KR20180059372A - Composition for preventing or treating porcine diarrhea using new pediococcus acidilactici - Google Patents

Abstract

The present invention relates to a novel composition for preventing or treating porcine diarrhea using Pediococcus acidilactici. The Pediococcus acidilactici is excellent in acid resistance and bile resistance, and is excellent in intestinal sticking ability, so that microbiological probiotics effectively stick to the intestines and can exhibit an excellent pathogen inhibiting effect. Therefore, the novel Pediococcus acidilactici can be provided as a composition for effectively preventing or treating porcine viral diarrhea.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating porcine diarrhea using a novel Pediococcus axicillus lacticus.

The present invention relates to P. acidilactici ACB26 (KCTC18512P) having excellent intestinal mucosa adhesive ability and a composition for preventing or treating porcine diarrhea using the same.

Diarrhea of livestock is caused by external environment such as rapid change of feed, stress, temperature change, and is divided into viral such as Escherichia coli and salmonella, and viral such as TGE and PED, and severe diarrhea is continued to cause absorption trouble of nutrients ingested And the water and electrolytes of body tissues are lost, resulting in imbalance of dehydration and electrolytes.

Diarrhea generally occurs in pigs with weak resistance. Infectious diarrhea is mainly seen in pre-infected piglets, and most of them cause severe damage due to growth disorder even if they are healed.

The prevention and treatment of diarrhea is mainly accompanied by vaccination and antibiotics. In particular, viral diarrhea causes massive mortality of piglets due to rapid spread of disease, but development of therapeutic substances is poor.

In Korea, the viral diarrheal diseases of pigs such as TJ, ROTA, and PIDI have been continuing to cause damage, but regulations on the use of synthetic antibiotics used as a countermeasure against bacterial diseases worldwide have been strengthened. The Ministry of Health and Welfare has been participating in the international antibiotic use regulation by reducing the number of veterinary drugs such as antibiotics that can be mixed into feeds from 53 to 25 in order to improve the safety of livestock products. In addition, as meat consumers are increasingly interested in health and preference for safe livestock products is increasing, concerns about the use of antibiotics indiscriminately are increasing.

Korean Patent Publication No. 10-2011-0055266 (Published May 25, 2011)

In the present invention, when a microorganism having an antimicrobial activity against a pathogenic microorganism is used as a probiotic agent, the intestinal adhesive ability is not verified even if the intestine is reached. In order to solve this problem, The present invention provides an effective pathogenic microorganism inhibiting microorganism prophylactic agent using P. acidilactici.

The present invention provides Pediococcus aceydyllacticity ACB26 (KCTC18512P) having antimicrobial activity against pathogenic bacteria causing swine diarrhea.

The present invention provides a pharmaceutical composition for preventing or treating porcine diarrhea comprising Pseudomonas aeruginosa (P. acidilactici ) as an active ingredient.

The present invention provides a feed additive containing Pseudomonas aeruginosa (P. acidilactici ) as an active ingredient and inhibiting pathogens causing swine diarrhea.

Further, the present invention provides a microbial agent for suppressing pathogens which contains an O Phedi Lactococcus aksi dill when rakti (P. acidilactici) as the active ingredient, cause the pig diarrhea.

According to the present invention, Akshil De Lactiti ACB26 was excellent in acid resistance and bile resistance, and was excellent in intestinal mucosal sticking ability. Thus, it was confirmed that the microbiological probiotics effectively adhered to the intestines and showed an excellent pathogenic inhibitory effect, Akshil De Lactiti ACB26 may be provided as a composition for the prevention or treatment of effective porcine viral diarrhea.

