초록 ▼

○ 연구결과
본 연구를 통하여 섬유소 분해 활성이 우수한 미생물 균주를 선발하고 이를 이용하여 고상발효 미생물 제제를 생산하였다.생산된 제품의 특성평가 및 동물실험 결과에 의하면 특히 단위동물에서 기존 효소제와 비교하여 증체량,사료효율,에너지 이용효율에서 보다 긍정적인 효과를 나타내었음을 검증하였다.이로써 섬유소 분해 촉진을 통한 소화계 질병예방 및 환경개선을 통한 생산비용 절감 및 농가소득증대를 꾀할 수 있고,항생제 대체제 시장의 확대를 도모하고 사료용 미생물제제 시장의 선점을 통한 수출증대효과를 볼 수 있을 것으로 기대된다

○ 연구결과
본 연구를 통하여 섬유소 분해 활성이 우수한 미생물 균주를 선발하고 이를 이용하여 고상발효 미생물 제제를 생산하였다.생산된 제품의 특성평가 및 동물실험 결과에 의하면 특히 단위동물에서 기존 효소제와 비교하여 증체량,사료효율,에너지 이용효율에서 보다 긍정적인 효과를 나타내었음을 검증하였다.이로써 섬유소 분해 촉진을 통한 소화계 질병예방 및 환경개선을 통한 생산비용 절감 및 농가소득증대를 꾀할 수 있고,항생제 대체제 시장의 확대를 도모하고 사료용 미생물제제 시장의 선점을 통한 수출증대효과를 볼 수 있을 것으로 기대된다.

Abstract ▼

Ⅳ. Research and development results
1. Development of excellent cellulolytic microorganims
a. Establishment of microbial selection methods
- Among the several methods such filter paper degradation, congo red staining, lysis of chromogenic cellulose and agar plates with dyed-cellulose, the l

