KR101841090B1 - A novel Enterococcus faecium and anti-bacterial use of the same - Google Patents

Abstract

The present invention relates to a novel enterococcus faecium strain having anti-bacterial vitality with respect to a gram-negative bacterium and an anti-bacterial composition including the strain as an effective component. According to the present invention, a donated E. faecium strain represents excellent anti-bacterial vitality against a gram-negative bacterium such as E. coli so that the E. faecium strain can be applied in manufacturing a probiotic for an animal including a person by using the excellent strain.

Description

The present invention relates to a novel Enterococcus faecium strain and an antimicrobial use thereof,

The present invention relates to a novel Enterococcus pseudomonas strain and its antibacterial use.

Worldwide regulation of the use of feed additive antibiotics to promote livestock growth has had a great impact on the growth and health of livestock, causing economic losses for farmers. Several researchers are looking for new ways to protect and promote livestock health. Probiotics among many candidates may be a good alternative to growth-promoting antibiotics.

Probiotics can be defined as microorganisms that have a positive effect on animal health when ingested in appropriate amounts. Probiotic microorganisms form communities in the intestines of animals to change intestinal microbial communities and contribute to animal health through various actions. Among the various characteristics of probiotics, the most important feature as an antibiotic substitute is the ability to inhibit pathogens. This ability helps prevent intestinal infections through the production of organic acids, bacteriocins, or directly to the immune function of the animal.

Enterococcus faecium (E. faecium) is a microorganism widely known as a probiotic for humans and animals. It is known that enterotoxin, a bacteriocin that produces this paper, is effective against Gram-negative bacteria such as Escherichia coli ( E. coli).

Korean Patent Publication No. 10-2016-0002650

The present invention has been made in view of the above needs, and an object of the present invention is to provide a strain having an excellent antimicrobial activity against Gram-negative bacteria.

Another object of the present invention is to provide a composition having an excellent antimicrobial activity against Gram-negative bacteria.

In order to solve the above problems, the present invention provides an Enterococcus faecium strain having an antibacterial activity against noxious bacteria and comprising a gene consisting of the nucleotide sequence of any one of SEQ ID NOs: 1 to 4.

Here, it is possible that the above-mentioned strain contains all the genes comprising the nucleotide sequences of SEQ ID NOS: 1 to 4.

The strain may have been deposited with Accession No. KACC 92186P.

In addition, the harmful bacteria can be gram negative bacteria.

In addition, the harmful bacteria may be E. coli.

The E. coli is preferably E. coli K88.

On the other hand, the present invention provides an antimicrobial composition comprising the above-mentioned strain as an active ingredient.

Here, the composition may be in the form of a food, a food additive, a feed additive, or a medicament.

The food may be selected from the group consisting of milk products, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, alcoholic beverage, Or more.

In addition, the present invention provides a probiotic composition comprising the above strain or a culture solution thereof as an active ingredient.

In addition, the present invention provides a method for producing an antimicrobial microbial preparation for Gram-negative bacteria comprising the step of culturing the above-mentioned strain.

Further, the present invention provides a method for treating Gram-negative bacteria by treating the strain or a culture solution thereof with Gram-negative bacteria.

The E. faecium strain screened and deposited according to the present invention exhibits excellent antimicrobial activity against E. coli Gram-negative bacteria, and the deposited strain contains a specific gene. Using this excellent strain, The present invention can be applied to the production of probiotics for animals including humans.

FIG. 1 shows clearing zone comparisons between the E. faecium strain JB00008 and the five antibacterial activity strains (JB00005, JB00034, JB00058, JB00063, and JB00066), which have excellent antimicrobial activity.
Figure 2 shows the survival curves of E. faecium JB00008 strain in liquid medium supplemented with (A) acidity (HCl, pH 3) and (B) bile salt (0.5%).
FIG. 3 shows the genomes of 11 genes of genome sequencing. The genome was annotated with RAST, and only the genotype of p <0.1 is shown by statistical analysis (T-test).
FIGS. 4A to 4D are sequences showing genetic sequence information specifically found in the E. faecium JB00008 strain among the 11 E. faecium strains to be compared.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention is capable of various modifications and various embodiments, and specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

 The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In the present application, the terms "comprises" or "having" and the like are used to specify that there is a feature, a number, a step, an operation, an element, a component or a combination thereof described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.

Hereinafter, the present invention will be described in more detail.