Figure 1 shows unknown bacterial identification results through multiplex PCR.
FIG. 2 is a porcine mucus binding assay method using a microplate reader.
FIG. 3 shows the results of analysis of porcine mucoadhesiveness using a microplate reader.
Figure 4 is a relative mucus adhesion assay.
Figure 5 shows the results of relative mucosal cohesion analysis.
FIG. 6 shows results of porcine mucus adhesion assay for protease treatment.
Figure 7 shows the results of IPEC-J2 adhesion assay.
Fig. 8 is a graph showing the results of measurement It is the result of ETEC K88 removal analysis using the axillary deactivation.
Fig. 9 is a graph showing the results of evaluation The results of the EHEC O157: H7 elimination analysis using the axide dextrose.
Fig. 10 shows the results of evaluating the growth activity of microorganisms having excellent intestinal mucosa sticking ability.
Fig. 11 shows the results of evaluating the acid resistance and bite resistance of microorganisms having excellent intestinal mucosa sticking ability.
12 is a schematic diagram showing a specification experiment schedule.
FIG. 13 shows the results of confirming the incidence of diarrhea and rash.
Fig. 14 shows the result of confirming TNF-a in serum.
Fig. 15 shows the results of confirming IL-10 in the blood.
FIG. 16 shows the result of confirming the microbial community in pig feces using Illumina Miseq (Macrogen, South Korea).
17 was obtained by using qRT-PCR in the feces (a) Pediococcus (C) F18, and (d) Intimin adducts, which are derived from E. coli.
Fig. 18 shows the results obtained by using (a) Bifidobacterium genus in feces using qRT-PCR. And (b) the relative amount of Lactobacillus species.

The present invention can provide Pediococcus acedylactacicide ACB26 having antimicrobial activity against pathogenic bacteria causing swine diarrhea.

The base sequence of the 16S rRNA gene of Pediococcus axilidyllacticus ACB26 can be represented by SEQ ID NO: 1, and the microorganism deposit number is KCTC18512P.

The pathogens exhibiting antimicrobial activity by the Pediococcus axilidyl lacticus ACB26 may be selected from the group consisting of enterotoxin E. coli (ETEC) K88 and intestinal hemorrhagic E. coli (EHEC) O157: H7.

In addition, the present invention can provide a Phedi O Lactococcus aksi dill rakti when pig diarrhea preventing or pharmaceutical composition containing (P. acidilactici) as an active ingredient.

The Pediococcus axilidyl lactis is selected from the group consisting of Pediococcus axilidyl lactic acid ACB5, Pediococcus axilidyl lactic acid ACB18, Pediococcus axilidyl lactic acid ACB26, Pediococcus axilidyl lactic acid ACB 27, Pediococcus axicil Lactic acid ACB44, and Pediococcus axicillus lacticus ACB48.

The Pediococcus axicillus lacticus is adhered to the intestinal mucous membrane of pigs and can inhibit disease induction upon entry into the intestines. More specifically, the pathogenic microorganisms include enterococci E. coli (ETEC) K88, Hemorrhagic E. coli (EHEC) O157: H7.

In addition, the present invention can provide a feed additive for inhibiting pathogen that contains an O Phedi Lactococcus aksi dill when rakti (P. acidilactici) as the active ingredient, cause the pig diarrhea.

In addition, the present invention can provide a microbial agent for suppressing pathogens which contains an O Phedi Lactococcus aksi dill when rakti (P. acidilactici) as the active ingredient, cause the pig diarrhea.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Pedio Caucus Akshil De Lactiti [ Pediococcus acidilactici  (P. acidilactici) Isolation and identification

620 colonies were isolated from pig feces using MRS agar plates as the lactic acid bacteria selection medium, and as shown in Table 1, Pediococcus Multiplex PCR was performed using a primer set that can be amplified by PCR specifically for axillary deactivation.

Using the primers, colonies suspended in MRS agar plates in a selective medium were subjected to PCR under the following conditions.

10 μl of 2 × I-Taq polymerase master mix (Intron, Korea), 1 μl of each primer and 2 μl of distilled water were added to a final volume of 20 μl by a colony, and PCR was carried out in the following manner.

PCR was confirmed by loading on 2% agarose gel, electrophoresis at 60 volts for 30 min, and UV detection with Gel Doc XR + (Bio Rad, USA).

As a result, as shown in Fig. 1, 48 colonies out of 620 colonies were found to be Pediococcus It was confirmed that it was an axillary dactylase, and the confirmed 48 pediococcus Axidil Lactissi and Pediococcus from our laboratory Axidyl lactis 175 (PC) and Pediococcus Genomic shuffling 1 (PG) was used for screening together with the axillary duct.

