonella typhimurium, and Streptococcus agalactiae. Wherein the strain is at least one selected from the group consisting of:
The method according to claim 1,
The strain is a Clostridium butyricum KNU-L09 strain (Accession No. KCTC 12975BP), which comprises the 16S rRNA base sequence of SEQ ID NO: 1.
The method according to claim 1,
Wherein the strain comprises at least one pathogenic microorganism antagonist gene selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5, a Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP) .
delete A culture of the above strain, a concentrate of said culture, a dried product of said culture, and a combination thereof, wherein said strain is selected from the group consisting of Clostridium butyricum KNU-L09 strain (Accession No. KCTC 12975BP) A microorganism preparation for dressing comprising one or more active ingredients.
7. A dressing composition comprising the microbial formulation of claim 5.
After culturing Clostridium butyricum strain KNU-L09 (Accession No. KCTC 12975BP) of claim 1, skim milk was added to the culture to a final concentration of 5-10% &Lt; / RTI &gt; to &lt; RTI ID = 0.0 &gt; -80 C &lt; / RTI &gt;

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