The present invention provides an Enterococcus faecium strain having an antibacterial activity against noxious bacteria and comprising a gene consisting of the nucleotide sequence of any one of SEQ ID NOS: 1 to 4.

Among the various characteristics of probiotics, the most important feature as an antibiotic substitute is the ability to inhibit pathogens. This ability helps prevent intestinal infections through the production of organic acids, bacteriocins, or directly to the immune function of the animal. Accordingly, it is very important to obtain a microorganism having a better pathogen inhibiting ability even in a microorganism species used as a probiotic agent.

The present inventors selected E. faecium strains excellent in the ability to inhibit Gram-negative bacteria among the pathogens suffering great damage to the pig specification industry (see Example 1). That is, the strains were isolated from samples such as chicken, chicken feces, soybean fermented foods (Chungkukjang, doenjang, soy sauce), pig feces and the like, and the physiochemical characteristics of the isolated strains were analyzed to determine acid resistance, bile acid resistance, The strains with excellent antimicrobial activity were identified.

Of the 143 E. enterococci strains, Enterococcus spp. (JB00008), which has a remarkable ability to inhibit E. coli, was selected (see Experimental Example 1) (See Experimental Example 4). In addition, the present invention provides a method for producing a microorganism capable of inhibiting pathogenic bacteria.

The Enterococcus spp. Strain (JB00008) according to the present invention thus selected is superior in its ability to inhibit E. coli basically and also has excellent ability to survive acid / bile acid (see Experimental Example 3). In addition, since the Enterococcus spp. Strain (JB00008) has a specific nucleotide sequence (SEQ ID NOS: 1 to 4) which is not possessed by other strains, the use of this nucleotide sequence significantly inhibits E. coli Excellent pseudomonas strains can be identified.

Accordingly, the strain according to the present invention can include all the genes comprising the nucleotide sequences of SEQ ID NOS: 1 to 4. When all the genes comprising the nucleotide sequences of SEQ ID NOS: 1 to 4 are used, it is possible to more accurately determine the pseudomonas strain having a remarkably excellent inhibitory ability against E. coli.

In addition, the strain may have been deposited with Accession No. KACC 92186P. The present inventors named the strain selected as Enterococcus faecium JB00008 strain and received deposit No. KACC 92186P on Aug. 04, 2017 by the Institute of Agricultural Biotechnology. The deposited strains are remarkably superior in their ability to inhibit E. coli than other strains, and have excellent acid / bile acid survival ability and contain all of the above-mentioned specific genes.

The strain of the present invention has excellent antibacterial activity against harmful microorganisms. In particular, the strain has a remarkably superior antimicrobial activity against noxious bacteria, preferably Gram-negative bacteria, more preferably Escherichia coli, most preferably E. coli K88 (K88 antigen-positive E. coli). In addition, the strain of the present invention is excellent in acid resistance and bile acid resistance and can play an important role as an active agent for animals.

Meanwhile, the present invention provides an antimicrobial composition comprising the strain of the present invention as an effective ingredient.

In addition, the composition is preferably in the form of a food, a food additive, a feed additive or a medicament.

The food may be selected from the group consisting of dairy, fermented milk, drink, meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, gum, ice cream, soup, beverage, alcoholic drink and vitamin complex Is more preferable.

The present invention also provides a probiotic composition comprising the strain of the present invention or a culture thereof as an active ingredient. At this time, the culture may contain both the concentrated liquid and the dried product of the culture liquid.

The Enterococcus faecium JB00008 strain or its composition for antimicrobial use according to the present invention is excellent in antimicrobial activity, acid resistance, and bile resistance against harmful microorganisms. Therefore, the Enterococcus faecium JB00008 strain of the present invention or its composition for antimicrobial use can be effectively used as a prophylactic agent, especially a human medicament, in a formulant, a lactic acid bacterium preparation, a feed additive and a health food. (Milk, soy milk, processed milk), fermented milk (liquid yogurt, yogurt yogurt), drinks, meat, sausage, bread, chocolates, candies and the like, preferably in the form of a food, a food additive, a feed additive or a medicament. , Snacks, confectionery, pizza, ramen, gums, ice cream, soup, beverages, alcoholic beverages and vitamin complexes.

The food of the present invention may contain a functional food. In the functional food of the present invention, in addition to the above-mentioned active ingredients, various auxiliary ingredients may be added as necessary. In the case of the functional food of the present invention, vitamins such as vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B12, folic acid, vitamin C, vitamin D3 and vitamin E, Iron, magnesium, potassium, zinc, etc., or lactic acid bacteria.

