WO2017073916A1 - Novel aeromonas salmonicida bacteriophage aer-sap-1, and use thereof for inhibiting aeromonas salmonicida proliferation - Google Patents

Novel aeromonas salmonicida bacteriophage aer-sap-1, and use thereof for inhibiting aeromonas salmonicida proliferation Download PDF

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WO2017073916A1
WO2017073916A1 PCT/KR2016/010957 KR2016010957W WO2017073916A1 WO 2017073916 A1 WO2017073916 A1 WO 2017073916A1 KR 2016010957 W KR2016010957 W KR 2016010957W WO 2017073916 A1 WO2017073916 A1 WO 2017073916A1
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bacteriophage
sap
bacteria
aer
bacteriophages
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French (fr)
Korean (ko)
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윤성준
전수연
권안성
송현민
엄태희
강상현
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주식회사 인트론바이오테크놀로지
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention is to prevent and treat the infection of the bacteriophage isolated from nature capable of killing the E. monmonix Salmonicida bacteria and the composition containing the composition containing the same as an active ingredient
  • the method relates to more specifically, a myobacterial bacteriophage Aer-SAP isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monas salmonisid bacteria. -1 (Accession No. KCTC 12814BP), and a method for preventing and post-infection treatment of Eromonas salmonisid bacteria using the composition comprising the bacteriophage as an active ingredient.
  • Aeromonas salmonicida a member of the Aeromonadaceae family, is a Gram-negative bacterium that is aerobic, aerobic, and non-motile, and is a bacterium that causes extensive bacterial septicemia or furunculosis in a variety of fish species.
  • Salmonid fish such as salmon and rainbow trout, have been reported as pathogenic bacteria causing high mortality diseases such as intestinal disease, but recently they are frequently detected in carp and fish. to be.
  • the Eromonas Salmonicida bacteria have a negative response to the indole production reaction, but have a positive reaction in the catalase and oxidase tests.
  • the suitable growth temperature of this bacterium is 22 to 25 ° C, but growth is possible even up to 34.5 ° C.
  • Eromonas infectious diseases caused by E. monmonix infections occur regardless of the size of the larvae from larvae to adult fishes, and the economic consequences of these diseases are very high. There is an urgent need to develop a plan that can be used. In particular, since safety as a food of aquatic products has become a major social concern in recent years, it is more preferable to be environmentally friendly.
  • Vaccine as a means to control the disease of aquaculture fish has been in full swing, but the type of vaccine has not yet diversified, so the variety of disease is diversified, and in order to cope with the increase in the incidence of mixed diseases, other diseases are combined with the vaccine. Control means must be further developed.
  • Bacteriophages are tiny microorganisms that infect bacteria, often called phage. Bacteriophages have the ability to infect bacteria and multiply inside bacterial cells, and kill off bacteria by destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria. Bacteriophage bacterial infections are highly specific, so there are only a few types of bacteriophages that can infect certain bacteria.
  • certain bacteriophages can infect only a specific category of bacteria, so that certain bacteriophages kill only certain bacteria and do not affect other bacteria.
  • the bacterial specificity of these bacteriophages provides antimicrobial effects only to the target bacteria and does not affect the environment or flora in fish.
  • Conventional antibiotics usually affect several kinds of bacteria at the same time. Bacteriophage only works for certain bacteria, so the use of bacteriophage does not cause normal bacterial total disturbances. Therefore, its use is very safe compared to the use of antibiotics, and the likelihood of side effects is relatively low.
  • Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer.
  • French bacteriologist d'Herelle discovered that some of the filtrates of ill feces dissolve Shigella dysenteriae . In the sense, they named it bacteriophage. Since then, bacteriophages have been found for many pathogenic bacteria such as dysentery, typhoid, and cholera.
  • bacteriophages Because of its special ability to kill bacteria, bacteriophages have been expected to be an effective way to combat bacterial infections since their discovery and many studies have been done. However, after the discovery of penicillin by Fleming, with the widespread use of antibiotics, research on bacteriophages has been limited to some Eastern European countries and the Soviet Union. However, since 2000, due to the increase of antibiotic-resistant bacteria, the limit of existing antibiotics appears, and as the possibility of developing an alternative to the existing antibiotics is highlighted, bacteriophage is attracting attention as an anti-bacterial agent.
  • bacteriophages have a very high specificity for bacteria. Due to this specificity, bacteriophages often exert an antimicrobial effect on only some strains, even if they belong to the same type of bacteria. In addition, the antibacterial activity of the bacteriophages may be different depending on the bacterial strain. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to secure effective control methods for specific kinds of bacteria. In order to develop an effective bacteriophage in response to the bacteriophage, it is naturally necessary to secure various useful bacteriophages (a variety of bacteriophages that can provide antibacterial effects against the bacteriophage). Furthermore, among the various useful bacteriophages obtained, it is also necessary to select bacteriophages that have comparative advantages in terms of the strength of the antimicrobial activity and the antimicrobial range.
  • the present inventors have developed a composition that can be used to prevent or treat the infection of the Eromonas Salmonicida bacteria by using bacteriophages isolated from nature capable of selectively killing the E.E.
  • bacteriophages isolated from nature capable of selectively killing the E.E.
  • the present invention was completed by developing a composition using the bacteriophage as an active ingredient, and then confirming that the composition can be effectively used for preventing and treating E. coli bacteria.
  • an object of the present invention is Myoviridae bacteriophage Aer-SAP isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monas Salmonisid bacteria. -1 (accession number KCTC 12814BP).
  • Another object of the present invention is to prevent the infection of the Eromonas Salmonicida bacteria comprising the isolated bacteriophage Aer-SAP-1 as an active ingredient that can kill the Eromonas Salmonicida bacteria and kill the E. It is to provide a composition which can be utilized for the purpose and a method for preventing infection of Eromonas salmonisid bacteria using the composition.
  • Another object of the present invention is to treat the infection of the E. monas Salmonicida bacteria comprising the isolated bacteriophage Aer-SAP-1 as an active ingredient capable of killing the E. monas Salmonicida bacteria to kill the E. It is to provide a composition which can be utilized for the treatment, and a method for treating infection of Eromonas salmonisid bacteria using the composition.
  • Still another object of the present invention is to provide a bath agent for the purpose of preventing and treating eromonas salmonisid infection using the compositions.
  • Still another object of the present invention is to provide a feed additive for the purpose of providing a specification effect through the prevention and treatment of the E. monas salmonisid infection using the compositions.
  • the present invention is Myobiridae bacteriophage Aer-SAP-1 (Accession No. KCTC) isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monmonix Salmonicida bacteria. 12814BP), and a method for preventing and treating infection of Eromonas salmonisid bacteria using a composition comprising the same as an active ingredient.
  • Bacteriophage Aer-SAP-1 was isolated by the inventors and deposited in the Korea Institute of Biotechnology and Microbial Resources Center on May 20, 2015 (Accession No. KCTC 12814BP).
  • the present invention also provides a bath and feed additive comprising a bacteriophage Aer-SAP-1 as an active ingredient that can be used to prevent or treat the infection of the Eromonas Salmonicida bacteria.
  • Bacteriophage Aer-SAP-1 included in the composition of the present invention effectively kills the bacteria E. monomonas Salmonida, and thus has an effect on the prevention (infection prevention) or treatment (infection treatment) of diseases caused by E. monmonas Salmonida. Indicates. Therefore, the composition of the present invention may be used for the purpose of prevention and treatment of choral disease and bacterial sepsis, which are representative diseases caused by E. monas salmonicida, but is not limited thereto.
  • treatment refers to (1) suppression of a disease caused by the bacterium Aeromonas salmonisid; And (2) all acts to alleviate the pathological condition of the disease caused by E. monas salmonicida bacteria.
  • isolated refers to the separation of bacteriophages from the natural state using various experimental techniques, and to securing specific characteristics that can be distinguished from other bacteriophages. This includes growing the bacteriophages for industrial use.
  • compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited to these. .
  • the composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
  • the composition of the present invention includes bacteriophage Aer-SAP-1 as an active ingredient.
  • the bacteriophage Aer-SAP-1 included at this time includes 1 ⁇ 10 1 pfu / ml to 1 ⁇ 10 30 pfu / ml or 1 ⁇ 10 1 pfu / g to 1 ⁇ 10 30 pfu / g, preferably 1 ⁇ . 10 4 pfu / ml to 1 ⁇ 10 15 pfu / ml or 1 ⁇ 10 4 pfu / g to 1 ⁇ 10 15 pfu / g.
  • compositions of the present invention may be prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. It may also be prepared by incorporation into a multi-dose container.
  • the formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • composition of the present invention may be implemented as a bath and feed additives.
  • Bacteriophages that can provide antimicrobial activity against other bacterial species can be added to the composition of the present invention in order to increase the efficiency in this application.
  • other types of bacteriophages having antimicrobial activity against the bacteria E. monmonas may also be added.
  • bacteriophages that have antimicrobial activity against E. monas salmonicida bacteria differ in terms of strength and antimicrobial range of antimicrobial activity, their proper combination can maximize the effect.
  • Infection prevention and treatment method of the E. monomonas Salmonishi bacteria using the composition comprising the bacteriophage Aer-SAP-1 of the present invention as compared to the conventional method based on chemicals such as antibiotics It can provide the advantage that the specificity for the bacteria is very high. This means that it can be used for the purpose of preventing or treating the infection of the E. monas salmonicida without affecting other useful floras, and its side effects are very low. In general, the use of chemicals, such as antibiotics will also damage the common flora bacteria, resulting in a decrease in the immunity of animals, resulting in various side effects.
  • the present invention can also provide the advantage of being very natural because it is used as an active ingredient of the composition to separate the bacteriophage already present in nature.
  • the bacteriophages may be separated against individual bacterial strains in terms of the strength of the antimicrobial activity and the antimicrobial range [strains of several bacterial strains belonging to the genus Eromonas Salmonisid]. Range of antibacterial activity.
  • bacteriophages can exert antimicrobial activity against some strains belonging to the same bacterial species.
  • the present invention is different from the other bacteriophages having antimicrobial activity against E. monas salmonisid. Can provide an effect. This makes a big difference in the effectiveness of industrial sites.
  • 1 is an electron micrograph of the bacteriophage Aer-SAP-1.
  • Figure 2 is an experimental result showing the killing ability of the bacteriophage Aer-SAP-1 against the E. monas Salmonida.
  • the transparent part is the lysate plaque formed by lysis of the bacteria under test.
  • TSB T ryptic S oy B roth
  • a rate medium Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; dextrose, 2.5 g / L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L
  • the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. The recovered supernatants were inoculated with 1 / 1,000 of E.
  • the drip experiment was conducted as follows. Inoculated with E. monomonas salmonicida in the ratio of 1 / 1,000 in TSB medium and then cultured with shaking at 25 °C overnight. The thus prepared 3 ml culture solution of the erosion Pseudomonas live monitor let bacteria (OD 600 is 1.5) TSA (T ryptic S oy A gar) plate medium (Casein Digest, 15 g / L; Soy bean digest, 5 g / L; NaCl , 5 g / L; agar, 15 g / L). The plated flat medium was left in a clean bench for about 30 minutes to allow the smear to dry.
  • Separation of pure bacteriophages was carried out using a filtrate in which the presence of bacteriophages with killing ability against E. monmonix Salmonida was confirmed. Separation of pure bacteriophage was carried out using a conventional Plaque assay. To explain this in detail, one of the lytic plaques formed in the lytic plaque assay was recovered using a sterilized tip, and then added to the E. monomonas salmonisid culture medium and incubated together at 25 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. The supernatant obtained was added to the culture of E.
  • Electron microscopic analysis was performed according to a conventional method. This is briefly described as follows. The solution containing pure bacteriophage was buried in a copper grid, subjected to reverse staining and drying with 2% uranyl acetate, and the form was photographed through a transmission electron microscope. Electron micrographs of purely isolated bacteriophages are shown in FIG. 1. Judging from the morphological features, the newly acquired bacteriophage belonged to the Myoviridae bacteriophage.
  • the solution containing pure bacteriophage identified in this way was subjected to the following purification process.
  • Eromonas Salmonicida culture medium was added to a volume of 1/50 of the total volume of the solution including the pure bacteriophage and then incubated again for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This procedure was repeated a total of five times to obtain a solution containing a sufficient number of bacteriophages.
