초록 ▼

○ 연구결과
목장원유와 신생아 및 성인에서 분변을 채취하여 1054개의 젖산균을 분리하였고, 37℃에서 18시간과 24시간 배양하여 응고된 균주가 각각 645개 균주와 682개 균주이었다. 면역증진 균주를 선발하기 위하여 반응성 질소종 분비능과 IL-1α 및 TNF시험을 거쳐 13종의 젖산균을 선발하였다. 선발된 젖산균을 현미경관찰, Gram stain, 당 발효실험 및 16S rRNA 염기서열 등의 일련의 생화학적, 유전학적 실험을 통하여 균을 동정하였다. 산 생성 속도와 면역활성능을 감안하여 5종의 젖산균을 선발하여, 모짜

○ 연구결과
목장원유와 신생아 및 성인에서 분변을 채취하여 1054개의 젖산균을 분리하였고, 37℃에서 18시간과 24시간 배양하여 응고된 균주가 각각 645개 균주와 682개 균주이었다. 면역증진 균주를 선발하기 위하여 반응성 질소종 분비능과 IL-1α 및 TNF시험을 거쳐 13종의 젖산균을 선발하였다. 선발된 젖산균을 현미경관찰, Gram stain, 당 발효실험 및 16S rRNA 염기서열 등의 일련의 생화학적, 유전학적 실험을 통하여 균을 동정하였다. 산 생성 속도와 면역활성능을 감안하여 5종의 젖산균을 선발하여, 모짜렐라 치즈의 종균으로서 적합한지의 여부를 검증하기 위하여 젖산균의 생장, 항생제 15종에 대한 내성시험, API ZYM kit를 이용한 효소활성 시험, 0.3% oxgall 첨가 배지에서의 담즙내성 시험, pH 내성시험, 식중독균에 대한 항균력 시험을 실시하였다.
한편, Vancomycin에 내성이 높아 위해논란이 있는 E. faecalis 683 균주를 배제하고 4종의 균주를 대상으로 배양온도 별(30℃, 35℃, 40℃), 균주의 접종량 별(0.1%, 0.5%, 1.0%, 2.0%)로 제조시간, 가공적성, 면역활성 및 수율 등을 감안하여 최적조건을 설정하였다. 이 중 가장 우수한 S. macedonicus 743 균주를 이용하여 제조공정을 설정하였고, 여기서 얻어진 모짜렐라 치즈가 국내 및 수입치즈와의 면역활성을 비교한 결과 우수한 것으로 나타났다. 이화학적 특성과 동물모델을 이용한 모짜렐라 치즈의 면역증진 효능평가 실험을 실시한 결과 S. macedonicus 743 균주로 제조된 모짜렐라 치즈의 경구투여는 복강 마크로파지의 활성에 관여하여 생체 내에서 면역기능을 증가시킬 수 있는 가능성이 있는 것으로 나타났다.

Abstract ▼

IV. Result of research and suggestion of application
1. Collection and isolation of lactic acid bacteria
a. Isolation of lactic acid bacteria from raw milk
Raw milk was collected from several farms under support of Raw milk testing lab. within the jurisdiction of Central, North, Western gui