In addition, among the functional foods of the present invention, health drinks may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the flavoring agent include natural sweetening agents such as tau martin and stevia extract, and synthetic sweetening agents such as saccharine and aspartame. Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.

The composition of the present invention may further comprise conventional pharmaceutical carriers and excipients in addition to the above-mentioned active ingredients. Such compositions may be prepared by heat drying or freeze-drying according to a conventional method for producing a probiotic composition, .

Examples of the diluent include lactose, corn starch, microcrystalline cellulose, and mannitol. The lubricant is preferably magnesium stearate or talc, and the binder The zero is preferably selected from polyvinylpyrrolidone or hydroxypropylcellulose. The disintegrant is preferably selected from carboxymethylcellulose calcium, sodium starch glycolate, polacrilin potassium or crospovidone, and the sweetener is selected from white sugar, fructose, sorbitol or aspartame. Sodium carboxymethylcellulose , Beta-cyclodextrin or xanthan gum, and the preservative is preferably selected from methyl parahydroxybenzoate, propyl p-hydroxybenzoate or potassium sorbate.

In addition, the present invention provides a method for producing a microorganism preparation for antifungal agents against harmful bacteria comprising culturing the strain. The method for culturing the strain of the present invention can be carried out according to a method commonly used in the art, preferably using a culture medium supplemented with L-cysteine, but not limited thereto.

In addition, the present invention provides a method for producing an antimicrobial microbial preparation for Gram-negative bacteria comprising the step of culturing the strain of the present invention.

The antimicrobial microbial preparation for harmful bacteria of the present invention can be produced using the deposited Enterococcus pseudomonas strain of the present invention as an active ingredient. The antimicrobial microbial preparation according to the present invention may be prepared as a solution, a powder, a suspension, a dispersion, an emulsion, an oil dispersion, a paste, a dust, a scattering material or a granule but is not limited thereto.

The present invention also provides a method for treating gram-negative bacteria by treating a strain or a culture solution thereof with Gram-negative bacteria.

The present invention provides a method for treating harmful bacteria by treating the strain or a culture solution thereof with harmful bacteria. The harmful bacteria are as described above.

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 present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Example 1: Isolation and Identification of Microorganisms

Chicken, chicken feces, soybean fermented food (Chungkukjang, soybean paste, soy sauce) and pig feces samples were obtained for identification of microbial isolates. The fecal samples were streaked on each Enterococcosel agar to isolate 1946 strains of lactic acid bacteria. Then, 143 strains of Enterococcus spp. Were obtained by PCR using E. faecium-specific primers in the lactic acid bacteria strain (E. faecium) strains were identified.

Experimental Example 1: Assessment of the ability to inhibit pathogens

Escherichia coli (E. coli) strain K88 was selected as a pathogen that has the greatest damage in pig farm to evaluate the pathogenic ability of E. faecium strains (Table 1). Agar diffusion assay was performed to measure the antimicrobial activity against the pathogens. E. faecium strains isolated and identified in Example 1 were cultured in BHI broth for 24 hours at 37 DEG C and then tubes containing E. faecium strains were centrifuged The supernatant was discarded and fresh BHI broth was added before dispensing on paper discs.

The pathogen was cultured for 24 hours and spread evenly on a 90 × 15 mm Petri dish filled with LB agar. After this, a paper disc with a diameter of 8 mm was placed on a Petri dish, and 60 μl of the prepared E. faecium culture was repeated twice on a paper disc (total of 120 μl treatment). After culturing for 24 hours at 37 ℃, the diameter of the clearing zone due to inhibition of pathogen growth was judged by the ability to inhibit the pathogen.

Pathogen Gram staining General lesion of pathogen Others K88 antigen-positive Escherichia coli Negative Food poisoning, gastroenteritis, urinary tract infection, neonatal meningitis, etc. Separation from Korea, causing diarrhea in pigs

* Table 1 shows the number of target pathogens

The 143 E.coli E. faecium strains obtained in Example 1 were evaluated for their antimicrobial activity against E. coli K88. The most excellent strain was E. faecium JB00008 strain (Fig. 1).

Experimental Example 2: Genome sequencing and assembly results

In order to compare the genomes, six strains (JB00008, JB00012, JB00023, JB00056, JB00057, JB00069) and five strains with low pathogen inhibitory ability (JB00005, JB00034, JB00058, JB00063, JB00066) were further screened for genome sequencing.