  • the supernatant obtained by the final centrifugation was filtered using a 0.45 ⁇ m filter followed by a conventional polyethylene glycol (PEG) precipitation process.
  • PEG polyethylene glycol
  • PEG and NaCl were added to 100 ml of the filtrate to be 10% PEG 8000 / 0.5 M NaCl, and then allowed to stand at 4 ° C. for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate.
  • the bacteriophage precipitate thus obtained was suspended in 5 ml of buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). This is called bacteriophage suspension or bacteriophage solution.
  • Example 2 bacteriophage Aer - SAP -1 genome isolation and sequencing
  • the genome of the bacteriophage Aer-SAP-1 was isolated as follows. Bacteriophage suspension obtained in the same manner as in Example 1 was used for dielectric separation. First, in order to remove DNA and RNA of the E. monas salmonisid bacteria, which may be included in the suspension, 200 U of each of DNase I and RNase A was added to 10 ml of the bacteriophage suspension, followed by standing at 37 ° C. for 30 minutes. In order to remove the activity of DNase I and RNase A after 30 minutes, 500 ⁇ l of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and allowed to stand for 10 minutes. The mixture was left at 65 ° C.
  • EDTA ethylenediaminetetraacetic acid
  • the genome thus obtained was subjected to genome sequencing of bacteriophage Aer-SAP-1 through Next generation sequencing analysis of Roche 454 GS Junior. Finally, the analyzed bacteriophage Aer-SAP-1 genome has a size of 162,883 bp and the entire genome sequence is set forth in SEQ ID NO: 1.
  • bacteriophage Aer-SAP-1 has an annular genome
  • the eromonas bacteriophage phiAS4 the eromonas bacteriophage Aes508, the eromonas bacteriophage Aes012, and the eromonas salmonicida bacteriophage 25 have a large difference in genome morphology.
  • the number of open reading frames (ORFs) on the bacteriophage Aer-SAP-1 genome was 234, whereas the Stenotropomonas bacteriophage IME13 had 182, indicating that they were different bacteriophages.
  • bacteriophage Aer-SAP-1 could be considered as a novel bacteriophage that has not been reported previously. With this fact, it is judged that bacteriophage Aer-SAP-1 can provide different antimicrobial effects from other bacteriophages previously reported from the fact that different kinds of bacteriophages have different strengths and ranges of antimicrobial activity.
  • the killing ability of the isolated bacteriophage Aer-SAP-1 against the E. monas salmonicida bacteria was investigated.
  • the killing ability was investigated by the drop test in the same manner as in Example 1 to determine the production of transparent rings.
  • Bacteriophage Aer-SAP-1 had the ability to kill 11 of the E. monmonix Salmonida bacteria. Representative experimental results are shown in FIG. 2.
  • the bacteriophage Aer-SAP-1 has a specific killing ability against the E. monas salmonisid, and it was confirmed that it can exert an antimicrobial effect against a number of eromonas Salmonida.
  • the bacteriophage Aer-SAP-1 can be used as an active ingredient of the composition for the purpose of preventing and treating the infection of Eromonas salmonisid.
  • Example 4 bacteriophage Aer - SAP -1's Erotica Let's Salmon For the prevention of bacterial infection Experimental Example
  • the bacteriophage Aer-SAP-1 of the present invention has the ability not only to inhibit the growth of the bacteriophages Salmonida, but also to kill the bacteriophage Aer-SAP-1. It can be concluded that the composition can be used as an active ingredient for the purpose of preventing infection of Sida.
  • Example 5 bacteriophage Aer - SAP With -1 Erotica Let's Salmon Infection prevention animal experiment
  • rainbow trout was used to investigate the effects of bacteriophage Aer-SAP-1 on the infection of E. monitis Salmonida.
  • Twenty five-week-old rainbow trout (average body length: 15.9 cm, average weight: 24.0 g) were divided into two groups, and then divided into two groups. The environment of the bath was controlled and the temperature of the laboratory containing the bath was kept constant. From the start of the experiment, the rainbow trout of the experimental group (bacteriophage treated group) were fed a feed containing 1 ⁇ 10 8 pfu / g of bacteriophage Aer-SAP-1 according to a conventional feed feeding method.
  • rainbow trout from the control group (no bacteriophage group) were fed with the same composition in the same manner without the bacteriophage Aer-SAP-1.
  • both the experimental and control groups included Emononas salmonisid bacteria at a feed level of 1 ⁇ 10 8 cfu / g for 2 days. Infection was induced.
  • the incidence of intestinal disease was investigated every day from the day after the feeding of the feed containing the two days of E. monmonix Salmonida, the day after the start of the test (the 9th day from the start of the test). Investigation of the incidence of intestinal disease was performed by measuring the size of the ulcer on the body surface.
  • Ulcer size measurements in the body surface are commonly used Ulcer size (US) score ⁇ normal (no ulcer): 0, mild ulcer (ulcer size: less than 0.5 cm): 1, strong ulcer (ulcer size: 0.5 cm or more): 2 ⁇ It was carried out in a manner to measure.
  • Body surface ulcer size measurement result (average value) date D9 D10 D11 D12 D13 D14 Control group (not administered bacteriophage) 0.45 0.5 0.65 0.7 0.7 0.7 Experimental group (bacteriophage administration) 0 0 0 0 0 0 0 0 0
  • the bacteriophage Aer-SAP-1 of the present invention is very effective in the prevention of an infectious disease caused by the bacterium Eromonas salmonisida.
  • Ulcer size measurements in the body surface are commonly used Ulcer size (US) score ⁇ normal (no ulcer): 0, mild ulcer (ulcer size: less than 0.5 cm): 1, strong ulcer (ulcer size: 0.5 cm or more): 2 ⁇ It was carried out in a manner to measure.
  • Body surface ulcer size measurement result (average value) date D8 D9 D10 D11 D12 D13 D14 Control group (not administered bacteriophage) 1.00 1.40 1.60 1.70 1.65 1.65 1.70 Experimental group (bacteriophage administration) 1.05 0.95 0.55 0.45 0.35 0.15 0.15
  • the bacteriophage Aer-SAP-1 of the present invention is very effective in the treatment of infectious diseases caused by the bacterium Eromonas salmonisida.
  • a feed additive was prepared using bacteriophage Aer-SAP-1 solution to include 1 ⁇ 10 8 pfu of bacteriophage Aer-SAP-1 per g of feed additive.
  • the method of preparing a feed additive was prepared by adding maltodextrin to the bacteriophage solution (50%, w / v) and then lyophilizing. Finally, it was ground to a fine powder form.
  • the drying process in the manufacturing process may be replaced by reduced pressure drying, warming drying, room temperature drying.
  • a feed additive without bacteriophage was used in place of the bacteriophage solution, using the buffer used to prepare the bacteriophage solution (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). It was prepared by.
  • Each of the two feed additives thus prepared was mixed with 250 times the fish feed for fish in a weight ratio to prepare the final two feeds.
  • the bacteriophage Aer-SAP-1 was prepared using a bacteriophage Aer-SAP-1 solution to contain 1 ⁇ 10 8 pfu of bacteriophage Aer-SAP-1 per ml of the bath.
  • the method of preparing a bath detergent is prepared by adding the above-mentioned bacteriophage Aer-SAP-1 solution so that 1 ⁇ 10 8 pfu of bacteriophage Aer-SAP-1 is included per 1 ml of the buffer used to prepare the bacteriophage solution. It was.
  • the buffer itself used in the preparation of the bacteriophage solution was used as it is.
  • the two types of baths thus prepared were diluted with 1,000 times water by volume and used as the final bath.
  • Example 7 and Example 8 Using the feed prepared in Example 7 and Example 8 and the bath was investigated whether the improvement of the specification results when breeding rainbow trout.
  • the survey was conducted in terms of mortality.
  • a total of 100 rainbow trout were divided into two groups (group-A fed; group-B treated with a bath) for 50 weeks.
  • Each group was divided into 25 subgroups, and each subgroup was divided into a small group (small group-1) to which bacteriophage Aer-SAP-1 was applied and a small group (small group-2) to which no bacteriophage was applied.
  • the rainbow trout for this test were five-week-old rainbow trout, and the rainbow trout from each subgroup were raised in separate tanks at regular intervals.
  • Each subgroup is divided and referred to as Table 4 below.
  • the feed prepared according to the feed preparation method described in Example 7 was fed according to the conventional feed feeding method according to the classification of Table 4, and in the case of the treatment of the bathing agent, the bathing bath described in Example 8
  • the bath preparation prepared according to the preparation method was treated according to the conventional bath treatment method according to the classification of Table 4.
  • the test results are shown in Table 5.
  • Mortality at Rainbow Trout Specimen Testing group Number of dead individuals / Number of test subjects % Mortality A-1 1/25 4.0 A-2 7/25 28.0 B-1 2/25 8.0 B-2 8/25 32.0

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Abstract

The present invention relates to: a Myoviridae bacteriophage Aer-SAP-1 (deposit number: KCTC 12814BP) isolated from nature, having the ability to specifically kill Aeromonas salmonicida and having the genome represented by SEQ ID NO: 1; and a method for preventing and treating an Aeromonas salmonicida infection by using a composition containing the same as an active ingredient.

Description

신규한 에로모나스 살모니시다 박테리오파지 Ａｅｒ-ＳＡＰ-1 및 이의 에로모나스 살모니시다 균 증식 억제 용도Novel Eromonas Salmonicida Bacteriophage Aerr-SAP-1 and Its Application to Suppressing the E.E.
본 발명은 에로모나스 살모니시다 균에 감염하여 에로모나스 살모니시다 균을 사멸시킬 수 있는 자연으로부터 분리한 박테리오파지 및 이를 유효성분으로 포함한 조성물을 이용한 에로모나스 살모니시다 균의 감염을 방지 및 처치하는 방법에 관한 것으로, 더욱 상세하게는 에로모나스 살모니시다 균을 특이적으로 사멸시킬 수 있는 능력을 갖는 서열번호 1로 표시되는 유전체를 갖는 것을 특징으로 하는 자연으로부터 분리한 미오비리대 박테리오파지 Aer-SAP-1(수탁번호 KCTC 12814BP), 및 상기 박테리오파지를 유효성분으로 포함하는 조성물을 이용한 에로모나스 살모니시다 균의 감염 방지 및 감염 후 처치 방법에 관한 것이다.The present invention is to prevent and treat the infection of the bacteriophage isolated from nature capable of killing the E. monmonix Salmonicida bacteria and the composition containing the composition containing the same as an active ingredient The method relates to more specifically, a myobacterial bacteriophage Aer-SAP isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monas salmonisid bacteria. -1 (Accession No. KCTC 12814BP), and a method for preventing and post-infection treatment of Eromonas salmonisid bacteria using the composition comprising the bacteriophage as an active ingredient.
에로모나스 과(Aeromonadaceae)에 속하는 에로모나스 살모니시다(Aeromonas salmonicida)는 그람 음성균이고, 통성혐기성이며 비운동성인 단간균으로서 다양한 어종에서 광범위하게 세균성 패혈증(Septicemia)이나 절창병(Furunculosis)을 일으키는 세균으로 알려져 있다. 특히, 이름에서 연상되듯이 과거에는 연어 및 무지개 송어 등의 연어과(Salmonid) 어류에서 절창병과 같은 치사율이 높은 질병을 유발하는 병원성 박테리아로 보고되었으나, 최근에 와서는 잉어과 어류 등에서도 자주 검출되고 있는 추세이다. 또한, 에로모나스 살모니시다 균은 인돌(Indole) 생성 반응에 대해서는 음성 반응을 보이나, 카탈라아제(Catalase) 및 옥시다아제(Oxidase) 시험에서는 양성 반응을 보이는 특징을 가진다. 이 세균의 적합 생장 온도는 22∼25℃이나, 최고 34.5℃에서도 생장이 가능하다. Aeromonas salmonicida , a member of the Aeromonadaceae family, is a Gram-negative bacterium that is aerobic, aerobic, and non-motile, and is a bacterium that causes extensive bacterial septicemia or furunculosis in a variety of fish species. Known as In particular, as the name suggests, in the past, Salmonid fish, such as salmon and rainbow trout, have been reported as pathogenic bacteria causing high mortality diseases such as intestinal disease, but recently they are frequently detected in carp and fish. to be. In addition, the Eromonas Salmonicida bacteria have a negative response to the indole production reaction, but have a positive reaction in the catalase and oxidase tests. The suitable growth temperature of this bacterium is 22 to 25 ° C, but growth is possible even up to 34.5 ° C.