< Example  2> Pedio Caucus Akshil De Lactiti [ Pediococcus acidilactici  (P. acidilactici) Of the intestinal mucosa

Using a microplate reader, 50 pediococcus In order to measure and quantify the absorbance value of microorganisms having excellent adhesive ability on a plate coated with porcine mucus after the staining of axilidyltaxy with crystal violet, 50 pediocytes Caucus Axidil lactis and Pediococcus as controls A total of 51 bacteria were evaluated together with KCTC by purchasing the DMS20284 (PK) strains of Axidyl lactis.

The porcine small intestine mucosa (Mucus) was diluted to contain 1 mg protein in HEPES-Hanks buffer and 200 μl of the suspension was fixed at 4 ° C overnight in a 96-well tissue culture plate (Costar).

The mucosa-fixed plate was washed twice with 250 μl HEPES-Hanks buffer to remove unbound protein.

Thereafter, the selected pedioccus Axidyl lactylate was added to each well at 10 ⁶ cfu, followed by incubation at 37 ° C for 1 hour.

After attaching the bacteria at 37 ° C for 1 hour and washing them with 250 μl of PBS three times to remove the unattached bacteria, the attached bacteria were fixed at 60 ° C for 20 minutes and then immersed in a crystal violet (100 μl / well, 0.1% solution) Lt; / RTI &gt; The wells were then washed five times with PBS to remove the remaining staining solution.

100 [mu] l citrate buffer (20 mmol / L; pH 4.3) was added to release the staining solution bound to the bacteria, and the absorbance was measured at 570 nm after 45 minutes incubation at room temperature.

The mucus stained without the addition of bacteria was used as a negative control, and the absorbance values of the negative control samples were subtracted from the absorbance values of the respective experimental samples to obtain the results.

As a result, as shown in FIG. 3, all the microbes showed higher binding values than the control PK. In particular, ACB5, ACB18, ACB26, ACB27, ACB44, ACB47 and ACB48 showed high binding values.

In order to confirm the correct numerical value, a relative mucus adhesion assay was performed to quantify the microorganisms before and after the microbial treatment on the plate coated with the porcine mucosa as the adhesion value as shown in FIG. Respectively.

Adhesion assays were performed in a 96-well polystyrene microtiter plate (SPL Life Sciences, Pocheon, Korea) using porcine ileum mucosa as a matrix.

10 mmol / L HEPES-Hanks (HH) buffer containing about 100 μl of mucosal solution (1 mg / ml) was fixed in the plate well for 1 hour and incubated overnight at 4 ° C.

The wells were then washed twice with 200 μl HH buffer and incubated with 100 μl bovine serum albumin (BSA; Sigma, 20 mg / ml) for 2 h at 4 ° C.

100 μl of the bacterial suspension (approx. 10 ⁶ CFU / ml) was washed and suspended in 10 mmol / L PBS buffer (pH 7) and added to the wells and incubated at 37 ° C for 1 hour.

The wells were then washed five times with 200 [mu] l of sterile citrate buffer to remove unbound bacteria.

In another experimental group, 200 μl of 0.5% (v / v) Triton X-100 was added to isolate the attached bacteria. The number of viable cells expressed in all cases of CFU / ml was determined by plating on MRS medium.

Each analysis was repeated two times or more each, and each time three times, and the percentage of adhesion was calculated by the following equation.

As a result, as shown in FIG. 5, twelve bacteria such as Pa ACB5, Pa ACB13, Pa ACB16, Pa ACB18, Pa ACB23, Pa ACB26, Pa ACB27, Pa ACB40, Pa ACB44, Pa ACB45, Pa ACB47, Pa ACB48 And porcine mucus binding assays were performed to confirm the presence of 12 bacterial strains such as Pa ACB4, Pa ACB11, Pa ACB19, Pa ACB20, Pa ACB22, Pa ACB25, Pa ACB28, Pa ACB42, Pa ACB46, , The adhesion value was 40% or more in the case of the strains having excellent mucosal adhesion ability and the difference was less than 30% in the case of the strains having poor adhesive ability.