As a result, there was no difference in the level of assembly as well as the basic genome characteristics such as genome size, GC content, and antimicrobial activity (Table 2).

JB00008 Ave. of High strains Ave. of Low stains Total length (bp) 3,378,187 3,258,049 2,935,876 Max contig length (bp) 133,497 127,370 121,819 Average contig length (bp) 15,639 14,045 17,354 No. of contigs 216 430 414 N50 (> = 500 nt scaffold) 44,064 44,887 48,911 GC content (%) 38.4 38.09 38.34

Table 2 shows the basic characteristics (assembly results) of the Draft genome.

Then, in order to confirm that the draft genome obtained from the genome sequencing result is E. faecium, the NCBI blast is compared with the E. faecium reference genome of the NCBI genome database As a result, it was confirmed that the E. faecium was 99% identity percentage.

Experimental Example 3: Survival experiment of acid / bile acid to evaluate ability as a probiotic agent

In order to utilize the strains as probiotics for human / livestock, the strains must survive in the digestive enzymes such as acid / bile acid which is a nutrient decomposition substance produced in the stomach or liver. Therefore, E. faecium JB00008 strain evaluated for its ability to inhibit pathogens was confirmed to be resistant to acid / bile acid-containing BHI broth.

JB00008 strain was cultivated in BHI broth for 24 hours and the number of bacteria was adjusted to about 1 × 10 ⁹ CFU / ml. For medium containing hydrochloric acid (HCI), 800 ml of BHI broth was adjusted to pH 3 by adding 38% HCl. For medium containing bile acid, 800 ml of BHI broth was added with Bile salts (Oxoid) 0.5% W / V Bile salts. 49.5 ml of each mixed solution was added to a 50 ml tube and 500 μl of JB00008 culture was inoculated. After shaking, the cells were incubated at 37 ° C in an incubator for 15 minutes and 30 minutes, The number of viable cells was measured by repeating 2 times.

As a result, the viable cell count of E. faecium JB00008 strain in BHI broth containing hydrochloric acid (HCI) was decreased due to the influence of HCI over time (15 minutes, 30 minutes) The mean survival rate was 82.16%, which was not significantly decreased (Fig. 2a). In the case of BHI broth containing bile salts, E. faecium JB00008 was found to have a two-fold average survival rate of 130.84% over time (15 minutes, 30 minutes), indicating that bile acid did not significantly affect JB00008 (FIG.

Experimental Example 4: Genome analysis

DNA of selected E. faecium strains was extracted and subjected to genome sequencing using Illumina HiSeq 2500 instrument. After the filtering process, the assembly was performed using a Ray assembler to create a draft genome. Draft genomes were subjected to Gene presence test and Single nucleotide polymorphism (SNP) analysis. The genome was annotated and analyzed using RAST.

Genome comparison result: JB00008 Specific gene

(JB00008, JB00012, JB00023, JB00056, JB00057, JB00069) and antibiotic activity group (Low antibacterial activity group) using RAST (Rapid Annotation using Subsystem Technology) ; JB00005, JB00034, JB00058, JB00063, JB00066) were analyzed and the genes that differed between the two groups were analyzed through the annotated results.

The results of FIG. 3 show the number of genes having a significant difference (p <0.1) between the two groups by heatmap. The more the number is, the darker the red color, while the blue is the darker the more the number is smaller. Glycerol and glycerol-3-phosphate uptake and utilization, chitinol, and other genes involved in DNA regulation, such as DNA repair, DNA processing cluster, and DNA topoisomerase, and N-acetylglucosamine utilization, lactose and galactose uptake and utilization, and E. faecium JB00008, a selective strain, was grouped with strains having excellent antimicrobial activity .

In the case of promoting the activity of live bacteria, Toxin or acid secreted by the pathogens or acid / bile acid secreted from the stomach-intestines are damaged due to high acidity or toxicity, thereby damaging the enzyme activity, protein and DNA secreted by the microorganisms. Do not let them or make mutations appear. However, as shown in FIG. 3, DNA repair and topoisomerase, which are highly expressed in a strain having excellent antimicrobial activity, can inhibit such growth inhibition or mutation through DNA repair such as DNA / RNA processing and repair. do. Therefore, it is considered that the genes can survive even in a harsh living environment due to the function of the genes expressed in the antibiotic-active strains such as Enterococcus faecium JB00008.

Using the results of annotation using the RAST, the genes that are specifically present in E. faecium JB00008 were searched through Orthologs gene presence analysis.