에로모나스 감염증은 세계최대 연어 양식국인 노르웨이를 포함한 유럽뿐만 아니라 북아메리카, 호주 등 전 세계적으로 발병 사례가 보고되고 있으며, 국내에서는 2010년에 경상남도 산청 지방의 양식장에서 우리나라 최초로 에로모나스 살모니시다의 쏘가리 감염 사례가 보고된 바 있다. 에로모나스 살모니시다 균에 감염된 어류의 일반적인 특징으로는 지느러미에 염증이 일어나고 지느러미가 너덜너덜해지기도 하나, 때때로 경미하거나 심각한 혼탁 현상만이 지느러미에 나타나는 경우도 있다. 또한, 2∼20 mm 길이의 화농성 종기 및 병변이 형성되기도 하고 2차 감염으로 곰팡이의 이상 증식이 발견되기도 한다. 내부적인 이상 증상으로는 표피, 아가미, 지느러미, 및 근육 조직에서 미량의 출혈, 체표 팽윤, 및 내부절창장염 등이 흔히 관찰된다. There have been reports of cases of Eromonas infectious disease not only in Europe, including Norway, which is the world's largest salmon farming country, but also in North America and Australia. Cases have been reported. Common features of fish infected with E. monmonix Salmonicida include fin inflamed fins and tattered fins, but sometimes only minor or severe turbidity appears in the fins. In addition, purulent boils and lesions of 2 to 20 mm in length are formed, and abnormal infection of the fungus is found as a secondary infection. Internal abnormalities are often observed in the epidermis, gills, fins, and muscle tissues with minor bleeding, body swelling, and internal colitis.
에로모나스 살모니시다 균 감염에 의한 에로모나스 감염증은 치어에서 성어까지 개체의 크기에 상관없이 발병하고 이에 따라 초래하는 경제적 피해가 매우 크기 때문에 에로모나스 살모니시다 균 감염을 예방하고 나아가 감염 처치에까지 활용될 수 있는 방안의 개발이 절실한 실정이다. 특히, 최근 수산물의 식품으로서의 안전성이 주요 사회적 관심사가 되고 있기 때문에 친환경적인 방안이면 더욱 바람직하다. Eromonas infectious diseases caused by E. monmonix infections occur regardless of the size of the larvae from larvae to adult fishes, and the economic consequences of these diseases are very high. There is an urgent need to develop a plan that can be used. In particular, since safety as a food of aquatic products has become a major social concern in recent years, it is more preferable to be environmentally friendly.
어류 양식 산업은 부족한 식량자원을 손쉽게 얻을 수 있다는 점에서 해마다 급속한 성장을 거듭하고 있다. 그러나 이러한 양식 산업의 성장이 증가 할수록 사료를 통한 주변 환경의 오염 역시 증가하고 있고 특히 사료에 포함되어 있는 많은 양의 항생제가 광범위하게 사용됨으로써 오히려 인류의 건강을 위협하기도 한다. 무지개 송어를 비롯한 어류 양식장에서는 세균성 질병의 치료 방법으로서 과다하게 항균 화학요법제인 항생제가 사용되고 있고, 이러한 결과로 다양한 약제에 대한 내성을 지닌 균이 빈번하게 나타나고 있어서, 어류 양식 어가에 상당한 경제적 손실을 입히고 있는 실정이다. 또한, 이러한 항생제의 과다한 사용은 국민 건강을 위협하고 나아가서는 양식 어류의 소비 심리를 위축시켜서 전반적인 수산업의 경쟁력 저하를 야기할 수 있다. 따라서 세균에 의한 어류 질병을 방지할 수 있고 또한 이들의 감염을 효과적으로 처치할 수 있는 방법의 개발이 절실하다 할 수 있다. The fish farming industry is growing rapidly each year in that it is easy to obtain scarce food resources. However, as the growth of these aquaculture industries increases, so does the pollution of the surrounding environment through the feed, and the widespread use of antibiotics in the feed also threatens human health. In the farms of rainbow trout and other fish farms, antibiotics, which are antimicrobial chemotherapeutic agents, are used excessively as a treatment method for bacterial diseases. As a result, bacteria that are resistant to various drugs frequently appear, which causes considerable economic loss to fish farms. There is a situation. In addition, excessive use of these antibiotics may threaten public health and, consequently, reduce the consumer sentiment of farmed fish, leading to a decline in overall fisheries competitiveness. Therefore, development of a method for preventing fish diseases caused by bacteria and effectively treating their infections is urgently needed.
최근에는 양식 어류의 질병제어를 위한 수단으로 백신(Vaccine) 개발 등이 본격화 되고 있으나 아직 백신종류가 다양화되지 못해 질병종류가 다양화 되고 혼합질병 발생 증가에 따른 대처를 위해서는 백신과 함께 또 다른 질병제어 수단이 추가적으로 개발되어야 한다. Recently, the development of vaccine (Vaccine) as a means to control the disease of aquaculture fish has been in full swing, but the type of vaccine has not yet diversified, so the variety of disease is diversified, and in order to cope with the increase in the incidence of mixed diseases, other diseases are combined with the vaccine. Control means must be further developed.
최근 세균성 질환의 대처 방안으로 박테리오파지(Bacteriophage)의 활용이 크게 주목을 받고 있다. 특히 자연친화적 방식의 선호로 인하여 박테리오파지에 대한 관심은 어느 때보다 높다고 할 수 있다. 박테리오파지는 세균에 감염하는 아주 작은 미생물로서 보통 파지(Phage)라고 줄여서 부르기도 한다. 박테리오파지는 박테리아에 감염(Infection)한 후 박테리아 세포 내부에서 증식을 하고, 증식 후 자손 박테리오파지들이 박테리아 밖으로 나올 때 숙주인 박테리아의 세포벽을 파괴하는 방식으로 박테리아를 사멸시키는 능력을 갖고 있다. 박테리오파지의 박테리아 감염 방식은 매우 특이성이 높아서 특정 박테리아에 감염할 수 있는 박테리오파지의 종류는 일부로 한정된다. 즉, 특정 박테리오파지는 특정 범주의 박테리아에만 감염할 수 있고 이로 인하여 특정 박테리오파지는 특정 박테리아만을 사멸시키며 다른 박테리아에는 영향을 주지 않는다. 이러한 박테리오파지의 세균 특이성은 목적으로 하는 세균에 대해서만 항균효과를 제공하고 환경이나 어류 내의 상재균들에는 영향을 초래하지 않는다. 통상적으로 기존의 항생제들은 여러 종류의 세균들에 대하여 동시에 영향을 끼쳤다. 박테리오파지는 특정 세균에 대해서만 작동하므로 박테리오파지 사용에 의해서 체내 정상균총 교란 등이 발생하지 않는다. 따라서 그 사용이 항생제 사용에 비교하여 매우 안전하고 그 만큼 부작용 초래 가능성이 상대적으로 크게 낮다. Recently, the use of bacteriophage as a countermeasure against bacterial diseases has attracted much attention. In particular, the interest in bacteriophages is higher than ever due to the preference of nature-friendly methods. Bacteriophages are tiny microorganisms that infect bacteria, often called phage. Bacteriophages have the ability to infect bacteria and multiply inside bacterial cells, and kill off bacteria by destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria. Bacteriophage bacterial infections are highly specific, so there are only a few types of bacteriophages that can infect certain bacteria. That is, certain bacteriophages can infect only a specific category of bacteria, so that certain bacteriophages kill only certain bacteria and do not affect other bacteria. The bacterial specificity of these bacteriophages provides antimicrobial effects only to the target bacteria and does not affect the environment or flora in fish. Conventional antibiotics usually affect several kinds of bacteria at the same time. Bacteriophage only works for certain bacteria, so the use of bacteriophage does not cause normal bacterial total disturbances. Therefore, its use is very safe compared to the use of antibiotics, and the likelihood of side effects is relatively low.
박테리오파지는 1915년 영국의 세균학자 Twort가 포도상구균(Micrococcus) 집락이 어떤 것에 의해 투명하게 녹는 현상에 대한 연구를 수행하면서 발견되었다. 또한, 1917년에는 프랑스의 세균학자 d'Herelle이 이질환자 변의 여과액 중에 적리균(Shigella dysenteriae)을 녹이는 작용을 가진 것이 있다는 것을 발견하고 이에 대한 연구를 통해 독립적으로 박테리오파지를 발견하였으며, 세균을 잡아먹는다는 뜻에서 박테리오파지라고 명명하였다. 이후 이질균, 장티푸스균, 콜레라균 등 여러 병원성 박테리아에 대한 박테리오파지가 계속적으로 발견되었다. Bacteriophage is a British bacteriologist Twort 1915 became discovered while conducting research on Staphylococcus aureus (Micrococcus) melting the colonies are transparent by any developer. In 1917, French bacteriologist d'Herelle discovered that some of the filtrates of ill feces dissolve Shigella dysenteriae . In the sense, they named it bacteriophage. Since then, bacteriophages have been found for many pathogenic bacteria such as dysentery, typhoid, and cholera.
박테리아를 사멸시킬 수 있는 특별한 능력으로 인하여 박테리오파지는 발견 이후 박테리아 감염에 대응하는 효과적 방안으로 기대를 모았으며 관련하여 많은 연구들이 있었다. 그러나 Fleming에 의해 페니실린이 발견된 이후, 항생제의 보급이 일반화되면서 박테리오파지에 대한 연구는 일부 동유럽 국가들 및 구소련에 한정되어서만 명맥이 유지되었다. 그런데 2000년 이후에 항생제 내성균의 증가로 인하여 기존 항생제의 한계성이 나타나고, 기존 항생제의 대체 물질로의 개발 가능성이 부각되면서 다시 박테리오파지가 항-박테리아제로 주목을 받고 있다.Because of its special ability to kill bacteria, bacteriophages have been expected to be an effective way to combat bacterial infections since their discovery and many studies have been done. However, after the discovery of penicillin by Fleming, with the widespread use of antibiotics, research on bacteriophages has been limited to some Eastern European countries and the Soviet Union. However, since 2000, due to the increase of antibiotic-resistant bacteria, the limit of existing antibiotics appears, and as the possibility of developing an alternative to the existing antibiotics is highlighted, bacteriophage is attracting attention as an anti-bacterial agent.
특히 최근 항생제 사용에 대한 정부 차원의 규제가 전 세계적으로 강화됨에 따라 박테리오파지에 대한 관심이 더욱 높아지고 있으며 산업적 활용 사례도 증가하고 있다.In particular, with the recent tightening of government-wide regulations on the use of antibiotics, interest in bacteriophages is increasing and industrial use cases are increasing.
앞에서 설명했듯이 박테리오파지는 세균에 대한 특이성이 매우 높다. 이러한 특이성으로 인하여 박테리오파지는 동일 종류에 속하는 세균들이라 할지라도 그 일부 주(Strain)에 대해서만 항균효과를 발휘하는 경우가 많다. 또한 대상 세균주에 따라 발휘되는 박테리오파지의 항균력 세기 자체도 다를 수 있다. 이러한 이유로 특정 종류의 세균에 대하여 효과적 제어법을 확보하려면 다양한 종류의 유용 박테리오파지의 확보가 필요하다. 에로모나스 살모니시다 균에 대응하여 효과적인 박테리오파지 활용법을 개발하기 위해서도 당연히 다양한 유용 박테리오파지들(에로모나스 살모니시다 균에 대하여 항균효과를 제공할 수 있는 여러 종류의 박테리오파지들)의 확보가 필요하고, 더 나아가 확보한 다양한 유용 박테리오파지들 중에서 항균력의 세기나 항균범위 측면에서 비교우위에 있는 박테리오파지의 선발도 필요하다.As mentioned earlier, bacteriophages have a very high specificity for bacteria. Due to this specificity, bacteriophages often exert an antimicrobial effect on only some strains, even if they belong to the same type of bacteria. In addition, the antibacterial activity of the bacteriophages may be different depending on the bacterial strain. For this reason, it is necessary to secure various kinds of useful bacteriophages in order to secure effective control methods for specific kinds of bacteria. In order to develop an effective bacteriophage in response to the bacteriophage, it is naturally necessary to secure various useful bacteriophages (a variety of bacteriophages that can provide antibacterial effects against the bacteriophage). Furthermore, among the various useful bacteriophages obtained, it is also necessary to select bacteriophages that have comparative advantages in terms of the strength of the antimicrobial activity and the antimicrobial range.