Mucus (Mucus) is a highly glycated protein, Pediococcus The porcine mucus adhesion effect by proteolytic enzyme treatment was examined to determine whether the mucoadhesive ability of axilidyl lactis is more affected by protein and carbohydrate.

First, the immobilized intestinal mucosa was treated with trypsin or protease K (0.8 mg / ml, 200 μl per well; Sigma Co.) to examine the effect of the proteolytic enzyme treatment on the bacterial adhesion of mucus.

BSA-treated (0.8 mg / ml, 200 μl per well) was used as a control. All plates were incubated at 37 ° C. for 2 hours, then incubated overnight at 4 ° C. and washed with HEPES-Hanks buffer.

200 [mu] l of solution was added to each well and incubated for 3 hours at 4 [deg.] C under dark conditions, and each well was washed with HEPES-Hanks buffer and then analyzed for adhesion.

As a result, as shown in FIG. 6, when protease K and trypsin were treated in comparison with the control group, BSA, the strains having excellent adhesive ability showed the% adhesion value of the BSA- .

From the above results, It has been confirmed that the ability of the pig mucosa adhesive possessed by axillary dactylis is mainly in the protein region of the mucosa.

&Lt; Example 3 > In porcine small intestinal epithelial cells, intestinal Confirm adhesive ability

Adhesion ability was confirmed using porcine small intestinal epithelium (IPEC-J2 cell) together with Pa-ACB26, Pa-ACB5, Pa-ACB18, Pa-ACB44, Pa-ACB27 and Pa- .

For evaluation of stickiness, IPEC-J2 cells grown after late confluence were seeded with respective Pediococcus Aksi dill were inoculated when rakti to 10 ⁸ CFU / ㎖, pathogens (Pathogen) to the firing of enterotoxins by using the F4, F18 Pim Calabria (fimbria) in the small intestine of a pig attached to the epithelial cells leads to diarrhea E. coli ( ETEC) K88 and intestinal hemorrhagic E. coli (EHEC) O157: H7 strains were inoculated.

The inoculated 6-well plate was incubated at 37 [deg.] C for 3 hours and then washed twice with PBS to remove unattached pediococcus Axilidylation and pathogens were removed.

Attached Pediococcus In order to analyze axillicylase and pathogens, the cell lysate obtained by treating 0.05% trypsin-EDTA with IPEC-J2 single membrane was serially diluted with 0.1% Triton-X100.

The serial dilutions obtained by the above method were inoculated into MRS agar and LB agar by injection flat plate method.

As a result, as shown in Fig. 7, the adhesion values of Pa ACB26, Pa ACB5, Pa ACB44, Pa ACB27 and Pa ACB23 were 40% or more, whereas those of PC and PG were 40% or less.

On the other hand, the pathogen removal effect of Pa ACB5, Pa ACB18, Pa ACB26, Pa ACB27, Pa ACB44 and Pa ACB48, which are excellent in intestinal mucosal sticking ability as described above, was confirmed in porcine mucosa and IPEC-J2.

(i) IPEC-J2 cells (10 &lt; ⁶ &gt; cells per well) Experimental group treated with ETEC K88 or EHEC O157: H7 (107 CFU / ml) for 2 hours after incubation for 2 hours with inocidyl lactylase (10 ⁸ CFU / ml). (ii) IPEC-J2 cells (10 &lt; ⁶ &gt; cells per well) Experiments in which axicidyl lactylase (10 ⁸ CFU / ml) and ETEC K88 or EHEC O157: H7 (10 ⁷ CFU / ml) were treated and incubated for 2 hours; And (iii) IPEC-J2 cells (10 ⁶ cells per well) and ETEC K88 or EHEC O157: H7 (10 ⁷ CFU / ml) (10 ⁸ CFU / ml) for 2 hours.

Each of the test groups was incubated under the above conditions and then washed twice with PBS to remove nonadhesive Pediococcus Axidyl lactylase and pathogens were removed, and IPEC-J2 cell monolayers were treated with 0.05% trypsin-EDTA.

