보고서 정보
주관연구기관 |
CTC바이오 CTC BIO CO., LTD. |
연구책임자 |
이재환
|
참여연구자 |
최대건
,
유승일
,
이수빈
,
박영준
,
박양순
,
김새롬
,
강봉조
,
전수연
,
전봉근
,
하현정
,
마은실
,
김문관
,
장영환
,
권안성
,
손지수
,
백형록
,
김종현
,
박석황
|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2016-06 |
과제시작연도 |
2015 |
주관부처 |
해양수산부 Ministry of Oceans and Fisheries |
등록번호 |
TRKO201800002347 |
과제고유번호 |
1525004789 |
사업명 |
수산실용화기술개발 |
DB 구축일자 |
2018-03-24
|
키워드 |
세균성 질병.양식.제어.천적.넙치.Bacterial disease.Aquaculture.Control.Natural predator.Paralichthys olivaceus.
|
DOI |
https://doi.org/10.23000/TRKO201800002347 |
초록
본 기술개발 사업 수행을 통해 넙치 양식에서 다발하는 세균성 질병의 예방에 유용한 박테리오파지들을 활용한 자연 친화적 제어 기술 및 이에 활용될 수 있는 제제를 개발하였음. 구체적으로, E. tarda, S. parauberis, S. iniae, V. anguillarum 등에 대응이 가능한 박테리오파지들을 확보하였으며, 이를 활용하여 넙치 양식장용 사료 첨가제인 벡터페이즈 A 제품을 개발하였음.
(출처 : 보고서 요약서 3p)
Abstract
▼
IV. Research Contents and Results
For this study, a development potential and product development of bacteriophage are prepared through interviews with many field experts and fish farmers, and cooperation of the host research organization and the first and second cooperative contractor to inv
IV. Research Contents and Results
For this study, a development potential and product development of bacteriophage are prepared through interviews with many field experts and fish farmers, and cooperation of the host research organization and the first and second cooperative contractor to investigate the usefulness of bacteriophage in bacterial diseases prevention and treatment of infections in the form of suitable formulations which can best be implemented.
According to the results of investigation, a major bacterial source of infection of Fish disease was a very high proportion of feed and farming water. Thus, a very important factor was preventing pollution through it because rare feed (MP) which accounts for about 80% is a large proportion of the total feed. The development of bacteriophage products applicable to the fishery industry was conducted based on the successful experience in development of bacteriophage-based product containing the bacteriophage which has a specific lytic activity against Salmonella Enteritidis.
With the help of the second partner institution (Jeju Ocean & Fisheries Research Institute), various bacterial strains to be used in screening of usable bacteriophages were collected. The target bacterial pathogens were bacteria causing Edwardsiellosis, Streptococcosis, or Vibriosis. Specifically, the target bacteria were Edwardsiella tarda, Streptococcus parauberis, Streptococcus iniae, Lactococcus garvieae, Vibrio anguillarum, and Vibrio ichthyoenteri.
For each target bacterial species, we collected more than 10 isolates and all isolates were identified by 16S rRNA sequencing. In addition, the optimal cultivating conditions were optimized for using them in the subsequent bacteriophage screening. We developed in vitro and in vivo efficacy evaluation methods. Specifically, overlay spot assay and top agar assay were developed as an in vitro efficacy evaluation method, and challenge test and field trial study were developed as an in vivo efficacy evaluation method.
We obtained lytic bacteriophage of Edwardsiella tarda and lytic bacteriophage of Streptococcus parauberis using the established methods in the first study year. In the second and the third study years, we isolated lytic bacteriophage of Vibrio anguillarum and lytic bacteriophage of Streptococcus iniae in the same way. Also, isolating usable bacteriophages from other cultivated fish species such as shrimp and salmon as well as flounder was conducted successfully. As a result, several usable bacteriophages were isolated.
For the isolated bacteriophages, isolation of pure monophage was performed. After isolation of pure monophage, development of optimized enrichment (proliferation) process was conducted for all pure isolated bacteriophages. The optimized enrichment processes for Streptococcus pararuberis bacteriophage Str-PAP-1, Edwardsiella tarda bacteriophage Edw-TAP-2, Streptococcus iniae bacteriophage Str-INP-1 and Vibrio anguillarum bacteriophage Vib-ANP-1 were developed. The conditions for the optimized enrichment processes were established by finding the best strain optimized for the proliferation process among the bacteriophage strains. The infection of bacteriophage was carried out for 1 hour with MOI = 0.01 and the highest bacteriophage titer was obtained when bacteriophage was multiplied for 4 hours. The enrichment process was scale-up to 50 L. For bacteriophage purification, two processes were developed based on PEG precipitation method and chromatography.