Ⅳ. Research and development results
1. Development of excellent cellulolytic microorganims
a. Establishment of microbial selection methods
- Among the several methods such filter paper degradation, congo red staining, lysis of chromogenic cellulose and agar plates with dyed-cellulose, the last one was chosen as most efficient and reliable method to screen out cellulolytic microorganisms.
b. Selection of excellent cellulolytic microorganism and its identification
- Using the prepared screening plates, we isolated 38 microorganisms from rumen and 10 microorganisms from fermented foods and stored frozen. The microorganism which was isolated from rumen was identified as Bacillus licheniformis, and the microorganism which was isolated from fermented food was Bacillus subtilis.
C. Research on Incresing cellulolytic activity and its efficiency
- In the 1st year studies, we selected two microorganisms (Bacillus subtilis, Bacillus licheniformis) and to enhance enzyme activity of those two microbes, we used the traditional mutation method (U.V. irradiation, EMS treatment) and genome shuffling.
The enzyme activity was increased by 25% for P11 which was treated with EMS, 100% for GS2 which was mutated by genome shuffling method.
d. Selection of cellulolytic gene
- We selected endoglucanse gene (Cel5) from cellulolytic bacteria, Eubacterium cellulosolvens which was ruminal microorganism isolated during the 1st year research.
And the selected endoglucanase gene was transformed into the E. coli and confirmed ints enzyme activity.
e. Development of microorganism with improved cellulolytic activity.
- The enzyme activity of selected Bacillus sp. #6 was 0.04U/ml and activity of avicelase and xylanase was negligible. Optimal pH and temperature was pH 5 and 50℃, respectively and CMCase enzyme activiety was 0.072U/ml at optimum condition.
- EMS-treated P11 was selected as the best mutant by screening with trypan blue plate.
The mutants P11's CMCase enzyme activity was 0.05U/ml at the condition of pH 6.6 and 37℃. And the optimal pH and temperature was pH 5 and 50℃, respectively.
f. Characterization for solid-state fermentation
- Optimal fermentation time for Bacillus sp. #6 was 72 hour and optimal substrate-to-water ratio was 1:1.
- When added 20% Bacillus sp. #6 to the forage, the dry matter digestibility was improved by 25.8%.
g. Test of efficacy of final developed microbial feed additives through animal experiment (in vitro, in situ, in vivo)
- The results of in vitro digestibility experiment showed significant difference between control and treatment with Bacillus #6 (P<0.05). There was also large difference in VFA production, which is believed due to increased A fraction of CHO and CP in the substrate during fermentation with the microbial additive treatment.
- There was significant difference in the in situ dry matter disappearance rate between the control group and Bacillus sp. #6 treatment group during the 9 hour to 48 hour incubation. Similar pattern was also observed in the protein disappearance rate with more than 20% differences (P<0.05).
- Judging from the amount of DNA isolated from in vitro rumen samples, the celluloytic bacteria and methanogen population was decreased.
- It seemed that the microbial product had bile acid tolerance at lower than 0.05% oxgall.
Bacillus sp. #6 has some degree of resistance compared with other probiotics such as L. bulgaricus and L. lactis which are sensitive at the low level of bile acid.
- When we checked the acid tolerance at the pH 2.5, and pH 3, the CFU at the acidic condition show similar compared with pH 5 which was optimal condition for exzyme activity of microbial feed additive.
- Supplementation of Bacillus sp.#6 did not gine any significant influences on apparent digestibility and nitrogen balance. Rumen fermentation and microbial population in the ruminal fluid and feces were not affected by the treatment with microbial feed additive.
- To evaluate the effect of microbial feed additive on the monogastric animal, we conducted feeding trial with broilers. And the results showed that the group which was treated with 0.1% fermentation enzyme had significant effect on body weight gain (BWG), feed conversion rate, true metabolize energy (TME) and its efficiency.