By comparison of 11 strains of E. faecium (JB00008, JB00012, JB00023, JB00056, JB00057, JB00069, JB00005, JB00034, JB00058, JB00063, JB00066) containing JB00008, 4 (SEQ ID NOS: 1 to 4) (Table 3, Figs. 4a to 4d). All of the above four genes (SEQ ID NOS: 1 to 4) were Hypothetical proteins in Annotation of RAST SEED viewer analysis. Hypothetical protein means a protein whose function or expression has not yet been revealed.

As a result, the above four genes (SEQ ID NOS: 1 to 4) showed that it could be used to distinguish JB00008 among various strains of E. faecium.

Orthologs JB
00005 JB
00008 JB
00012 JB
00023 JB
00034 JB
00056 JB
00057 JB
00058 JB
00063 JB
B00066 JB
00069 SEQ ID NO: 1 0 One 0 0 0 0 0 0 0 0 0 SEQ ID NO: 2 0 One 0 0 0 0 0 0 0 0 0 SEQ ID NO: 3 0 One 0 0 0 0 0 0 0 0 0 SEQ ID NO: 4 0 One 0 0 0 0 0 0 0 0 0

* Table 3 shows that among the 11 E. faecium strains analyzed, only the Sequence 4 species specifically detected in the JB00008 strain

Experimental Example 5: Antibiotic resistance and pathogenicity analysis

The European Food Safety Authority (EFSA) guidelines require the presence of pathogenic genes IS16, Esp, hyl, and gelE as safety assessments for the use of Enterococcus faecium as an animal feed additive. have. In order to be used as a probiotic for feed and human, antibiotic resistance and the presence of a pathogenic gene are important for the safety of the bacterium. Therefore, we compared antibiotic resistance genes and pathogenicity genes with pathogenic gene database and antibiotic resistance gene database (ARDB) and E. faecium JB00008.

Antibiotic resistance and pathogenicity analysis

1) Search for antibiotic resistance genes

To confirm the safety (antibiotic resistance gene and pathogenicity gene) as a feed additive, 768 genes of ARDB (Antibiotic Resistance Genes Database) were compared with each other. Antibiotic resistance genes, including the most problematic Vancomycin resistance gene in E. faecium, were not detected in all strains (Table 4).

^a ND not detected

* Table 4 shows the results of confirmation of the vancomycin resistance gene of E. faecium strains

2) Pathogenicity gene search

88 genes, known as Enterococcus virulence factor, were analyzed with genomes of E. faecium strains to confirm the presence of VF gene. As a result, it was confirmed that IS16, Esp, hyl and gelE were not present in all strains including JB00008 strain (Table 5).

^a ND not detected

* Table 5 shows the presence of pathogenic genes in E. faecium isolates

Experimental Example 6: Comparison of functional characteristics between strains

Genomic annotation was performed on 11 strains that had been sequenced using RAST, and we identified genes that appeared in strains with excellent pathogen - inhibiting ability.

There were 14 genes that were statistically expressed (P <0.05) more frequently than strains with poor pathogen inhibition ability. These genes were mainly DNA / RNA repair and processing related genes, L-rhamnose utilization, specific nutrients such as lactose and galactose uptake and utilization And E. faecium JB00008 was clustering with a strain having an excellent pathogen-inhibiting ability (Fig. 4).

1) Comparison of functional characteristics between strains: Lactose and Galactose uptake and utilization

Lactose is a disaccharide that is a combination of galactose and glucose. It is also called lactose and lactose, and it is produced mainly from mammalian milk. E. faecium, which had a high antimicrobial activity, rapidly accelerated bacterial growth by using lactose and galactose and increased the production of antimicrobial substances such as lactic acid production and Bacteriocin by the proliferation of bacteria, . As it is used as a probiotic, it has an excellent ability to degrade lactose and galactose.

2) Comparison of functional characteristics between strains: DNA / RNA repair and processing gene

DNA repair and processing genes are a function of repairing or repairing exogenous genes when they are imported or damaged, and are essential for maintaining the integrity of the genome, and furthermore, the normal functioning of the cells. Therefore, E. faecium strains, which have high antimicrobial activity including E. faecium JB00008, survive in external attacks such as pathogens and attack host pathogens by these mechanisms, It is thought that it played a role of going.

Although the present invention has been shown and described with respect to certain preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the following claims It will be apparent to those skilled in the art.

