초록 ▼

□ 과제명 : 사료 첨가용 생균제의 현장 활용증진 기술개발
▶ 연구목적
○ 항생제를 사용하지 않은 무항생제 축산물에 대한 소비자 수요에 대응
○ 농업기술센터에서 미생물 생산 및 농가 보급사업의 애로점 해결
○ 생균제 품질관리 규격 가이드라인 보완 및 생균제 현장적용 기술 개발
▶ 주요연구내용
○ 1세부과제명 : 농업기술센타용 생균제의 경제적 생산 및 이용기술 개발
- 농가보급용 저비용 배양 배지 개발
· 유산균 및 바실러스용(특허)
· 농업기술센터 지도사 교육에 활용
- 혼합미생

□ 과제명 : 사료 첨가용 생균제의 현장 활용증진 기술개발
▶ 연구목적
○ 항생제를 사용하지 않은 무항생제 축산물에 대한 소비자 수요에 대응
○ 농업기술센터에서 미생물 생산 및 농가 보급사업의 애로점 해결
○ 생균제 품질관리 규격 가이드라인 보완 및 생균제 현장적용 기술 개발
▶ 주요연구내용
○ 1세부과제명 : 농업기술센타용 생균제의 경제적 생산 및 이용기술 개발
- 농가보급용 저비용 배양 배지 개발
· 유산균 및 바실러스용(특허)
· 농업기술센터 지도사 교육에 활용
- 혼합미생물에서 유산균 선택 배지 개발
· 발효사료의 품질 측정에 활용
- 액상미생물 농가 보급기술
· 유산균, 바실러스, 효모 계절별 농가 유통기간 제시
- 미생물 원균 보존방법 및 기간 제시
- 미생물 활용기술 농가 보급을 위한 지도사 실습 교육 추진
· 95개소 농업기술센터 107명 지도사(2박 3일)
- 농가보급 액상 유산균 여름철 유통기간 연장
· 탄산나트륨 0.5% 첨가시 유통기간을 약 4 → 12일로 연장
- 농가보급 액상 미생물 3종 혼합유통시 미생물 생존기간
· 약 28일간 농가 유통 가능
- 농가형 발효사료 제조 및 품질 분석
· 유산균수 : 배합사료>옥분> 미강
· 유기산 : 미강> 배합사료>옥분
○ 1협동과제명 : 유통생균제의 유효검증 시스템 및 농가현장이용 기술개발
- 유통 생균제의 미생물 품질 확인
· 미생물 균 수 및 등록균주 확인
- 미생물 확인을 위한 방법 개선
· 기존 선택배지의 개선 및 분자생물학적 방법 개발
- 유통생균제의 저장성 강화 방법
· 규산염제의 활용
- 생균제의 효과검증을 위한 in vitro 방법 개발
- 생균제의 가축사료내 첨가량 가이드라인 조사
- 사일리지 제조시(호모+헤테로형 유산균 혼합)
· 수용성 탄수화물 생성 : 총체보리 > 이탈리안 라이그라스
· 휘발성지방산 생성 : 총체보리 > 이탈리안 라이그라스
· 병원성 미생물 억제제력 : 총체보리 > 이탈리안 라이그라스
○ 2협동과제명 : 축산전용 생균제 현장적용 기술개발
- 발효양파에서 유산균 선발
· 소화력효소, 항균, 항산화 활성 평가
※ L. plantarum SK1990(특허출원)
- 양파즙을 배지원으로 하는 선발 유산균 배양 방법 확립
· 양파즙 첨가 농도 설정
· 주요영양원 설정
※ 최적배지결정 및 배양 공정 최적화 확립 (특허출원)
- 육계의 생산성 향상 및 항생제 대체 효과 : 양파발효유산균 0.5% 첨가
- 자돈의 생산성 향상 및 항생제 대체 효과 : 양파발효유산균 0.1%첨가

Abstract ▼

The study of the first paragraph was conducted to develop economical optimum medium composition for the mass production of Lactobacillus plantarum and Lactobacillus reuteri, a probiotics for livestock. Medium ingredients factors were selected with the basis of MRS broth (Difco) composition and total