2. Development of production technique of microbial feed additives containing high cellulase activity through the solid-state fermentation.
a. Establishment the microbial fermentation process
- Through the comparison of enzyme activity of Aspergillus oryzae, Aspergillus niger and trichoderma reesei after liquid and solid state fermentation, the enzyme activity of solid state fermentation was higher than liquid one. And wheat bran with high carbon source (C/N ratio: 20) had high degree of enzyme secretion.
b. Research of dietary fiber content of each feed
- The total NSP content of the cell membrane was high in the wheat bran which had high crude fiber content, and the content of pentosan and hemicelluloses was also high.
In the soybean meal, total content of NSP was lowest and pentosan content was relatively high. And in the case of corn, The hemicellulose content was high.
- For the efficient cellulase production, wheat bran was selected as optimal substrate for solid-state fermentation because of its high content of cellulose and hemicellulose.
c. Establishment of enzyme activity assay
- Three assay methods having different procedures for detecting reducing sugars were compared to find best method.
- The assay which use disodium hydrogen phosphate-citric acid buffer and DNS solution was chosen for the standard method in our project, because of its ability to express the prepared samples' enzyme activity.
d. Development of solid-state fermentation process
- From the resutls of fermentation according to the type of substrate with Bacills sp. #6 and mutant P11 and B. licheniformis G7 isolated from the rumen, SW media (soybean meal + wheat bran, 6:4 and C/N ratio= 10) was chosen for the coptimal media composition.
- The optimum level of carbon sources such as cellulose, cellobise and lactose was about 5%.
- The cellulase activity was increased by 50% when urea was added as nitrogen source.
- In the form of fermentation type, at the aerobic fermentation condition, mutant P11 had higher activity, and Bacillus sp. #6 was proper conditate for anaerobic fermentation condition and had more than 30% higher activity than mutant P11.
- Although the optimal water content was determined as 65%, we chose 50% because of the problems in mass production.
- For the determination of optimal fermentation condition, 40℃ for mutant P11, 35℃ for Bacillus sp. #6, and other factors (amount of inoculums = 10⁶CFU/g, more than pH 5, fermentation for 60 hour) was applied for the test conditons of pilot scale production.
f. Characterizaiton of selected microorganism
- There was little effect on the number of mutant P11 even at 100℃ for 10min. On the other hand, the enzyme activity was stable after treatment with 60℃ for 10min.
However after treatment at higher than 70℃, loss of enzyme activity was detected.
And after treatment with 100℃ for 10 min, 50% level of enzyme activity was observed.
g. Establishment of mass production process
- Through the result of conducting preliminary research with each selected microorganisms for the mass production, B. subtilis #6 was finally chosen as condidate because of its maintenance of high cellulase activity at about 1,500 U/g.
- The fermentation of B. subtilis #6 alone had higher enzyme activity than fermentation together with B. licheniformis G7.
- Optimal fermentation time was 42 hours in subsequent studies so that in the mass production, fermentation time will be able to be shortened from 60 hours to 42~45 hours.
- Sufficient aeration was an essential culture condition for B. subtilis #6 for the production of cellulase.
h. Test of developed product's stability
- Though the cellulase activity was approximately 87% compared to the initial level, the number of microorganism was maintained about 10⁹ cfu/g after 6 months of storage.
ｉ. Comparison with other commercial products
- Bacillus sp. #6 produced by poilot scale processing had more than 80% higher enzyme activity compared with other probiotic samples. And in terms of microbial population of Bacillus sp. #6, the degree 4.0×10⁹CFU/g was absolutely high. So, solid-state fermentation product of Bacillus sp.#6 is a far superior product than other products.