Attached Pediococcus For the analysis of axillidyl lactis and pathogens, serial dilutions of the cell lysate were continuously inoculated with MRS agar and LB agar by injection flat-plate method with 0.1% Triton-X100.

As a result, as shown in Fig. 8, Pa ACB18, Pa ACB26, Pa ACB27 and Pa ACB44 inhibited, compete and displace ETEC K88 in comparison with PBS and control PK in porcine mucus and IPEC-J2 cells, 9, it was confirmed that Pa ACB5, Pa ACB26, Pa ACB27 and Pa ACB44 can effectively remove EHEC O157: H7 from PBS and control PK.

Pa ACB26, Pa ACB27 and Pa ACB44, which are excellent in intestinal adhesive ability, have excellent ability to remove ETEC K88 and EHEC O157: H7, which are capable of infecting small intestine, It can be suggested that axillary dactitis may be effective against diarrhea.

&Lt; Example 4 > Confirmation of growth activity of microorganisms having excellent intestinal adhesive ability

Pa ACB26, Pa ACB27, and Pa ACB44, which are excellent in intestinal sticking ability and excellent in pathogen removal effect, are incubated in MRS culture medium with O / N and then inoculated into 50 ml fresh MRS culture medium at a level of 0.1% And incubated at 37 ° C. for 16 hours. The number of viable cells was measured, and a growth curve was prepared based on the measured number of viable cells.

As a result, as shown in FIG. 10, the doubling time of the sample was calculated using the concentration and duration of the exponential phase (log phase) of 2 to 8 h. As a result, Pa ACB26 55.25 minutes, Pa ACB27 was 55.82 minutes, and Pa ACB44 was 57.03 minutes. The Pediococcus Acidyllactic acid showed a short proliferation time of 55 to 57 minutes. However, as the difference between the samples was not found to be large, an additional evaluation of growth activity did not proceed.

&Lt; Example 5 > Identification of acid resistance and biliary properties of microorganisms with excellent intestinal adhesive properties

Pa ACB26, Pa ACB27, and Pa ACB44, which are excellent in intestinal adhesiveness and excellent pathogen removal effect, are incubated in MRS culture medium with O / N and then inoculated into simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) at a level of 1% And incubated at 30 min and 1 h intervals. The cells were continuously diluted and plated on MRS agar. After incubation for 16 hours at 37 ° C, the number of viable cells was measured.

As a result, as shown in Fig. 11, the acid tolerance and biliary cholesterol were superior to those of the control group PK, and the number of viable cells was increased in SIF.

From the above results, when Pa ACB26, Pa ACB27 and Pa ACB44 are fed to pigs, they are delivered at a high survival rate into the intestines, effectively secrete useful substances through intestinal sticking and colonization, and have a beneficial effect on pigs through pathogen removal .

<Example 6> Confirmation of effect of microorganism probiotic agent on the experiment

1. Specification experiment using 4-week-old piglet

1-1. Sample Preparation

ACB26, ACB27, and ACB44 mixed with feed were incubated overnight in 1 L MRS medium. Cells were washed with 300 ml of 0.85% NaCl at 500 rpm at 4500 rpm, 15 min, 4 ° C, Lt; 0 &gt; C.

After the supernatant was removed, the supernatant was removed, and the supernatant was removed by centrifugation at 4000 rpm, 10 min, and 4 ° C after 20% glucose washing. The supernatant was removed, and the supernatant was suspended in 100 ml of 20% skim milk and lyophilized overnight at 120 ° C.

1-2. Specification experiment and sampling method

Pa-ACB26 (T1), Pa-ACB27 (T2) and Pa-ACB44 (T3) of 1 g (2 × 10 ⁹ CFU / g) per kg of feed for four weeks were used in 72 weeks of age And weighing and blood and fecal sampling were performed at intervals of two weeks as shown in Table 2.

In addition, the daily feed intake (ADFI) and feed conversion ratio (FCR) were measured.