The study of the first paragraph was conducted to develop economical optimum medium composition for the mass production of Lactobacillus plantarum and Lactobacillus reuteri, a probiotics for livestock. Medium ingredients factors were selected with the basis of MRS broth (Difco) composition and total 15 ingredient variables were as following: sucrose, glucose, molasses, yeast extract, corn steep liquor, soy peptone, dipotassium phosphate, manganese chloride, magnesium chloride, tween 80, sodium chloride, sodium acetate, ammonium citrate, sodium sulphate and ferrous sulphate, Plackett Burman design consisted of 20 runs was employed for the analysis of ingredient effects on cell growth of L. plantarum and L. reuteri. As a result, sucrose, glucose, molasses, yeast extract, corn steep liquor, soy peptone, sodium acetate and ammonium citrate showed positive effect on the growth of L. plantarum. In the growth of L. reuteri, yeast extract, soy peptone, K₂PHO₄ and tween 80 showed positive effect. Positive effects were found from sucrose, yeast extract and soy peptone in the integrated analysis of effects of was formulated ad following: sucrose(200 g/L), glucose(5.0 g/L), soy peptone(11.0/L), yeast extract(5.0 g/L), K₂PHO₄(0.2 g/L), CH₃COONa(2 g/L) and MgCl₂(0.02 g/L).
The second paragraph is the studies of validation system of commerical probiotics and application in livestocks farm. First study was performed to measure substantial differences probitics information marked on commercial probiotics by examining qualities of microbial products with selective media and molecular biological methods. For the experiment, 11 commercial probiotics (6 probiotics and 5 fermented milks) were purchased and lactic acid bacterium selective media and specific primers were made. The number of lactic acid bacteria was measured on Lactobacillus anaerobic MRS agar with vancomycin and bromocresol green (LAMVAB), acidified de Man, Rogosa and Sharpe agar with triphenyl tetrazolim chloride (AMRSA). For exact determination of probiotics, polymerase chain reaction (PCR)-based method used 16S universal primer and lactobacilli genus specific primer mixtures and determined the lactic acid bacteria. For the result of the experiment, the number of lactic acid bacteria in commercial probiotics as well as fermented milks was lower than what they said to have. As a result of Lactobacilli specific primer PCR analysis, 5 probiotics and 3 fermented milks formed the band, thus the existence of lactic acid bacteria and difference of lactobacilli groups in the products were confirmed. The remaining 1 probiotics and 2 fermented milks didn' t show any band, which means lactic acid bacteria that are supposed be were not there. This study have a potential to provide molecular biological guidelines for quality control of probiotics and can be used for base data of animal supplementary feeds and fermented milk. Quantitative Real-time PCR was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from10₂ to 10₁₁. A standard curve of plasmid DNA copy number also found using the real time PCR.
The slope value, Y-axis intercept and R2 value were -3.346, 33.18 and 0.993, respectively in the first method. For second method, the slope value, Y-axis intercept and R2 were -3.321, 31.10 and 0.995, respectively. PCR inhibitor couldn' t express the detection curve over 108 copy number with much early DNA in both of the method. DNA extract from probiotics had diluted without pre-culture and 16 products have amplified in both of the methods.
The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method.
Measured probiotics value and log copies values were shown a little distinction among the methods. Finally it is decided that both of the method has some good impact for analysis, but furthermore research is needed to confirm the best method. Total mixed ration(TMR) and fermented feed has been used with a great interested by farmers. In order to increase the feed quality on inoculated probiotics we conducted to determine the quality of Italian rye grass (IRG) and whole crop barley (WCB) with addition of homofermentative and heterofermentative LAB during fermentation. Three strains of homofermentative LAB (L. plantarum, L. acidophilus, and L. casei) and heterofermentative LAB (L. reuteri, L. buchneri and L. diolivorans) were used in this study. L. casei and L. reuteri had the highest propionic acid production, thus they were used in fermenting the forages. The forages were fermented using monoculture and co-culture of L. casei and L. reuteri for 5, 15, 30, 45 and 60 days ensiling. Addition of LAB lowers the pH of the silages but as the incubation time becomes longer, pH also becomes higher. Percent moisture, ammonia nitrogen (NH₃-N), lactic and acetic acid is comparatively higher in IRG than WCB. Percent NDF is higher in WCB than IRG. Propionic acid production was detected at days 30, 45 and 60 of WCB while butyric acid was detected only in IRG control at days 30, 45, and 60. Lactobacilli count was significantly lower on day 45 using IRG. Water soluble carbohydrate was significantly higher in WCB than IRG on the entire days of ensiling.