목차 Contents

• 표지 ... 1
• 제출문 ... 2
• 요약문 ... 3
• Summary(영문요약문) ... 10
• CONTENTS(영문목차) ... 17
• 목차 ... 20
• 제1장. 연구개발과제의 개요 ... 23
• 제2장. 국내외 기술개발 현황 ... 24
• 제1절. 본 연구관련 국내외 기술수준 비교 ... 24
• 제2절. 특허분석 ... 24
• 제1항. 특허분석 범위 ... 24
• 제2항. 특허분석에 따른 본 연구과제와의 관련성 ... 25
• 제3절. 논문분석 ... 26
• 제1항. 논문분석 범위 ... 26
• 제2항. 논문분석에 따른 본 연구과제와의 관련성 ... 26
• 제4절. 제품 및 시장 분석 ... 27
• 제1항. 생산 및 시장현황 ... 27
• 제3장. 연구개발 수행 내용 및 결과 ... 28
• 제1절. 미생물 선발방법 확립 ... 28
• 제1항. Observation ... 28
• 제2항. Congo-red staining ... 28
• 제3항. Chromogenic cellulose method ... 28
• 제2절. 섬유소 분해 활성 우수 미생물 선발 ... 29
• 제1항. 반추동물의 반추위에서 섬유소분해 활성이 우수한 박테리아 선발 ... 29
• 제2항. 식품군에 존재하는 섬유소 분해 활성 우수균주 선발 ... 32
• 제3절. 미생물 발효 방법에 따른 효소발현 특성 ... 35
• 제1항. 액상발효 및 고상발효를 통한 효소활성 비교 ... 35
• 제4절. 원료별 dietary fiber분획조사 및 고상발효용 배지선정 ... 38
• 제1항. 원료별 일반성분 및 dietary fiber분획 조사 ... 38
• 제2항. 미생물 발효 및 대사물질 생산에 최적인 곡물배지조성 선정 ... 39
• 제5절. 효소활성 분석법 확립 ... 41
• 제1항. Cellulase 분석법-1:사료공정서 분석법 ... 41
• 제2항. Cellulase 분석법 2 ... 42
• 제3항. Cellulase 분석법 3 ... 44
• 제4항. 효소활성 분석결과 ... 46
• 제6절. 섬유소 분해 활성 증가 미생물 개발 ... 47
• 제1항. 실험 목표 ... 47
• 제2항. 실험 재료 및 방법 ... 47
• 제3항. 결과 및 고찰 ... 50
• 제7절. 섬유소 분해효소 유전자 선발 ... 57
• 제1항. 반추동물의 반추위로부터 섬유소 분해 균주선발 ... 57
• 제2항. 섬유소 분해효소 유전자 분리 및 분석 ... 60
• 제8절. 효소활성이 개선된 형질전환 균주 개발 ... 62
• 제1항. 사용균주 및 plasmid ... 62
• 제2항. Cellulase gene cloning 및 형질전환 균주의 확인 ... 63
• 제3항. CMCase 활성 측정 ... 63
• 제4항. DNA 분리와 조작 ... 63
• 제5항. 형질전환 균주 확인 및 효소역가 확인 ... 63
• 제6항. 균주 활용방안 ... 66
• 제9절. 고상발효를 위한 최종 미생물 선발 및 특성 평가 ... 66
• 제1항. 섬유소 분해력이 높은 자연계 미생물 선발 및 동정 ... 66
• 제2항. 고전적 mutation을 이용한 우수 섬유소 분해 균주 개발 ... 68
• 제3항. 선발된 균주의 섬유소 분해 특성 조사 ... 69
• 제4항. 고상발효를 통한 섬유소 분해 효소의 생산 ... 74
• 제10절. 선발균주별 pilot scale 공정 개발을 위한 기초자료 조사 ... 79
• 제1항. C/N ratio에 따른 선발균주의 고체발효 특성 ... 79
• 제2항. 탄소원의 종류에 따른 고체발효 특성 ... 82
• 제3항. Mineral 첨가에 따른 영향 ... 86
• 제4항. 질소원의 종류에 따른 발효특성 ... 86
• 제5항. 탄소원 및 질소원의 동시 처리가 효소활성에 미치는 영향 ... 91
• 제6항. 발효형태에 따른 효소활성 변화 ... 91
• 제7항. 고체배지의 초기 수분함량에 따른 발효특성 ... 93
• 제8항. 발효 온도에 따른 영향 ... 94
• 제9항. 종균 접종량에 따른 효소활성 변화 ... 95
• 제10항. 초기 pH에 따른 영향 조사 ... 96
• 제11항. 최적 발효시간 설정 ... 96
• 제12항. 최적 고체발효조건 결정 ... 97
• 제11절. 최적조건에서의 pilotscale발효특성 평가 ... 98
• 제1항. 액상종균의 발효조건 결정 ... 98
• 제2항. 최적 발효조건에서의 발효특성 평가 ... 100
• 제3항. 경쟁제품 비교평가 ... 101
• 제12절. 선발 미생물의 특성평가 ... 103
• 제1항. 내열성 평가 ... 103
• 제2항. 제품 저장성 테스트 ... 103
• 제13절. 대량 고체발효 공정 확립 ... 104
• 제1항. 실험 목표 ... 104
• 제2항. 재료 및 방법 ... 104
• 제3항. 결과 ... 106
• 제4항. 고찰 ... 109
• 제14절. 저장안정성 평가 ... 109
• 제1항. 목적 ... 109
• 제2항. 재료 및 방법 ... 110
• 제3항. 결과 ... 110
• 제15절. 고상발효 미생물제제의 효능평가 ... 111
• 제1항. 실험 목표 ... 111
• 제2항. 실험 재료 및 방법 ... 111
• 제3항. 결과 ... 114
• 제4항. 고찰 ... 121
• 제16절. 고상발효 미생물 제제의 반추동물 대사 실험 ... 123
• 제1항. 실험 목표 ... 123
• 제2항. 실험 재료 및 방법 ... 123
• 제3항. 결과 ... 125
• 제4항. 고찰 ... 131
• 제17절. 단위동물 사양실험을 통한 고상발효제제의 효과 평가 ... 132
• 제1항. 실험 목표 ... 132
• 제2항. 실험 1:섬유소 분해활성이 우수한 미생물제제의 급여가 육계의 성장,도체중 및 장내균총에 미치는 영향 ... 133
• 제3항. 실험 2:섬유소 분해활성이 우수한 미생물제제의 강제급여가 소화율에 미치는 영향 측정 ... 135
• 제4항. 고찰 ... 138
• 제4장 목표달성도 및 관련분야에의 기여도 ... 139
• 제5장. 연구개발 성과 및 성과활용 계획 ... 141
• 제1절. 개발기술의 산업화 방향 및 기대효과 ... 141
• 제2절. 특허출원 내용 ... 141
• 제3절. 과제기간 중 논문실적 ... 142
• 제4절. 제품 및 시장분석 측면 ... 144
• 제6장. 연구개발과정에서 수집한 해외과학기술정보 ... 145
• 제1절. 균주의 활성증가 방안 탐색 및 활용 ... 145
• 제2절. 액상발효 및 고상발효 공정 비교 ... 146
• 제7장 참고문헌 ... 149
• 끝페이지 ... 156