As a result, the ADG and ADFI values of the T1 group treated with Pa ACB26 for 14-27 days were the highest, though there was no significant increase in the group as compared to the control group as shown in Table 3.

In addition, as shown in Table 4, diarrheal incidence of 4 weeks was the highest in all groups from 0 to 7 days, but T1 group showed the lowest incidence of diarrhea at 14-21 days and 4th week.

On the other hand, the incidence rate of 4-week ran- dom was high between early and 14th and 21st day as shown in Table 5, but it was confirmed that the incidence of T1 group was the lowest at 0% in the 4th week of the last experiment.

As shown in FIG. 13, the diarrhea and the incidence of the incidences of the diarrhea and the incidences of the incidences of the diarrhea and the incidences of the incidences of the incidences of the diarrhea were examined.

In addition, TNF-a, a representative proinflammatory cytokine in the blood, was measured using an ELISA.

First, the sampled blood was transferred to an SST tube (BD Vacutainer), centrifuged at 13,000 rpm, 5 min, 4 ° C, and serum was transferred to a new tube and stored at 120 ° C until the experiment. Blood cytokine analysis was performed using the Porcine TNF-alpha Quantikine ELISA Kit PTA00, Porcine IL-10 Quantikine ELISA Kit P1000 (R & D Systems, UK) as described by the manufacturer and was tested for inflammatory cytokines, (anti-inflammatory cytokine).

As a result, as shown in FIG. 14, it was confirmed that all of them were similar without any significant difference compared to the control group. As a result of measurement by ELISA, IL-10 which is a typical anti-inflammatory cytokine in blood, But it was confirmed that there was no significant difference compared to the control group.

2. Analysis of fecal microorganisms

Microbial communities in the feces of pigs sampled using Illumina Miseq (Macrogen, South Korea) were identified.

First, DNA was extracted from a 250 mg fecal sample taken from each group using the NucleoSpin ™ Soil Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions and stored at -20 ° C. until further analysis.

The V4 region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified from the entire extracted DNA.

PCR amplification was performed using Takara Ex-taq polymerase (Takara Bio, Shiga, Japan) and universal primers (forward direction: 5'-GGACTACHVGGGTTTCTAAT-3 'and reverse direction: 5'-GTGCCAGCMGCCGCGGTAA-3' After one cycle at 94 ° C for 3 minutes, 45 cycles at 94 ° C, 1 minute at 55 ° C and 1.5 minutes at 72 ° C, and finally one cycle at 72 ° C for 10 minutes.

DNA libraries were constructed using the NEBNext Ultra DNA Library Prep Kit (Illumina, New England Biolabs, Ipswich, Mass., USA) in a slightly modified manner from the manufacturer's instructions.

The size selection step of the adapter-coupled DNA and purification steps was replaced by PCR product purification using the QIAquick PCR purification kit (Qiagen, Valencia, Calif., USA) and adapter and index primers were purchased from Illumina Kit (New England Biolabs, Ipswich, MA , USA) using NEBNext Multiplex Oligos.

After sequencing, the library was constructed using Illumina MiSeq 2 × 250 bp paired-end sequencing (Macrogen, Seoul, Republic of Korea), and the microbial communities were analyzed using Quantitative Insights in Microbiology Ecology (QIIME) version 1.9.0 .org), and the microorganism taxa was expressed as a percentage of the total 16S rRNA gene sequence.

As a result, as shown in FIG. 16, it was confirmed that the distinction of microbial communities was more distinct than that of the microbial community groups.

On the other hand, in order to identify specific species, the qRT-PCR method was used to identify pediococcus The level of E. coli with axilidyltaxy and F4, F18 and Intimin attached molecules was determined and compared to the control group to determine the presence in each experimental group.

QRT-PCR was performed using the primers shown in Table 6 above.

(1 pmol / μl), 6 μl of the reverse primer (1 pmol / μl), 0.5 μl of gDNA (1 pmol / μl), and 10 μl of the primer A sample (6 ng / ul) was prepared so as to have a final volume of 20 μl, and PCR was carried out under the following conditions.