이에, 본 발명자들은 에로모나스 살모니시다 균을 선택적으로 사멸시킬 수 있는 자연으로부터 분리된 박테리오파지를 이용하여 에로모나스 살모니시다 균의 감염을 방지 또는 처치하는 데에 활용될 수 있는 조성물을 개발하고, 또 이 조성물을 이용하여 에로모나스 살모니시다 균의 감염을 방지 또는 처치하는 방법을 개발하고자 노력한 끝에, 이에 적합한 박테리오파지를 자연으로부터 분리하고 이 분리된 박테리오파지를 타 박테리오파지와 구별하여 특정 지을 수 있도록 유전체(Genome)의 유전자 서열을 확보한 후 상기 박테리오파지를 유효성분으로 한 조성물을 개발한 다음 이 조성물이 에로모나스 살모니시다 균의 감염 방지 및 처치에 효과적으로 활용될 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors have developed a composition that can be used to prevent or treat the infection of the Eromonas Salmonicida bacteria by using bacteriophages isolated from nature capable of selectively killing the E.E. In addition, after trying to develop a method for preventing or treating an infection of E. monmonix using this composition, it is possible to isolate a suitable bacteriophage from nature and to separate the bacteriophage from other bacteriophages. After securing the gene sequence of Genome), the present invention was completed by developing a composition using the bacteriophage as an active ingredient, and then confirming that the composition can be effectively used for preventing and treating E. coli bacteria.
따라서 본 발명의 목적은 에로모나스 살모니시다 균을 특이적으로 사멸시킬 수 있는 능력을 갖는 서열번호 1로 표시되는 유전체를 갖는 것을 특징으로 하는 자연으로부터 분리한 미오비리대(Myoviridae) 박테리오파지 Aer-SAP-1(수탁번호 KCTC 12814BP)을 제공하는 것이다.Therefore, an object of the present invention is Myoviridae bacteriophage Aer-SAP isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monas Salmonisid bacteria. -1 (accession number KCTC 12814BP).
본 발명의 또 다른 목적은 에로모나스 살모니시다 균에 감염하여 에로모나스 살모니시다 균을 사멸시킬 수 있는 분리 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 에로모나스 살모니시다 균의 감염을 방지하는 데에 활용 가능한 조성물 및 이 조성물을 이용한 에로모나스 살모니시다 균의 감염 방지 방법을 제공하는 것이다.Another object of the present invention is to prevent the infection of the Eromonas Salmonicida bacteria comprising the isolated bacteriophage Aer-SAP-1 as an active ingredient that can kill the Eromonas Salmonicida bacteria and kill the E. It is to provide a composition which can be utilized for the purpose and a method for preventing infection of Eromonas salmonisid bacteria using the composition.
본 발명의 또 다른 목적은 에로모나스 살모니시다 균에 감염하여 에로모나스 살모니시다 균을 사멸시킬 수 있는 분리 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 에로모나스 살모니시다 균의 감염을 처치하는 데에 활용 가능한 조성물 및 이 조성물을 이용한 에로모나스 살모니시다 균의 감염 처치 방법을 제공하는 것이다.Another object of the present invention is to treat the infection of the E. monas Salmonicida bacteria comprising the isolated bacteriophage Aer-SAP-1 as an active ingredient capable of killing the E. monas Salmonicida bacteria to kill the E. It is to provide a composition which can be utilized for the treatment, and a method for treating infection of Eromonas salmonisid bacteria using the composition.
본 발명의 또 다른 목적은 상기 조성물들을 이용한 에로모나스 살모니시다 균 감염 방지 및 처치 목적의 약욕제를 제공하는 것이다.Still another object of the present invention is to provide a bath agent for the purpose of preventing and treating eromonas salmonisid infection using the compositions.
본 발명의 또 다른 목적은 상기 조성물들을 이용한 에로모나스 살모니시다 균 감염 방지 및 처치를 통한 사양 효과 제공 목적의 사료첨가제를 제공하는 것이다.Still another object of the present invention is to provide a feed additive for the purpose of providing a specification effect through the prevention and treatment of the E. monas salmonisid infection using the compositions.
본 발명은 에로모나스 살모니시다 균을 특이적으로 사멸시킬 수 있는 능력을 갖는 서열번호 1로 표시되는 유전체를 갖는 것을 특징으로 하는 자연으로부터 분리한 미오비리대 박테리오파지 Aer-SAP-1(수탁번호 KCTC 12814BP), 및 이를 유효성분으로 포함하는 조성물을 이용한 에로모나스 살모니시다 균의 감염 방지 및 처치 방법을 제공한다.The present invention is Myobiridae bacteriophage Aer-SAP-1 (Accession No. KCTC) isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monmonix Salmonicida bacteria. 12814BP), and a method for preventing and treating infection of Eromonas salmonisid bacteria using a composition comprising the same as an active ingredient.
박테리오파지 Aer-SAP-1은 본 발명자들에 의해 분리된 후 2015년 5월 20일자로 한국생명공학연구원 미생물자원센터에 기탁되었다(수탁번호 KCTC 12814BP).Bacteriophage Aer-SAP-1 was isolated by the inventors and deposited in the Korea Institute of Biotechnology and Microbial Resources Center on May 20, 2015 (Accession No. KCTC 12814BP).
또한, 본 발명은 에로모나스 살모니시다 균의 감염을 방지 또는 처치하는 데에 활용될 수 있는 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 약욕제 및 사료첨가제를 제공한다.The present invention also provides a bath and feed additive comprising a bacteriophage Aer-SAP-1 as an active ingredient that can be used to prevent or treat the infection of the Eromonas Salmonicida bacteria.
본 발명의 조성물에 포함되는 박테리오파지 Aer-SAP-1은 에로모나스 살모니시다 균을 효과적으로 사멸시키므로 에로모나스 살모니시다 균에 의해 유발되는 질병의 예방(감염 방지)이나 치료(감염 처치)에 효과를 나타낸다. 따라서 본 발명의 조성물은 에로모나스 살모니시다 균에 의해 유발되는 대표적인 질병인 절창병 및 세균성 패혈증에 대한 예방 및 치료 목적으로 활용될 수 있으나, 이에 한정되는 것은 아니다.Bacteriophage Aer-SAP-1 included in the composition of the present invention effectively kills the bacteria E. monomonas Salmonida, and thus has an effect on the prevention (infection prevention) or treatment (infection treatment) of diseases caused by E. monmonas Salmonida. Indicates. Therefore, the composition of the present invention may be used for the purpose of prevention and treatment of choral disease and bacterial sepsis, which are representative diseases caused by E. monas salmonicida, but is not limited thereto.
본 명세서에서 사용된 “처치” 또는 “치료”라는 용어는 (1) 에로모나스 살모니시다 균에 의해 유발된 질환의 억제; 및 (2) 에로모나스 살모니시다 균에 의해 유발된 질환의 병적 상태를 경감시키는 모든 행위를 의미한다.As used herein, the term "treatment" or "treatment" refers to (1) suppression of a disease caused by the bacterium Aeromonas salmonisid; And (2) all acts to alleviate the pathological condition of the disease caused by E. monas salmonicida bacteria.
본 명세서의 “분리” 또는 “분리된”은 자연 상태로부터 여러 실험 기법을 활용하여 박테리오파지를 분리하는 것과 타 박테리오파지와 구별하여 특정 지을 수 있는 특징들을 확보하는 일을 지칭하며, 이에 더하여 생물공학기술로 박테리오파지를 산업적으로 활용할 수 있게끔 증식시키는 것도 포함한다.As used herein, “isolated” or “isolated” refers to the separation of bacteriophages from the natural state using various experimental techniques, and to securing specific characteristics that can be distinguished from other bacteriophages. This includes growing the bacteriophages for industrial use.
본 발명의 조성물에 포함되는 약제학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산칼슘, 알지네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로오스, 폴리비닐피롤리돈, 셀룰로오스, 물, 시럽, 메틸 셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but are not limited to these. . The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.
본 발명의 조성물에는 박테리오파지 Aer-SAP-1이 유효성분으로 포함된다. 이때 포함되는 박테리오파지 Aer-SAP-1은 1× 101 pfu/㎖ 내지 1× 1030 pfu/㎖ 또는 1× 101 pfu/g 내지 1× 1030 pfu/g로 포함되며, 바람직하게는 1× 104 pfu/㎖ 내지 1× 1015 pfu/㎖ 또는 1× 104 pfu/g 내지 1× 1015 pfu/g로 포함된다.The composition of the present invention includes bacteriophage Aer-SAP-1 as an active ingredient. The bacteriophage Aer-SAP-1 included at this time includes 1 × 10 1 pfu / ml to 1 × 10 30 pfu / ml or 1 × 10 1 pfu / g to 1 × 10 30 pfu / g, preferably 1 ×. 10 4 pfu / ml to 1 × 10 15 pfu / ml or 1 × 10 4 pfu / g to 1 × 10 15 pfu / g.
본 발명의 조성물은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수도 있다. 이때 제형은 오일 또는 수성 매질 중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캡슐제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수도 있다.The compositions of the present invention may be prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. It may also be prepared by incorporation into a multi-dose container. The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
본 발명의 조성물은 활용 방식에 따라, 이에 국한되지 않지만, 약욕제 및 사료첨가제로 구현될 수 있다. The composition of the present invention, depending on the mode of utilization, but is not limited to this, may be implemented as a bath and feed additives.
이러한 활용 목적에서의 효율성을 높이기 위하여 다른 세균종에 대하여 항균활성을 제공할 수 있는 박테리오파지들이 본 발명의 조성물에 추가될 수 있다. 또한, 에로모나스 살모니시다 균에 대하여 항균활성을 갖는 다른 종류의 박테리오파지들도 추가될 수 있다. 비록 에로모나스 살모니시다 균에 대하여 항균활성을 갖는 박테리오파지라 하더라도 항균력의 세기나 항균범위 측면에서 차이가 있으므로 이들의 적절한 조합은 그 효과를 극대화 할 수 있다.Bacteriophages that can provide antimicrobial activity against other bacterial species can be added to the composition of the present invention in order to increase the efficiency in this application. In addition, other types of bacteriophages having antimicrobial activity against the bacteria E. monmonas may also be added. Although bacteriophages that have antimicrobial activity against E. monas salmonicida bacteria differ in terms of strength and antimicrobial range of antimicrobial activity, their proper combination can maximize the effect.
본 발명의 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 조성물을 이용한 에로모나스 살모니시다 균의 감염 방지 및 처치 방법은 기존의 항생제 등의 화학물질에 기반을 둔 방식에 비하여 에로모나스 살모니시다 균에 대한 특이성이 매우 높다는 장점을 제공할 수 있다. 이는 다른 유용한 상재균에는 영향을 주지 않으면서도 에로모나스 살모니시다 균의 감염 방지 또는 처치 목적으로 사용할 수 있음을 의미하며, 이의 사용에 따른 부작용이 매우 적다. 통상적으로 항생제 등의 화학물질을 사용하면 일반 상재균들도 피해를 함께 입게 되어 결과적으로 동물의 면역력 저하 등을 초래하여 다양한 부작용이 나타난다. 한편, 본 발명은 자연계에 이미 존재하는 박테리오파지를 분리하여 조성물의 유효성분으로 사용하기 때문에 매우 자연 친화적이라는 장점 또한 제공할 수 있다. 한편, 박테리오파지는 항균활성을 발휘할 수 있는 세균종이 같다 하더라도 항균효과 발휘에 있어 항균력의 세기나 항균범위[에로모나스 살모니시다 균에 속하는 여러 세균 주(Strain)의 측면에서 개별 세균 주에 대하여 박테리오파지의 항균활성이 발휘되는 범위. 통상적으로 박테리오파지는 같은 세균 종(Species)에 속하는 일부 세균 주(Strain)에 대하여 항균활성을 발휘할 수 있음. 즉, 같은 세균 종에 속한다 하더라도 개별 세균 주에 따라 박테리오파지에 대한 감수성에서 차이가 있을 수 있음] 측면에서 차이가 있으므로 본 발명은 에로모나스 살모니시다 균에 대한 항균력을 갖는 타 박테리오파지에 비교하여 차별적 항균효과를 제공할 수 있다. 이는 산업현장 활용 시에 그 효과에 있어 큰 차이를 제공한다.Infection prevention and treatment method of the E. monomonas Salmonishi bacteria using the composition comprising the bacteriophage Aer-SAP-1 of the present invention as compared to the conventional method based on chemicals such as antibiotics It can provide the advantage that the specificity for the bacteria is very high. This means that it can be used for the purpose of preventing or treating the infection of the E. monas salmonicida without affecting other useful floras, and its side effects are very low. In general, the use of chemicals, such as antibiotics will also damage the common flora bacteria, resulting in a decrease in the immunity of animals, resulting in various side effects. On the other hand, the present invention can also provide the advantage of being very natural because it is used as an active ingredient of the composition to separate the bacteriophage already present in nature. On the other hand, even though bacteriophages have the same bacterial species that can exert their antimicrobial activity, the bacteriophages may be separated against individual bacterial strains in terms of the strength of the antimicrobial activity and the antimicrobial range [strains of several bacterial strains belonging to the genus Eromonas Salmonisid]. Range of antibacterial activity. Generally, bacteriophages can exert antimicrobial activity against some strains belonging to the same bacterial species. That is, even if they belong to the same bacterial species, there may be a difference in sensitivity to bacteriophages according to individual bacterial strains]. Therefore, the present invention is different from the other bacteriophages having antimicrobial activity against E. monas salmonisid. Can provide an effect. This makes a big difference in the effectiveness of industrial sites.