This study is still on going. Co-culture of L. casei and L. reuteri is the best culture for fermentation of forage while WCB has higher water soluble carbohydrates and produces higher propionic acid than IRG. For feed purposes, probiotics are applied as additives to by-products feed, protein concentrates, etc. The majority of the scientific experiments in the literature had been performed with pig and ruminants. Probiotics microorganisms are recommended at concentrations of 10⁸-10⁹ CFU/kg feed. However, depending on the category and age of the animal, the application rate can range widely.
The study of the third paragraph is a development of technology for the improvement and application of animal probiotics. L. plantarum was selected as an excellent probiotic based on antioxidant activity and antimicrobial activity against animal pathogenic bacteria.
To develop economical animal probiotics with L plantarum. Domestic onions to be chiefly produced were used as medium ingredient of L. plantarum. The addition levels of onion juice were investigated on the growth yield of L. plantarum. The levels were increased from 20% to 60% with fixing MRS to 20% of medium. The viable cells of L. planatrum were increased depending on the addition levels of onion juice in medium. In growth curve, inhibitory effect caused by addition of onion were not found in run 3 (MRS 20%, onion juice 20%), run 4 (MRS 20%, onion juice 40%) and run 5 (MRS 20%, onion juice 60%) compared to that in run 2 (MRS 20%, onion juice 0%). From the results of the investigation of fermentation profiles using 5 L fermenter, new medium was composed of 600 g/L of onion juice, 15 g/L of sucrose, 5 g/L of yeast extract, 0.1 g/L of K₂HPO₄,0.01g/LMgCl₂ and 1 g/L of CH3COONa. Finally new feed additive was developed by the antibacterial activity of L. plantarum and antioxidant activity of onion. In feeding trial of fermented onion juice to broiler, there was no significant difference among the experimental groups in feed intake, body weight, weight gain, feed efficiency and feed conversion rate. The performance of group fed 0.5% fermented onion was similar to antibiotic fed group. In large intestine and other organs, carcass characteristics, performance index and meat quality, no significant effect was found. The carcass characteristic of 0.5% fermented onion fed group showed similar to that of antibiotic fed group. In blood analysis, 0.5% fermented onion fed group showed significantly higher GPT values than that of antibiotic fed group. As a result, it was suggested that fermented onion can improve the productivity of broiler as alternative antibiotics. Particularly, decreased yellowness in color of breast meat compared to antibiotic fed group showed the possibility of chicken meat with low fat content. In the investigation of effect of fermented onion on the performance of weaned piglet, there was no significant effect in feed intake. However, the group fed 0.1% fermented onion showed increased performance similar to that of antibiotic fed group. Average daily gain in the group fed 0.1% fermented onion was significantly higher than antibiotic fed group but similar feed conversion ratio, suggesting the possibility of alternative antibiotics. In the analysis of feces bacteria, the group fed 0.5% fermented onion showed decreased E. coli counts in second week of trials and the highest counts of Lactobacilli in fourth week of trails compared to the control. The fermented onion juice using isolated L. plantarum showed a good potential of livestock feed additive.

목차 Contents

• 제 출 문 ... 1
• 요 약 문 ... 2
• SUMMARY ... 3
• 제1장 서 론 ... 6
• 제1절 연구개발의 필요성 ... 6
• 제2장 국내외 기술개발 현황 ... 6
• 제1절 국내 기술동향 ... 6
• 제2절 국외 기술동향 ... 7
• 제3장 연구개발수행 내용 및 결과 ... 8
• 제1절 농업기술센터용 생균제의 경제적 생산 및 이용기술 개발 ... 8
• 제2절 유통생균제의 유효접종 시스템 및 농가현장이용 기술개발 ... 19
• 제3절 축산전용 생균제 현장적용 기술개발 ... 39
• 제4장 연구개발목표 달성도 및 대외기여도 ... 68
• 제1절 목표대비 달성도 ... 68
• 제2절 정량적 성과 ... 69
• 제5장 연구개발결과의 활용계획 ... 73
• 제6장 중요 변동사항 ... 73
• 제7장 참고문헌 ... 74
• 사료 첨가용 생균제의 현장 활용증진 기술개발 ... 78
• 주요 결과 요약서 ... 79