IV. Result of research and suggestion of application
1. Collection and isolation of lactic acid bacteria
a. Isolation of lactic acid bacteria from raw milk
Raw milk was collected from several farms under support of Raw milk testing lab. within the jurisdiction of Central, North, Western guidance division (part of Kyoung Gi Do and Gang Won Do)in Seoul Dairy Cooperation, Provincial institute for livestock promotion(Jeonbuk, Kyoungbuk, Jeju). 1054 bacteria were isolated from modified MRS media. 598 bacteria were further isolated after the microbes were innoculated on 10% the reconstituted skim milk followed by incubation at 37℃ for 24hr.
b. Isolation of lactic acid bacteria from infant feces
45 new-born infant feces were collected under support of Soon Chun Hyang Hospital and Hangang Sungshim Hospital. 67 bifidobacteria were isolated and 42 coagulated lactic acid bacteria were isolated after incubation at 37℃ for 18 and 24hr.
c. Isolation of lactic acid bacteria from adults feces 50 adult feces were collected from staffs and their family members of KFRI.
91 bifidobacteria and 87 lactic acid bacteria were isolated. 42 coagulated lactic acid bacteria were isolated after incubation at 37℃ for 18 and 24hr.
d. Collection of lactic acid bacteria having milk coagulation capacity 645 and 682 bacteria were further isolated after the microbes were inoculated on 10% the reconstituted skim milk followed by incubation at 37℃ for 18hr and 24hr, respectively.
2. Selection of lactic acid bacteria having immunomodulating activity
Microbes possessing IL-1α secreting activity over 500pg/ml counted 285, and those over 2500pg/ml counted 245. Microbes producing nitric oxide over 10uM counted 19, while those over 40uM counted 1. Microbes possessing TNF secreting activity over 1000pg/ml counted 151, while those over 2000pg/ml counted 146. 13 microbes were selected lastly.
3. Identification of lactic acid bacteria
Identification of lactic acid bacteria having immunomodulating activity were Lactobacillus plantarum 4 strains, Lactobacillus fermentum 2 strains, Lactobacillus acidophilus 1 strains, Lactobacillus sakei 1 strain, Streptococcus thermophilus 1 strains, Enterococcus faecalis 2 strains, Streptococcus salivarius 1 strain, Streptococcus macedonicus 1 strain as a result of Gram staining, fractography, sporogenesis, aerobic and anaerobic growth, catalase formation, growth in 15℃ and 45℃, gas formation from glucose, ammonia formation from arginine, sugar fermentation test and 16S rRNA sequencing.
4. Characteristics of selected lactic acid bacteria
Among 13 species of lactic bacteria with immuno modulating activity selected from the 1st year study, 5 species of lactic bacteria(Lactobacillus fermentum 450, Streptococcus salivarius O39, Enterococcus faecalis 683, Streptococcus macedonicus 743, Lactobacillus fermentum 782) was selected regarding acid producing speed and immunomodulating activity and then investigated the possibility of starter culture for the production of Mozzarella cheese.
The optimum temperature of 4 kinds of lactic acid bacteria and S salivarius O39 was 40℃ and 37℃, respectively. E. faecalis 683 has comparatively strong antibiotic resistance but S. salivarius O39 and L. fermentum 782 has weak antibiotic resistance. It was possible to understand the safety of selected 5 lactic bacteria by the confirmation of no β-glucuronidase activity transferring benzopyrene to calcinogen. Among 5 lactic bactria, S. salivarius O39 and E. faecalis 683 has shown higher bile juice resistance than L. fermentum 782, S. macedonicus 743, L. fermentum 782. S. salivarius O39, E. faecalis 683 and L. fermentum 782 has shown higher pH resistance than L. fermentum 450 and S.macedonicus 743. on the other hand, S. macedonicus 743 has shown weak pH resistance but strongest antibiotic resistance.
5. Optimization of cultivation condition for the Mozzarella cheese production Optimum temperature(30℃, 35℃, 40℃) and dorsage(0.1%, 0.5%, 1.0%, 2.0%) of L. fermentum 450 and S. salivarius O39 has studies with the removal of E. faecalis 683 of high resistancre on the Vancomycin. In case of cheese making with the stasrter culture of L. fermentum 450, incubation at 40℃ with 1～2% addition or 30℃ with 2.0% addition was the best condition for the process control, immuno activity and yield. In case of cheese making with the starter culture of S. salivarius O39 , incubation at 35℃ with 0.1～1% addition was the best for the process control and cheese yield on the other hand incubation at 30℃ with 2.0% addition was the best for the immuno activity.