도 1은 박테리오파지 Aer-SAP-1의 전자현미경 사진이다. 1 is an electron micrograph of the bacteriophage Aer-SAP-1.
도 2는 박테리오파지 Aer-SAP-1의 에로모나스 살모니시다 균에 대한 사멸능을 보여주는 실험 결과이다. 투명한 부분은 시험대상 박테리아가 용균되어 결과적으로 형성된 용균반이다.Figure 2 is an experimental result showing the killing ability of the bacteriophage Aer-SAP-1 against the E. monas Salmonida. The transparent part is the lysate plaque formed by lysis of the bacteria under test.
이하, 실시예에 의거하여 본 발명을 보다 구체적으로 설명하지만, 이들 실시예는 본 발명의 예시일 뿐이며 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, although an Example demonstrates this invention more concretely, these Examples are only illustrations of this invention, The scope of the present invention is not limited to these Examples.
실시예Example 1:  One: 에로모나스Erotica 살모니시다Let's Salmon 균을 사멸시킬 수 있는 박테리오파지의 분리 Isolation of Bacteriophage Can Kill Bacteria
에로모나스 살모니시다 균을 사멸시킬 수 있는 박테리오파지의 선별에는 자연 환경으로부터 확보된 시료들을 이용하였다. 한편, 박테리오파지 분리에 사용된 에로모나스 살모니시다 균은 본 발명자들에 의해 미리 분리되어 에로모나스 살모니시다 균으로 동정(Identification)된 것이다. In order to screen the bacteriophages capable of killing the E. monmonix, the samples obtained from the natural environment were used. On the other hand, the eromonas Salmonida bacteria used in the bacteriophage separation was previously identified by the present inventors (Identification).
박테리오파지 분리 과정을 상세히 설명하면, 수집된 시료를 에로모나스 살모니시다 균을 1/1,000 비율로 접종한 TSB(Tryptic Soy Broth) 배지(카제인 다이제스트, 17 g/L; 소이빈 다이제스트, 3 g/L; 덱스트로스, 2.5 g/L; NaCl, 5 g/L; 디포타슘 포스페이트, 2.5 g/L)에 함께 첨가한 다음 25℃에서 3-4시간동안 진탕배양 하였다. 배양 후, 8,000 rpm에서 20분간 원심분리하여 상등액을 회수하였다. 회수된 상등액에 에로모나스 살모니시다 균을 1/1,000 비율로 접종한 다음 25℃에서 3-4시간동안 또 다시 진탕배양 하였다. 박테리오파지가 시료에 포함되어 있었을 경우 박테리오파지의 수(Titer)가 증가될 수 있도록 이러한 과정을 총 5회 반복하였다. 5회 반복 후에 배양액을 8,000 rpm에서 20분간 원심분리 하였다. 원심분리 후, 회수된 상등액을 0.45 ㎛의 필터를 이용하여 여과를 실시해 주었다. 얻어진 여과액을 사용한 통상의 점적 실험(Spot assay)을 통하여 에로모나스 살모니시다 균을 사멸시킬 수 있는 박테리오파지가 있는지를 조사하였다.More specifically the bacteriophage separation process, let Pseudomonas erotic the collected sample live monitor strain of 1 / 1,000 a TSB (T ryptic S oy B roth) inoculated at a rate medium (Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; dextrose, 2.5 g / L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L) together and then shaken at 25 ° C. for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. The recovered supernatants were inoculated with 1 / 1,000 of E. monmonix Salmonida, and then shaken again at 25 ° C. for 3-4 hours. When the bacteriophage was included in the sample, the process was repeated five times in order to increase the number of bacteriophages (Titer). After five repetitions, the culture was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. The usual spot assay using the obtained filtrate examined whether bacteriophages capable of killing the bacterium bacterium eromonas.
상기 점적 실험은 다음과 같이 실시되었다. TSB 배지에 에로모나스 살모니시다 균을 1/1,000 비율로 접종한 다음 25℃에서 한밤동안 진탕배양 하였다. 이렇게 하여 준비된 에로모나스 살모니시다 균의 배양액 3 ㎖(OD600이 1.5)을 TSA(Tryptic Soy Agar) 평판배지(카제인 다이제스트, 15 g/L; 소이빈 다이제스트, 5 g/L; NaCl, 5 g/L; 아가, 15 g/L)에 도말(Spreading)하였다. 도말한 평판 배지를 클린벤치(Clean bench)에서 약 30분 정도 방치하여 도말액이 건조되게 하였다. 건조 후 앞에서 준비한 여과액 10 μl를 에로모나스 살모니시다 균이 도말된 평판 배지 위에 점적하였다. 이를 30분 정도 방치하여 건조시켰다. 건조 후 점적한 평판 배지를 25℃에서 하루 동안 정치 배양한 다음 여과액이 떨어진 위치에 투명환(Clear zone)이 생성되는가를 조사하였다. 투명환이 생성되는 여과액의 경우가 에로모나스 살모니시다 균을 사멸 시킬 수 있는 박테리오파지가 포함되어 있다고 판단할 수 있다. 이러한 조사를 통하여 에로모나스 살모니시다 균에 대한 사멸능을 가진 박테리오파지를 포함한 여과액을 확보할 수 있었다.The drip experiment was conducted as follows. Inoculated with E. monomonas salmonicida in the ratio of 1 / 1,000 in TSB medium and then cultured with shaking at 25 ℃ overnight. The thus prepared 3 ㎖ culture solution of the erosion Pseudomonas live monitor let bacteria (OD 600 is 1.5) TSA (T ryptic S oy A gar) plate medium (Casein Digest, 15 g / L; Soy bean digest, 5 g / L; NaCl , 5 g / L; agar, 15 g / L). The plated flat medium was left in a clean bench for about 30 minutes to allow the smear to dry. After drying, 10 μl of the filtrate prepared above was dipped onto a plate medium plated with E. monitis salmonisid. It was left to dry for 30 minutes. After drying, the plated medium was incubated for one day at 25 ° C., and then a clear zone was formed at the position where the filtrate was separated. In the case of the filtrate in which the transparent ring is formed, it can be judged that the bacteriophage capable of killing the bacteria of E. monmonis. Through this investigation, it was possible to secure a filtrate including bacteriophages having an ability to kill E. monmonix Salmonida.
에로모나스 살모니시다 균에 대한 사멸능을 가진 박테리오파지의 존재가 확인된 여과액을 이용하여 순수 박테리오파지의 분리를 실시하였다. 순수 박테리오파지의 분리에는 통상의 용균반 분석(Plaque assay)을 이용하였다. 이를 자세히 설명하면, 용균반 분석에서 형성된 용균반 하나를 멸균된 팁을 이용하여 회수한 다음 이를 에로모나스 살모니시다 균 배양액에 첨가해 주어 4-5 시간 동안 25℃에서 함께 배양하였다. 배양 후 8,000 rpm에서 20분간 원심분리하여 상등액을 얻었다. 얻어진 상등액에 50분의 1의 부피로 에로모나스 살모니시다 균 배양액을 첨가해 준 다음 다시 25℃에서 4-5 시간 배양해 주었다. 박테리오파지의 수를 증가시키기 위하여 이러한 과정을 최소 5회 이상 실시한 다음 최종적으로 8,000 rpm에서 20분간 원심분리하여 상등액을 얻었다. 얻어진 상등액을 사용하여 다시 용균반 분석을 실시하였다. 통상 순수 박테리오파지의 분리가 상기 과정의 1회로 달성되지 않기 때문에 이때 형성된 용균반을 이용하여 앞 단계를 전체적으로 다시 반복하였다. 이와 같은 과정을 최소 5회 이상 실시하여 순수한 박테리오파지를 포함한 용액을 확보하였다. 통상적으로 순수 박테리오파지의 분리는 형성된 용균반의 크기 및 모양이 모두 유사하게 될 때까지 반복하였다. 그리고 최종적으로는 전자현미경을 통하여 박테리오파지의 순수 분리 여부를 확인하였다. 전자현미경 분석에서 순수 분리가 확인될 때까지 앞에 기술한 과정을 반복하였다. 전자현미경 분석은 통상의 방법에 따라 실시하였다. 이를 간단히 설명하면 다음과 같다. 순수한 박테리오파지를 포함한 용액을 구리 격자(Copper grid)에 묻히고 2% 우라닐 아세테이트(Uranyl acetate)로 역염색법(Negative staining)과 건조를 수행한 후 투과전자현미경을 통하여 그 형태를 촬영하였다. 순수 분리한 박테리오파지의 전자현미경 사진이 도 1에 제시되어 있다. 형태적 특징으로 판단할 때 신규 확보된 박테리오파지는 미오비리대(Myoviridae) 박테리오파지에 속함을 확인할 수 있었다.Separation of pure bacteriophages was carried out using a filtrate in which the presence of bacteriophages with killing ability against E. monmonix Salmonida was confirmed. Separation of pure bacteriophage was carried out using a conventional Plaque assay. To explain this in detail, one of the lytic plaques formed in the lytic plaque assay was recovered using a sterilized tip, and then added to the E. monomonas salmonisid culture medium and incubated together at 25 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. The supernatant obtained was added to the culture of E. monomonas salmonisid bacteria in a volume of 1/50 and then incubated at 25 ° C. for 4-5 hours. In order to increase the number of bacteriophages, this procedure was performed at least five times, and finally, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. Using the obtained supernatant, lysis plate analysis was performed again. Since the separation of the pure bacteriophage is not usually achieved in one step of the above process, the previous step was repeated again using the lysate formed. This procedure was carried out at least five times to obtain a solution containing pure bacteriophage. Typically, separation of pure bacteriophage was repeated until both the size and shape of the lysate formed were similar. And finally, the electron microscope confirmed the pure separation of bacteriophage. The procedure described above was repeated until pure separation was confirmed by electron microscopy analysis. Electron microscopic analysis was performed according to a conventional method. This is briefly described as follows. The solution containing pure bacteriophage was buried in a copper grid, subjected to reverse staining and drying with 2% uranyl acetate, and the form was photographed through a transmission electron microscope. Electron micrographs of purely isolated bacteriophages are shown in FIG. 1. Judging from the morphological features, the newly acquired bacteriophage belonged to the Myoviridae bacteriophage.