The morphological characteristics of the purely isolated bacteriophages were analyzed by Transmission Electron Microscope (TEM) anaysis and the genetic characteristics of the purely isolated bacteriophages were analyzed by the Next Generation Sequencing (NGS). In addition, we examined the acid tolerance, salt tolerance, freezing/thawing stability, and thermal stability. Generally, bacteriophages tested were stable over pH 3.0 and less than 50 °C, and exhibited very good salt tolerance. The freezing/thawing stability of bacteriophages were assessed by titration of viable bacteriophages after bacteriophages were frozen at - 20 °C for eight hours and thawed. As a result, it was confirmed to be stable with no change in titer. The safety of bacteriophages to the flounder were assessed using the immersion test and the dipping tank, etc. Bacteriophages tested were safe to fish.
By using a cocktail formulation mixed with 4-kinds of bacteriophages, anhydrous glucose, functional glucose and maltodextrin (MD) were selected as appropriate excipients for aquatic farming. Then, the prototype containing anhydrous glucose, BacterPhage A, was developed in the second study year. The BacterPhage A contained bacteriophages Str-PAP-1 and Edw-TAP-2. In addition, BacterPhage A plus was developed using bacteriophages Str-INP-1 and Vib-ANP-1, which were isolated in the second study year and the third study year.
Using this prototype (BacterPhage A), we conducted field trials. In this case, the prototype was applied to MP (moisture pellet) feed. In addition, for application to EP (Extruded Pellet), the “after coating” method was developed in cooperation with Jeju Federation of Fisheries Cooperatives. The field trials were conducted at the three test ports of 3 farms for 17 weeks. The mortality for 17-week test period between bacteriophage-supplemented group (test group) and normal feed fed group (control group) was examined. As a result, the percentage of the cumulative mortality in the test group was 10% lower than that of control group (control group: 25%; test group: 16-18%). It was confirmed that the cost of antibiotics used in farm decreased, which seemed to be replaced antibiotic with bacteriophage. By using the bacteriophage, it is anticipated that the antibiotic-relating problems will be reduced.
In conclusion, we developed the bacteriophage-based in-feed antibiotic alternative (BacterPhage A) using Streptococcus pararberis bacteriophage and Edwardsiella tarda bacteriophage that were developed in the second study year of this project. In the near future, we will develop the version-up product of BacterPhage A using Streptococcus iniae bacteriophage and Vibrio anguillarum bacteriophage that were isolated in the second or the third study year of this project.
(출처 : SUMMARY 9p)
목차 Contents
- 표지 ... 1
- 제출문 ... 2
- 보고서 요약서 ... 3
- 요 약 ... 4
- SUMMARY ... 8
- CONTENTS ... 12
- 목차 ... 13
- 제 1 장 연구개발과제의 개요 ... 14
- 제 2 장 국내외 기술개발 현황 ... 32
- 제 3 장 연구개발 수행 내용 및 결과 ... 39
- 제 1 절 박테리오파지 분리에 사용할 박테리아 확보 ... 39
- 제 2 절 박테리오파지의 유효성 및 안전성 평가법 개발 ... 45
- 제 3 절 넙치 세균성 어병 (에드워드병,비브리오병, 연쇄구균증) 제어용 유용 박테리오파지 확보 ... 52
- 제 4 절 분리 박테리오파지의 유효성 평가 ... 55
- 제 5 절 순수 분리 박테리오파지의 표준 증식공정 개발 ... 68
- 제 6 절 순수 분리 박테리오파지의 표준 정제공정 개발 ... 104
- 제 7 절 박테리오파지의 특성 규명 및 제품화 대상 박테리오파지의 선별 ... 108
- 제 8 절 박테리오파지의 원제 및 사료첨가제 시제품 제조 ... 127
- 제 9 절 박테리오파지의 안전성 평가 및 현장적용시험 ... 138
- 제 10 절 최종 시제품 개발 ... 164
- 제 4 장 목표달성도 및 성과 ... 167
- 제 5 장 연구개발결과의 활용계획 ... 176
- 제 6 장 참고문헌 ... 178
- 끝페이지 ... 182
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