In case of cheese making with the starter culture of S. macedonicus 743, incubation at 32℃ with 2% addition was the best for the process control and immunomodulating activity with minimum processing time. on the other hand cheese making with the starter culture of L. fermentum 782, incubation at 35℃ with 2% addition was the best for the process control, immunomodulating activity and cheese yield.
6. Designing of cheese making process
2% of S. macedonicus 743 starter culture was added on the 32℃ milk after pasteurization at 63℃ 30 minutes or 72～73℃, 15 seconds and agitated for 4～5 minutes after addition of 230㎕/Kg of rennet. Curd was made by the setting the milk 25～35 minutes after addition of 0.01～0.02% Calcium chloride. Cutting the curd was done at 0.1～0.12% acidity(81 minutes later) and heating the cheese vat up to 43℃. Whey was removed at the acidity of 0.17～0.18% by the agitation for 53 minutes and continue to cheddaring for 210 minutes up to the acidity of 0.6～0.65% followed by the stretching at 72～75℃ and moulding at 65～70℃ and then cool down to 5℃. Salting was done with the solution of 18～20 Brix at 12℃ for 20 minutes and drying condition was 80-90% RH at 10℃ for 2～3 day.
7. Physico-chemical characteristics
Based on the comparison of immunomudulating activity of Mozzarella cheese made of S. macedonicus 743 starter and commercial starter, S. macedonicus 743 starter added cheese was higher than commercial starter cheese. And Mozzarella cheese made of mixed starer of S. macedonicus 743 and commercial starter has shown excellent immunomodulating activity. Mozzarella cheese made of S. macedonicus 743 starter has revealed higher protein and total amino acids and low fat contents than that of commercial starter. And Mozzarella cheese made of S. macedonicus 743 starter has revealed higher non protein nitrogen but lower water soluble nitrogen, amino nitrogen, organic acids and yield than that of commercial starter.
8. Animal experiments
We investigated the immunomodulatory action of Streptococcus macedonicus 743 in male C57BL/6 mice. Mice were treated with Mozarella Cheese added three kinds of fed with S. macedonicus 743, FD-DVS TCC-3 and S. macedonicus 743 : FD-DVS TCC-3(1:1) for 7 weeks, respectively. This study was performed to investigate the immunomodulative effects of S. macedonicus 743 in mice, using in vivo experiments. Three kinds of cytokines (IL-1β, IL-6, TNF-α) production by peritoneal machrophages cultured with experimental fed was orally administrated into mice, and isolated macrophages were used as experimental model. In vivo experiment, six weeks old mice were fed. macedonicus 743, FD-DVS TCC-3 and S. macedonicus 743 : FD-DVS
TCC-3(1:1) was orally administrated everyday for seven weeks. For the basic data, body weight and physiological parameters such as organ weight and spleen index were observed. No significant differences of body gain weight were recorded in various groups of animals. The production of cytokine (IL-1β, IL-6, TNF-α) which was secreted by macrophages was detected by ELISA assay using the cytokine kit. S. macedonicus 743 group significantly increased the production of TNF-α compared to the control group. The IL-1β production was more significantly enhanced the threated with S. macedonicus 743 group than skim milk group.
The result of this study may suggest that the supplementation of S. macedonicus 743 can regulating the cytokine production capacity by the activated macrophages in mice. As reported in this study, S. macedonicus 743 showed a stimulatory effect on immune functions in mice.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 9
• CONTENTS ... 14
• 목차 ... 15
• 제 1 장 연구개발과제의 개요 ... 16
• 제 2 장 국내외 기술개발 현황 ... 18
• 제 1 절. 치즈의 시장 현황 ... 18
• 제 2 절. 국내외 기술 현황 ... 21
• 제 3 장 연구개발수행 내용 및 결과 ... 25
• 제 1 절 재료 ... 25
• 제 2 절 방법 ... 26
• 제 3 절 연구결과 및 고찰 ... 40
• 1. 젖산균 분리 및 수집 ... 40
• 2. 면역증진 균주 선발 ... 41
• 3. 면역증진 균주 동정 ... 76
• 4. 선발 젖산균의 특성조사 ... 82
• 5. 모짜렐라치즈 제조를 위한 최적 배양조건 확립 ... 99
• 6. 제조공정 설정 ... 121
• 7. 이화학적 특성 조사 ... 123
• 8. 동물모델을 이용한 모짜렐라 치즈의 면역증진 효능평가 ... 135
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 143
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 145
• 제 6 장 연구개발과정에서 수집한 해외과학 기술정보 ... 146
• 제 7 장 참고문헌 ... 156
• 끝페이지 ... 160