이런 방식으로 확인된 순수 박테리오파지를 포함한 용액은 다음의 정제 과정을 거쳤다. 순수 박테리오파지를 포함한 용액 전체 부피의 50분의 1의 부피로 에로모나스 살모니시다 균 배양액을 첨가해 준 다음 다시 4-5 시간 배양하였다. 배양 후 8,000 rpm에서 20분간 원심분리하여 상등액을 얻었다. 충분한 수의 박테리오파지가 포함된 액을 얻기 위해 이러한 과정을 총 5회 반복하였다. 최종 원심분리로 얻어진 상등액을 0.45 μm의 필터를 이용하여 여과한 다음 통상의 폴리에틸렌 글리콜(Polyethylene Glycol; PEG) 침전 과정을 실시하였다. 구체적으로, 여과액 100 ㎖에 10% PEG 8000/0.5 M NaCl이 되게 PEG와 NaCl을 첨가한 다음 4℃에서 2-3시간 동안 정치한 후, 8,000 rpm에서 30분간 원심분리하여 박테리오파지 침전물을 얻었다. 이렇게 얻어진 박테리오파지 침전물을 완충액(Buffer; 10 mM Tris-HCl, 10 mM MgSO4, 0.1% Gelatin, pH 8.0) 5 ㎖로 부유시켰다. 이를 박테리오파지 부유액 또는 박테리오파지 액이라 지칭한다.The solution containing pure bacteriophage identified in this way was subjected to the following purification process. Eromonas Salmonicida culture medium was added to a volume of 1/50 of the total volume of the solution including the pure bacteriophage and then incubated again for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This procedure was repeated a total of five times to obtain a solution containing a sufficient number of bacteriophages. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter followed by a conventional polyethylene glycol (PEG) precipitation process. Specifically, PEG and NaCl were added to 100 ml of the filtrate to be 10% PEG 8000 / 0.5 M NaCl, and then allowed to stand at 4 ° C. for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. The bacteriophage precipitate thus obtained was suspended in 5 ml of buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). This is called bacteriophage suspension or bacteriophage solution.
이렇게 하여 정제된 순수 박테리오파지를 확보할 수 있었고, 이 박테리오파지를 박테리오파지 Aer-SAP-1로 명명한 뒤, 2015년 5월 20일자로 한국생명공학연구원 미생물자원센터(수탁번호 KCTC 12814BP)에 기탁하였다. In this way, purified pure bacteriophage was secured, and this bacteriophage was named as bacteriophage Aer-SAP-1 and deposited on May 20, 2015 at the Korea Research Institute of Bioscience and Biotechnology (Accession Number KCTC 12814BP).
실시예Example 2: 박테리오파지  2: bacteriophage AerAer -- SAPSAP -1의 유전체 분리 및 서열 분석-1 genome isolation and sequencing
박테리오파지 Aer-SAP-1의 유전체를 다음과 같이 분리하였다. 유전체 분리에는 실시예 1에서와 같은 방법으로 얻어진 박테리오파지 부유액을 이용하였다. 먼저 부유액에 포함되어 있을 수 있는 에로모나스 살모니시다 균의 DNA와 RNA를 제거하기 위해, 박테리오파지 부유액 10 ㎖에 DNase I과 RNase A를 각각 200 U씩 첨가한 다음 37℃에서 30분간 방치하였다. 30분 방치 후에 DNase I과 RNase A의 활성을 제거하기 위해, 0.5 M 에틸렌디아민테트라아세트산(Ethylenediaminetetraacetic acid; EDTA) 500 μl를 첨가한 다음 다시 10분간 정치시켰다. 그리고 이를 추가로 10분간 65℃에 정치시킨 다음 박테리오파지 외벽을 와해시키기 위해 proteinase K(20 ㎎/㎖) 100 μl를 첨가한 후 37℃에서 20분간 반응시켰다. 그 후 10% 도데실 황산 나트륨염(Sodium dodecyl sulfate; SDS) 500 μl를 첨가한 다음 다시 65℃에서 1시간 동안 반응시켰다. 1 시간 반응 후, 이 반응액에 25:24:1의 구성비를 갖는 페놀(Phenol) : 클로로포름(Chloroform) : 이소아밀알코올(Isoamylalcohol)의 혼합액 10 ㎖을 첨가해 준 후 잘 섞어 주었다. 그리고는 이것을 13,000 rpm에서 15분간 원심분리하여 층이 분리되게 한 다음 분리된 층들 중에서 위층을 취하여 여기에 1.5 부피비의 이소프로필 알코올(Isopropyl alcohol)을 첨가한 다음 13,000 rpm에서 10분간 원심분리하여 유전체를 침전시켰다. 침전물을 회수한 후 침전물에 70% 에탄올(Ethanol)을 첨가한 다음 다시 13,000 rpm에서 10분간 원심분리하여 침전물의 세척을 실시하였다. 세척된 침전물을 회수하고 진공 건조 시킨 다음 100 μl의 물에 녹였다. 상기 과정을 반복하여 박테리오파지 Aer-SAP-1의 유전체를 다량 확보하였다. The genome of the bacteriophage Aer-SAP-1 was isolated as follows. Bacteriophage suspension obtained in the same manner as in Example 1 was used for dielectric separation. First, in order to remove DNA and RNA of the E. monas salmonisid bacteria, which may be included in the suspension, 200 U of each of DNase I and RNase A was added to 10 ml of the bacteriophage suspension, followed by standing at 37 ° C. for 30 minutes. In order to remove the activity of DNase I and RNase A after 30 minutes, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and allowed to stand for 10 minutes. The mixture was left at 65 ° C. for 10 minutes, and then 100 μl of proteinase K (20 mg / ml) was added to disintegrate the bacteriophage outer wall, followed by reaction at 37 ° C. for 20 minutes. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reaction at 65 ° C. for 1 hour. After the reaction for 1 hour, 10 ml of a mixture of phenol (Phenol): chloroform (Isoamylalcohol) having a composition ratio of 25: 24: 1 was added to the reaction solution, and the mixture was mixed well. After centrifugation at 13,000 rpm for 15 minutes to separate the layers, take the upper layer from the separated layers, add 1.5 volume ratio of Isopropyl alcohol, and centrifuge at 13,000 rpm for 10 minutes. Precipitated. After recovering the precipitate, 70% ethanol (Ethanol) was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried in vacuo and dissolved in 100 μl of water. The process was repeated to secure a large amount of the genome of bacteriophage Aer-SAP-1.
이렇게 얻어진 유전체는 ㈜천랩에서 Roche 454 GS Junior 기기의 차세대 염기서열 분석(Next generation sequencing analysis)을 통하여 박테리오파지 Aer-SAP-1의 유전체 서열분석을 실시하였다. 최종적으로 분석된 박테리오파지 Aer-SAP-1 유전체는 162,883 bp의 크기를 가지며, 전체 유전체 서열은 서열번호 1로 제시되어 있다. The genome thus obtained was subjected to genome sequencing of bacteriophage Aer-SAP-1 through Next generation sequencing analysis of Roche 454 GS Junior. Finally, the analyzed bacteriophage Aer-SAP-1 genome has a size of 162,883 bp and the entire genome sequence is set forth in SEQ ID NO: 1.
확보된 박테리오파지 Aer-SAP-1의 유전체 서열을 기반으로 Web상의 BLAST(http://www.ncbi.nlm.nih.gov/BLAST/)를 이용하여 기존에 알려진 박테리오파지 유전체 서열과의 상동성(Similarity)을 조사해 보았다. BLAST 조사 결과, 박테리오파지 Aer-SAP-1의 유전체 서열은 에로모나스 박테리오파지 phiAS4의 서열(Genbank Accession No. HM452125.1), 스테노트로포모나스(Stenotrophomonas) 박테리오파지 IME13의 서열(Genbank Accession No. JX306041.1), 에로모나스 박테리오파지 Aes508의 서열(Genbank Accession No. JN377894.1), 에로모나스 박테리오파지 Aes012의 서열(Genbank Accession No. JN377895.1), 및 에로모나스 살모니시다 박테리오파지 25의 서열(Genbank Accession No. DQ529280.1)과 비교적 높은 상동성을 가지고 있는 것으로 확인되었다(Query coverage/identity: 각각 순서대로 96%/97%, 94%/96%, 89%/97%, 93%/97%, 및 93%/98%). 그러나 박테리오파지 Aer-SAP-1은 환형의 유전체를 가짐에 반하여 에로모나스 박테리오파지 phiAS4, 에로모나스 박테리오파지 Aes508, 에로모나스 박테리오파지 Aes012, 및 에로모나스 살모니시다 박테리오파지 25는 선형의 유전체를 가져 유전체 형태에서 큰 차이가 있었으며, 박테리오파지 Aer-SAP-1 유전체 상의 개방형해독틀(Open Reading Frame, ORF)의 개수가 234개임에 반하여 스테노트로포모나스 박테리오파지 IME13은 182개를 가지고 있어 서로 상이한 박테리오파지임을 확인할 수 있었다. Based on the obtained genome sequence of bacteriophage Aer-SAP-1, homology with previously known bacteriophage genome sequence using BLAST on the web (http://www.ncbi.nlm.nih.gov/BLAST/) I checked). As a result of the BLAST investigation, the genome sequence of bacteriophage Aer-SAP-1 was sequenced from the genotype of Eromonas bacteriophage phiAS4 (Genbank Accession No. HM452125.1), and the sequence of Stenotrophomonas bacteriophage IME13 (Genbank Accession No. JX306041.1 ), The sequence of Eromonas bacteriophage Aes508 (Genbank Accession No. JN377894.1), the sequence of Eromonas bacteriophage Aes012 (Genbank Accession No. JN377895.1), and the sequence of Eromonas salmonicida bacteriophage 25 (Genbank Accession No. DQ529280 .1) and relatively high homology with each other (Query coverage / identity: 96% / 97%, 94% / 96%, 89% / 97%, 93% / 97%, and 93%, respectively) / 98%). However, while bacteriophage Aer-SAP-1 has an annular genome, the eromonas bacteriophage phiAS4, the eromonas bacteriophage Aes508, the eromonas bacteriophage Aes012, and the eromonas salmonicida bacteriophage 25 have a large difference in genome morphology. In addition, the number of open reading frames (ORFs) on the bacteriophage Aer-SAP-1 genome was 234, whereas the Stenotropomonas bacteriophage IME13 had 182, indicating that they were different bacteriophages.
이러한 사실에 근거하여 박테리오파지 Aer-SAP-1은 기존에 보고된 바 없는 신규한 박테리오파지라고 판단할 수 있었다. 이러한 사실과 함께 통상적으로 박테리오파지의 종류가 다르면 제공할 수 있는 항균력의 세기 및 항균범위가 다르다는 사실로부터 박테리오파지 Aer-SAP-1은 기존에 보고된 다른 박테리오파지들과는 다른 항균효과를 제공해 줄 수 있다고 판단하였다. Based on this fact, bacteriophage Aer-SAP-1 could be considered as a novel bacteriophage that has not been reported previously. With this fact, it is judged that bacteriophage Aer-SAP-1 can provide different antimicrobial effects from other bacteriophages previously reported from the fact that different kinds of bacteriophages have different strengths and ranges of antimicrobial activity.
실시예Example 3: 박테리오파지  3: bacteriophage AerAer -- SAPSAP -1의 -1's 에로모나스Erotica 살모니시다Let's Salmon 균에 대한  Against fungi 사멸능Death 조사 Research
분리된 박테리오파지 Aer-SAP-1의 에로모나스 살모니시다 균에 대한 사멸능을 조사하였다. 사멸능 조사에는 실시예 1에서와 같은 방법으로 점적 실험을 통하여 투명환 생성 여부를 조사하였다. 사멸능 조사에 사용되어진 에로모나스 살모니시다 균은 본 발명자들에 의해 분리되어 에로모나스 살모니시다 균으로 동정된 것들로 총 13종이었다. 박테리오파지 Aer-SAP-1은 실험에 대상이 된 에로모나스 살모니시다 균 중 11종에 대하여 사멸능을 갖고 있었다. 대표적 실험 결과가 도 2에 제시되어 있다. 한편, 박테리오파지 Aer-SAP-1의 에드워드시엘라 타르다(Edwardsiella tarda), 비브리오 안길라룸(Vibrio anguillarum), 비브리오 익티오엔테리(Vibrio ichthyoenteri), 락토코커스 가르비에(Lactococcus garvieae), 및 스트렙토코커스 파라우베리스(Streptococcus parauberis)에 대한 사멸능 조사도 별도 실험으로 실시하였는데, 그 결과로 박테리오파지 Aer-SAP-1은 이들 균종들에 대해서는 사멸능을 갖고 있지 않았다.The killing ability of the isolated bacteriophage Aer-SAP-1 against the E. monas salmonicida bacteria was investigated. The killing ability was investigated by the drop test in the same manner as in Example 1 to determine the production of transparent rings. There were a total of 13 types of eromomonas Salmonicida bacteria used for investigation of killing ability and identified by the present inventors as Eromonas Salmonicida bacteria. Bacteriophage Aer-SAP-1 had the ability to kill 11 of the E. monmonix Salmonida bacteria. Representative experimental results are shown in FIG. 2. Edwardsiella tarda of the bacteriophage Aer-SAP-1 and Vibrio anguillarum ), Vibrio ichthyoenteri , Lactococcus garvieae ), and Streptococcus parauberis ) was also examined as a separate experiment. As a result, bacteriophage Aer-SAP-1 had no killing ability against these species.