Agricultural Biotechnology Research Institute KACC92186P 20170906

<110> KNU-Industry Cooperation Foundation <120> A novel Enterococcus faecium and anti-bacterial use of the same <130> PA-D2017-20 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 750 <212> DNA <213> Enterococcus faecium <220> <221> gene &Lt; 222 > (1) <223> Sequence 1 <400> 1 atgtatgatt atgtttatat ggatgataat ggtgaactaa gatatttaaa ttattacagc 60 attaaaaatc tggcaaagca attagctaga gtaacgaacc aaaagaaagt aagcttagat 120 aggacaagaa aaattcttta tcatatgatt gttactgaga ttgttaaaaa gaaaccagaa 180 gcagaaattc ctaaggatat atttaatggt ttgatactgg aacaaaagaa agattgcact 240 aagctaagaa cgagtaatat ctatgaacca acaaatttga cagatgaaaa cacagcagtt 300 agtgaaggaa tagctaaggt tttaagagaa aataaagtaa cagtcggtag tttaagttat 360 gaattggtat atagactttt tggagaagaa aaggcttatc aggattttcc acaatcttat 420 aaaccacttg ttgaacggaa aaagataaag atgagtagaa cagacaacaa cctaagccaa 480 gcaagtgaga agttagaaaa atcagcagtt agtataatta tgagagagat tgaagaaaag 540 ggttatgtat acgaaaagga tgttatttct aaggtagcta aagcaagaag attaaaagtt 600 cgtgacactg cagaaagatt taagaaacta agagcggata tatacaataa atatggtttg 660 aatacgtgta gactgactaa agaattgtat aatcagttag agattaagga gaagtattca 720 tctaagaatg ttatatataa aatgaattag 750 <210> 2 <211> 177 <212> DNA <213> Enterococcus faecium <220> <221> gene <222> (1). (177) <223> Sequence 2 <400> 2 atgacagatg gagcttcaca aggacttttt gtaatagtag cggtggtaat ttttggtatt 60 tttgtattaa tcgcctatat cctattccgt gagacattac aacctaaatt agctgaaata 120 ttcagtaaag cgacaactga tgcaggagca agcttgaatt atacacctgc tgggtaa 177 <210> 3 <211> 696 <212> DNA <213> Enterococcus faecium <220> <221> gene &Lt; 222 > (1) .. (696) <223> Sequence 3 <400> 3 atggataaaa ttaatgaagt agaagttttg cctagagatg agaattttac ttataaaaac 60 ggatataagt cttgggtaag ttctattttt attgatattc gtaattcaac agagttgata 120 gaaaataata gcgagcttaa ggtagcaaaa gtaattcgtg gctttacttc agaaattatt 180 gaaatattaa gaaaagatac ggataagttg aaagaaatcg gcatacgagg tgactgtgtt 240 tatgcaatat attcttcacc agcgcaaaaa gatatttacg atcttacaga tagagcctat 300 tatgtaaata cgtatttaaa aatgctaaat tctttacttt ctaaaagaaa cttacccaag 360 ataacagcag gaatcggttt ggcaacctcc caaacattag tagttaaagc aggtagacat 420 tcttcaggaa ttaataattt ggtgtggatt ggagaatcag tttcaatggc ttgtaattta 480 gctaatattg ctaataaaga aatagatgat tccatactac tttctagcaa ttttcattat 540 aattattcta aatatataga acaatatgcc caagaagaaa aaaagacatt gaattattat 600 aaacgatgtg aacatgaacg ctatgggacg ttttatcatg ctagtattat tatgtcagct 660 tttaataaat ggataaaaga cgacatgcct gtataa 696 <210> 4 <211> 123 <212> DNA <213> Enterococcus faecium <220> <221> gene <222> (1). (123) <223> Sequence 4 <400> 4 ttgcaacaga accatttaac tggtattaca gacgaagaca tgcacgataa aaaagttcat 60 gacgtatatc aattagtttc taccgagatc attaatagaa ttgaggaatt tgaaaatgaa 120 taa 123

Claims (12)

Has antibacterial activity against Gram-negative bacteria E. coli K88,
Enterococcus faecium strain, deposited with Accession No. KACC 92186P.
delete delete delete delete The method according to claim 1,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14,
11. An animal prothrombin agent composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
delete delete A probiotic composition comprising the strain of claim 1 or a culture thereof as an active ingredient.
A method for producing an antimicrobial microbial preparation for Gram-negative bacteria comprising culturing the strain of claim 1.


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