As a result, as shown in Fig. 17, in comparison with the control group, The results showed that axillary ductility was significantly higher and T1 was higher at 4th week. F4, F18, and intimin, representative adherent molecules of enterotoxin E. coli, showed less T1 in the 2nd and 4th weeks than the control group .

As a result of confirming the contents of the fungi in the feces, It was confirmed that Bifidobacterium spp. And Lactobacillus spp. Were increased in all of the experimental groups treated with axetil de lactis.

From the above results, it was confirmed that the ACB26 selected in the present invention lowers the incidence of diarrhea of 4 to 8 weeks old weeds, inhibits infectious E. coli, which is a harmful microorganism, and increases the intestinal beneficial bacteria, May be provided as a composition for preventing or treating viral diarrhea.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Korea Biotechnology Research Institute KCTC18512P 20161116

<110> SNU R & DB FOUNDATION <120> Composition for preventing or treating porcine diarrhea using new          pediococcus acidilactici <130> ADP-2017-0422 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 645 <212> RNA <213> pediococcus acidilactici ACB26 16s rRNA <400> 1 cgaagtgagt ggcggacggg tgagtaacac gtgggtaacc tgcccagaag caggggataa 60 cacctggaaa cagatgctaa taccgtataa cagagaaaac cgcctggttt tcttttaaaa 120 gatggctctg ctatcacttc tggatggacc cgcggcgcat tagctagttg gtgaggtaac 180 ggctcaccaa ggcgatgatg cgtagccgac ctgagagggt aatcggccac attgggactg 240 agacacggcc cagactccta cgggaggcag cagtagggaa tcttccacaa tggacgcaag 300 tctgatggag caacgccgcg tgagtgaaga agggtttcgg ctcgtaaagc tctgttgtta 360 aagaagaacg tgggtgagag taactgttca cccagtgacg gtatttaacc agaaagccac 420 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttat ccggatttat 480 tgggcgtaaa gcgagcgcag gcggtctttt aagtctaatg tgaaagcctt cggctcaacc 540 gaagaagtgc attggaaact gggagacttg agtgcagaag aggacagtgg aactccatgt 600 gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaa 645

Claims (9)

Pediococcus axicillilitirus ACB26 (KCTC18512P) with antimicrobial activity against pathogens causing pig diarrhea. [Claim 4] The Pediococcus axilidylationase ACB26 according to claim 1, wherein the nucleotide sequence of the 16S rRNA gene of Pediococcus axysilylationase ACB26 is represented by SEQ. [Claim 4] The Pediococcus acepidyl lacticus ACB26 according to claim 1, wherein the pathogen is selected from the group consisting of enterotoxin E. coli (ETEC) K88 and intestinal hemorrhagic E. coli (EHEC) O157: H7. Phedi O Lactococcus aksi dill when rakti (P. acidilactici) the pig diarrhea preventing or pharmaceutical composition containing as an active ingredient. [5] The method according to claim 4, wherein the Pediococcus axydilactaci is selected from the group consisting of Pediococcus axilidyl lactylase ACB5, Pediococcus axilidyl lactic acid ACB18, Pediococcus axilidyl lactic acid ACB26, Pediococcus axilidyl lactylcyste ACB27, Pediococcus axilidyllactic acid ACB44, and Pediococcus axilidyllactic acid ACB48. The pharmaceutical composition for preventing or treating swine diarrhea according to claim 1, [Claim 5] The pharmaceutical composition according to claim 4, wherein the Pediococcus acedillus lactis is adhered to a porcine intestinal mucosa to remove pathogens causing swine diarrhea. The pharmaceutical composition according to claim 6, wherein the pathogen is selected from the group consisting of enterotoxin E. coli (ETEC) K88 and intestinal hemorrhagic E. coli (EHEC) O157: H7. Phedi O Lactococcus aksi dill rakti when feed additive for inhibiting the pathogen to the (P. acidilactici) and containing as an active ingredient, cause the pig diarrhea. Phedi O Lactococcus aksi dill microbial agent to inhibit the pathogen, and which contains a rakti when (P. acidilactici) the active ingredient, cause the pig diarrhea.

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