이상의 결과로 박테리오파지 Aer-SAP-1은 에로모나스 살모니시다 균에 대하여 특이적인 사멸능을 가지며, 다수의 에로모나스 살모니시다 균에 대하여 항균 효과를 발휘할 수 있음을 확인할 수 있었다. 이는 박테리오파지 Aer-SAP-1이 에로모나스 살모니시다 균의 감염 방지 및 처치 목적의 조성물의 유효성분으로 활용 가능함을 의미한다.As a result, the bacteriophage Aer-SAP-1 has a specific killing ability against the E. monas salmonisid, and it was confirmed that it can exert an antimicrobial effect against a number of eromonas Salmonida. This means that the bacteriophage Aer-SAP-1 can be used as an active ingredient of the composition for the purpose of preventing and treating the infection of Eromonas salmonisid.
실시예Example 4: 박테리오파지  4: bacteriophage AerAer -- SAPSAP -1의 -1's 에로모나스Erotica 살모니시다Let's Salmon 균의 감염 예방에 대한  For the prevention of bacterial infection 실험예Experimental Example
9 ㎖의 TSB 배지를 담은 하나의 튜브에 1× 108 pfu/㎖ 수준의 박테리오파지 Aer-SAP-1 액 100 μl를 넣어주고, 다른 하나의 9 ㎖의 TSB 배지를 담은 튜브에는 동량의 TSB 배지만을 추가로 첨가하였다. 그 다음에 각 튜브에 600 nm에서 흡광도가 약 0.5 정도가 되도록 에로모나스 살모니시다 균의 배양액을 넣어 주었다. 에로모나스 살모니시다 균을 첨가한 후 튜브들을 25℃의 배양기에 옮겨 진탕배양하면서 에로모나스 살모니시다 균의 성장 상태를 관찰하였다. 표 1에 제시된 바와 같이, 박테리오파지 Aer-SAP-1 액을 첨가해 준 튜브에서는 에로모나스 살모니시다 균의 성장 억제가 관찰된 반면에 박테리오파지 액을 첨가하지 않은 튜브에서는 에로모나스 살모니시다 균의 성장 억제가 관찰되지 않았다. 100 μl of 1 × 10 8 pfu / ml bacteriophage Aer-SAP-1 solution was added to one tube containing 9 ml TSB medium, and the same amount of TSB medium was added to the other tube containing 9 ml TSB medium. Additionally. Then, the culture solution of E. monmonix Salmonida was added to each tube so that the absorbance was about 0.5 at 600 nm. After the addition of the Eromonas Salmonicida bacteria, the tubes were transferred to a 25 ° C. incubator and shaken to observe the growth state of the Eromonas Salmonicida bacteria. As shown in Table 1, growth inhibition of E. monomonas salmonisid was observed in the tube to which the bacteriophage Aer-SAP-1 solution was added, whereas growth of E. monomonas Salmonida was observed in the tube without the bacteriophage solution. No inhibition was observed.
에로모나스 살모니시다 균의 성장 억제Inhibition of growth of Eromonas Salmonisid
구분division OD600 흡광도 값OD 600 absorbance value
배양 0분Incubation 0 minutes 배양후 60분60 minutes after incubation 배양후 120분120 minutes after incubation
박테리오파지 액 미첨가No bacteriophage solution added 0.5020.502 0.8630.863 1.4651.465
박테리오파지 액 첨가Add bacteriophage solution 0.5020.502 0.2540.254 0.1320.132
이 결과로부터 본 발명의 박테리오파지 Aer-SAP-1이 에로모나스 살모니시다 균의 성장을 저해할 뿐만 아니라 사멸까지 시키는 능력이 있음을 확인할 수 있었고, 이로부터 박테리오파지 Aer-SAP-1이 에로모나스 살모니시다 균의 감염을 방지하는 목적의 조성물 유효성분으로 활용될 수 있다고 결론지을 수 있었다. From this result, it can be confirmed that the bacteriophage Aer-SAP-1 of the present invention has the ability not only to inhibit the growth of the bacteriophages Salmonida, but also to kill the bacteriophage Aer-SAP-1. It can be concluded that the composition can be used as an active ingredient for the purpose of preventing infection of Sida.
실시예Example 5: 박테리오파지  5: bacteriophage AerAer -- SAPSAP -1을 이용한 With -1 에로모나스Erotica 살모니시다Let's Salmon 균의 감염 예방 동물실험 Infection prevention animal experiment
무지개 송어를 이용하여 박테리오파지 Aer-SAP-1의 에로모나스 살모니시다 균 감염 예방 효과를 조사하였다. 생후 5주령의 무지개 송어(평균 체장: 15.9 cm, 평균 체중: 24.0 g) 20마리를 한 그룹으로 하여 총 두 그룹으로 나눈 후 수조에서 분리 사육하면서 14일간 실험을 실시하였다. 수조의 주위환경은 통제하였고, 수조가 있는 실험실의 온도는 일정하게 유지시켰다. 실험 개시일부터 실험군(박테리오파지 투여군)의 무지개 송어들에게는 1× 108 pfu/g의 박테리오파지 Aer-SAP-1을 포함하고 있는 사료를 통상적인 사료 급이 방식에 따라 급이하였다. 반면에 대조군(박테리오파지 미투여군)의 무지개 송어들에게는 박테리오파지 Aer-SAP-1이 포함되지 않은 동일 조성의 사료를 동일한 방식으로 급이하였다. 시험개시일로부터 7일째가 되는 날부터 실험군과 대조군 모두 2일간 1× 108 cfu/g 수준으로 에로모나스 살모니시다 균을 급이하는 사료에 포함시켜 하루 2회씩 급이하여 에로모나스 살모니시다 균 감염을 유도하였다. 2일간의 에로모나스 살모니시다 균을 포함하고 있는 사료 급이 시행 다음날(시험 개시일로부터 9일째가 되는 날)부터 매일 모든 시험동물들을 대상으로 절창병 발생 상태를 조사하였다. 절창병 발생 상태 조사는 체표의 궤양 크기를 측정하는 방식으로 실시하였다. 체표의 궤양 크기 측정은 통상 사용되는 Ulcer size(US) score{정상(궤양 없음): 0, 약한 궤양(궤양 크기: 0.5 cm 미만): 1, 강한 궤양(궤양 크기: 0.5 cm 이상): 2}를 측정하는 방식으로 실시하였다. Rainbow trout was used to investigate the effects of bacteriophage Aer-SAP-1 on the infection of E. monitis Salmonida. Twenty five-week-old rainbow trout (average body length: 15.9 cm, average weight: 24.0 g) were divided into two groups, and then divided into two groups. The environment of the bath was controlled and the temperature of the laboratory containing the bath was kept constant. From the start of the experiment, the rainbow trout of the experimental group (bacteriophage treated group) were fed a feed containing 1 × 10 8 pfu / g of bacteriophage Aer-SAP-1 according to a conventional feed feeding method. On the other hand, rainbow trout from the control group (no bacteriophage group) were fed with the same composition in the same manner without the bacteriophage Aer-SAP-1. From the 7th day from the start of the test, both the experimental and control groups included Emononas salmonisid bacteria at a feed level of 1 × 10 8 cfu / g for 2 days. Infection was induced. The incidence of intestinal disease was investigated every day from the day after the feeding of the feed containing the two days of E. monmonix Salmonida, the day after the start of the test (the 9th day from the start of the test). Investigation of the incidence of intestinal disease was performed by measuring the size of the ulcer on the body surface. Ulcer size measurements in the body surface are commonly used Ulcer size (US) score {normal (no ulcer): 0, mild ulcer (ulcer size: less than 0.5 cm): 1, strong ulcer (ulcer size: 0.5 cm or more): 2} It was carried out in a manner to measure.
시험 결과, 2일간의 에로모나스 살모니시다 균을 포함하고 있는 사료 급이 다음 날부터 절창병의 임상증상을 보이는 개체가 대조군 수조에서 확인되었다. 그 결과는 표 2와 같았다.As a result of the test, the feed grade containing the two days of E. monomonas salmonicida was identified in the control tank from the next day. The results were shown in Table 2.
체표 궤양 크기 측정 결과 (평균치)Body surface ulcer size measurement result (average value)
날짜date D9D9 D10D10 D11D11 D12D12 D13D13 D14D14
대조군(박테리오파지 미투여)Control group (not administered bacteriophage) 0.450.45 0.50.5 0.650.65 0.70.7 0.70.7 0.70.7
실험군(박테리오파지 투여)Experimental group (bacteriophage administration) 00 00 00 00 00 00
이 결과로부터 본 발명의 박테리오파지 Aer-SAP-1이 에로모나스 살모니시다 균을 원인으로 하는 감염 질환의 예방에 매우 효과적이라는 것을 확인할 수 있었다. From these results, it was confirmed that the bacteriophage Aer-SAP-1 of the present invention is very effective in the prevention of an infectious disease caused by the bacterium Eromonas salmonisida.
실시예Example 6: 박테리오파지  6: bacteriophage AerAer -- SAPSAP -1을 이용한 With -1 에로모나스Erotica 살모니시다Let's Salmon 균의 감염 질환  Fungal diseases 처치예Procedure
에로모나스 살모니시다 균에 의해 절창병이 유발된 무지개 송어에서의 박테리오파지 Aer-SAP-1의 처치 효과를 조사하였다. 생후 5주령의 무지개 송어(평균 체장: 15.8 cm, 평균 체중: 23.9 g) 40마리를 한 그룹으로 하여 총 두 그룹으로 나눈 후 수조에서 분리 사육하면서 14일간 실험을 실시하였다. 수조의 주위환경은 통제하였고, 수조가 있는 실험실의 온도는 일정하게 유지시켰다. 실험 개시일로부터 5일째 되는 날부터 3일간 1× 108 cfu/g 수준으로 에로모나스 살모니시다 균을 포함하고 있는 사료를 하루 2회씩 통상적인 사료 급이 방식으로 급이하였다. 에로모나스 살모니시다 균을 포함하고 있는 사료 급이 마지막 날부터 절창병의 임상증상을 보이는 개체가 두 수조 모두에서 확인되었다. 3일간의 에로모나스 살모니시다 균을 포함하고 있는 사료 급이 시행 다음날(시험 개시일로부터 8일째가 되는 날)부터 실험군(박테리오파지 투여군)의 무지개 송어들에게는 1× 108 pfu/g의 박테리오파지 Aer-SAP-1을 포함하고 있는 사료를 통상적인 사료 급이 방식에 따라 급이하였다. 반면에 대조군(박테리오파지 미투여군)의 무지개 송어들에게는 박테리오파지 Aer-SAP-1이 포함되지 않은 동일 조성의 사료를 동일한 방식으로 급이하였다. 시험 개시일로부터 8일째가 되는 날부터는 매일 모든 시험동물들을 대상으로 절창병 발생 상태를 조사하였다. 절창병 발생 상태 조사는 체표의 궤양 크기를 측정하는 방식으로 실시하였다. 체표의 궤양 크기 측정은 통상 사용되는 Ulcer size(US) score{정상(궤양 없음): 0, 약한 궤양(궤양 크기: 0.5 cm 미만): 1, 강한 궤양(궤양 크기: 0.5 cm 이상): 2}를 측정하는 방식으로 실시하였다. The effect of bacteriophage Aer-SAP-1 treatment on rainbow trout induced by Eromonas salmonicida was investigated. Forty five-week-old rainbow trout (mean body length: 15.8 cm, average body weight: 23.9 g) were divided into two groups, which were divided into two groups. The environment of the bath was controlled and the temperature of the laboratory containing the bath was kept constant. Feed containing the E. monas salmonisid bacteria was fed twice a day in a conventional feed system at a level of 1 × 10 8 cfu / g for 3 days from the 5th day from the start of the experiment. In both tanks, individuals with clinical symptoms of cholinergic disease were identified from the last day of feed containing Emononas salmonisida. 1 x 10 8 pfu / g of bacteriophage Aer- to rainbow trout from the experimental group (the bacteriophage group) from the day after the feeding (containing the eighth day from the start of the test). The feed containing SAP-1 was fed according to a conventional feed feeding method. On the other hand, rainbow trout from the control group (no bacteriophage group) were fed with the same composition in the same manner without the bacteriophage Aer-SAP-1. From the 8th day after the start of the test, all the test animals were examined for the incidence of intestinal disease every day. Investigation of the incidence of intestinal disease was performed by measuring the size of the ulcer on the body surface. Ulcer size measurements in the body surface are commonly used Ulcer size (US) score {normal (no ulcer): 0, mild ulcer (ulcer size: less than 0.5 cm): 1, strong ulcer (ulcer size: 0.5 cm or more): 2} It was carried out in a manner to measure.
그 결과는 표 3과 같았다.The results were shown in Table 3.
체표 궤양 크기 측정 결과 (평균치)Body surface ulcer size measurement result (average value)
날짜date D8D8 D9D9 D10D10 D11D11 D12D12 D13D13 D14D14
대조군(박테리오파지 미투여)Control group (not administered bacteriophage) 1.001.00 1.401.40 1.601.60 1.701.70 1.651.65 1.651.65 1.701.70
실험군(박테리오파지 투여)Experimental group (bacteriophage administration) 1.051.05 0.950.95 0.550.55 0.450.45 0.350.35 0.150.15 0.150.15
이 결과로부터 본 발명의 박테리오파지 Aer-SAP-1이 에로모나스 살모니시다 균을 원인으로 하는 감염 질환의 처치에도 매우 효과적이라는 것을 확인할 수 있었다. From these results, it was confirmed that the bacteriophage Aer-SAP-1 of the present invention is very effective in the treatment of infectious diseases caused by the bacterium Eromonas salmonisida.
실시예Example 7: 사료첨가제 및 사료의 제조 7: Preparation of feed additives and feed
박테리오파지 Aer-SAP-1 액을 이용하여 사료첨가제 1 g당 1× 108 pfu의 박테리오파지 Aer-SAP-1이 포함되도록 사료첨가제를 제조하였다. 사료첨가제의 제조 방법은 박테리오파지 액에 말토덱스트린을 첨가(50%, w/v)한 다음에 동결건조시켜 제조하였다. 최종적으로 고운 가루 형태로 분쇄하였다. 상기 제조 과정 중의 건조 과정에는 감압 건조, 가온 건조, 상온 건조도 대체 가능하다. 대조 실험을 위해, 박테리오파지가 포함되지 않은 사료첨가제도 박테리오파지 액 대신에 박테리오파지 액의 제조 시에 사용한 완충액(Buffer; 10 mM Tris-HCl, 10 mM MgSO4, 0.1% Gelatin, pH 8.0)을 사용하는 방식으로 제조하였다.A feed additive was prepared using bacteriophage Aer-SAP-1 solution to include 1 × 10 8 pfu of bacteriophage Aer-SAP-1 per g of feed additive. The method of preparing a feed additive was prepared by adding maltodextrin to the bacteriophage solution (50%, w / v) and then lyophilizing. Finally, it was ground to a fine powder form. The drying process in the manufacturing process may be replaced by reduced pressure drying, warming drying, room temperature drying. For the control experiment, a feed additive without bacteriophage was used in place of the bacteriophage solution, using the buffer used to prepare the bacteriophage solution (Buffer; 10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). It was prepared by.
이렇게 제조된 2종의 사료첨가제 각각을 중량비로 250배의 양어용 생사료와 혼합하여 최종 2종의 사료를 제조하였다. Each of the two feed additives thus prepared was mixed with 250 times the fish feed for fish in a weight ratio to prepare the final two feeds.
실시예Example 8:  8: 약욕제의Bath 제조 Produce
박테리오파지 Aer-SAP-1 액을 이용하여 약욕제 1 ㎖당 1× 108 pfu의 박테리오파지 Aer-SAP-1이 포함되도록 약욕제를 제조하였다. 약욕제의 제조 방법은 박테리오파지 액 제조 시에 사용하는 완충액 1 ㎖당 1× 108 pfu의 박테리오파지 Aer-SAP-1이 포함되도록 상기 박테리오파지 Aer-SAP-1 액을 첨가하여 잘 혼합해 주는 방식으로 제조하였다. 대조 실험을 위해, 박테리오파지가 포함되지 않은 약욕제로는 박테리오파지 액의 제조 시에 사용한 완충액 자체를 그대로 사용하였다.The bacteriophage Aer-SAP-1 was prepared using a bacteriophage Aer-SAP-1 solution to contain 1 × 10 8 pfu of bacteriophage Aer-SAP-1 per ml of the bath. The method of preparing a bath detergent is prepared by adding the above-mentioned bacteriophage Aer-SAP-1 solution so that 1 × 10 8 pfu of bacteriophage Aer-SAP-1 is included per 1 ml of the buffer used to prepare the bacteriophage solution. It was. For the control experiment, as a bath agent that does not contain bacteriophage, the buffer itself used in the preparation of the bacteriophage solution was used as it is.
이렇게 제조된 2종의 약욕제는 부피비로 1,000배의 물로 희석하여 최종적인 약욕제로 사용하였다. The two types of baths thus prepared were diluted with 1,000 times water by volume and used as the final bath.
실시예Example 9: 무지개 송어 사육에서의 사양 효과 확인 9: Confirmation of Specification Effect in Rainbow Trout Breeding
실시예 7 및 실시예 8에서 제조한 사료와 약욕제를 이용하여 무지개 송어 사육 시의 사양 결과 개선 여부에 대하여 조사해 보았다. 특히 본 조사는 폐사율 관점에서 실시되었다. 총 100 마리의 무지개 송어를 50 마리씩 한 그룹으로 총 2개 그룹(사료로 급이한 그룹-A; 약욕제로 처치한 그룹-B)으로 나누어 4주간 시험을 실시하였다. 각 그룹은 다시 25마리로 구성되는 소그룹으로 나누어지며 각 소그룹은 박테리오파지 Aer-SAP-1이 적용된 소그룹(소그룹-①) 및 박테리오파지가 적용되지 않은 소그룹(소그룹-②)으로 나누었다. 본 시험에 대상이 된 무지개 송어는 5주령의 무지개 송어 치어였으며, 각 시험 소그룹의 무지개 송어는 일정 간격을 두고 위치한 격리된 각각의 수조에서 사육되었다. 각 소그룹은 다음의 표 4와 같이 구분되고 지칭되었다. Using the feed prepared in Example 7 and Example 8 and the bath was investigated whether the improvement of the specification results when breeding rainbow trout. In particular, the survey was conducted in terms of mortality. A total of 100 rainbow trout were divided into two groups (group-A fed; group-B treated with a bath) for 50 weeks. Each group was divided into 25 subgroups, and each subgroup was divided into a small group (small group-①) to which bacteriophage Aer-SAP-1 was applied and a small group (small group-②) to which no bacteriophage was applied. The rainbow trout for this test were five-week-old rainbow trout, and the rainbow trout from each subgroup were raised in separate tanks at regular intervals. Each subgroup is divided and referred to as Table 4 below.
무지개 송어 사양 시험에서의 소그룹 구분 및 표시Small Group Identification and Marking in Rainbow Trout Specimen
적용apply 소그룹 구분 및 표시Small Group Separation and Marking
박테리오파지 Aer-SAP-1 적용Application of bacteriophage Aer-SAP-1 박테리오파지가 적용되지 않음Bacteriophage is not applicable
사료로 급이한 그룹A group fed at a feed A-①A-① A-②A-②
약욕제로 처치한 그룹Group treated with a bath B-①B-① B-②B-②
사료 급이의 경우에는 실시예 7에서 설명한 사료 제조 방식에 따라 제조한 사료를 표 4의 구분에 따라 통상적인 사료 급이 방식을 따라 급이 하였으며, 약욕제 처치의 경우에는 실시예 8에서 설명한 약욕제 제조 방식에 따라 제조한 약욕제를 표 4의 구분에 따라 통상적인 약욕제 처치 방식에 따라 처치하였다. 시험 결과가 표 5에 제시되어 있다. In the case of feed feeding, the feed prepared according to the feed preparation method described in Example 7 was fed according to the conventional feed feeding method according to the classification of Table 4, and in the case of the treatment of the bathing agent, the bathing bath described in Example 8 The bath preparation prepared according to the preparation method was treated according to the conventional bath treatment method according to the classification of Table 4. The test results are shown in Table 5.
무지개 송어 사양 시험에서의 폐사율Mortality at Rainbow Trout Specimen Testing
그룹group 폐사개체수/시험개체수Number of dead individuals / Number of test subjects 폐사율(%) % Mortality
A-①A-① 1/251/25 4.04.0
A-②A-② 7/257/25 28.028.0
B-①B-① 2/252/25 8.08.0
B-②B-② 8/258/25 32.032.0
이상의 결과로 본 발명에 따라 제조된 사료의 급이와 본 발명에 따른 약욕제 처치가 무지개 송어 사육에서의 폐사율 감소에 효과가 있음을 확인할 수 있었다. 이로부터 본 발명의 조성물의 적용이 무지개 송어의 사양 결과 개선에 효과적이라는 결론을 내릴 수 있었다.As a result, it was confirmed that the feeding of the feed prepared according to the present invention and the bath treatment according to the present invention are effective in reducing mortality in rainbow trout breeding. From this it was concluded that the application of the composition of the present invention is effective in improving the specification results of rainbow trout.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
기탁기관명: KCTCDepositary Name: KCTC
수탁번호: KCTC 12814BPAccession number: KCTC 12814BP
수탁일자: 20150520Trust Date: 20150520
Figure PCTKR2016010957-appb-I000001
Figure PCTKR2016010957-appb-I000001

Claims (5)

  1. 자연으로부터 분리된 에로모나스 살모니시다 균을 특이적으로 사멸시킬 수 있는 능력을 갖는 서열번호 1로 표시되는 유전체를 갖는 것을 특징으로 하는 미오비리대 박테리오파지 Aer-SAP-1(수탁번호 KCTC 12814BP).Myobiridae bacteriophage Aer-SAP-1 (Accession No. KCTC 12814BP), characterized in that it has a genome represented by SEQ ID NO: 1 having the ability to specifically kill the E. monas salmonisid bacteria isolated from nature.
  2. 제1항의 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 에로모나스 살모니시다 균의 감염 방지 및 처치용 조성물.Claim 1 bacteriophage Aer-SAP-1 as an active ingredient, the composition for preventing and treating the infection of the E. monas salmonicida bacteria.
  3. 제2항에 있어서, 상기 조성물은 약욕제 또는 사료첨가제 제조 용도로 사용되는 것을 특징으로 하는 에로모나스 살모니시다 균의 감염 방지 및 처치용 조성물.The method of claim 2, wherein the composition is a composition for preventing and treating infection of Eromonas salmonicida bacteria, characterized in that it is used for manufacturing a bath or feed additive.
  4. 제2항 또는 제3항에 의한 박테리오파지 Aer-SAP-1을 유효성분으로 포함하는 조성물을 사람을 제외한 동물에 투여하는 단계를 포함하는, 에로모나스 살모니시다 균에 의한 감염을 방지 또는 처치하는 방법.A method for preventing or treating an infection caused by E. monas salmonisid, comprising administering to a non-human animal a composition comprising the bacteriophage Aer-SAP-1 according to claim 2 or 3 as an active ingredient. .
  5. 제4항에 있어서, 상기 조성물이 약욕제 또는 사료첨가제 용도로 사람을 제외한 동물에 투여되는 것을 특징으로 하는 에로모나스 살모니시다 균에 의한 감염을 방지 또는 처치하는 방법.The method according to claim 4, wherein the composition is administered to animals other than humans for use as a bath or feed additive.
PCT/KR2016/010957 2015-10-28 2016-09-30 Novel aeromonas salmonicida bacteriophage aer-sap-1, and use thereof for inhibiting aeromonas salmonicida proliferation WO2017073916A1 (en)

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