초록 ▼

▣ BIFIDUS균주의 배양학적 특성 연구
- 쌀/과채류 발효 적합성 검증
- 균의 성장율, 발효패턴등 배양학적 연구
- Growth Factor 검증
▣곡과류 발효용 BIFIDUS 균주의 선발
- 한국인유래의 amylolytic BIFIDUS를 분리하고 제품발효 우수균주 선발
- 면역능력검정 system 개발
(Michigan대학, Dr. Pestka와 공동연구)
▣ Amylase 생산 BIFIDUS균을 이용한 쌀 당화/발효 시스템 개발
- 쌀 당화 시스템개발
- 쌀/사과박 발

▣ BIFIDUS균주의 배양학적 특성 연구
- 쌀/과채류 발효 적합성 검증
- 균의 성장율, 발효패턴등 배양학적 연구
- Growth Factor 검증
▣곡과류 발효용 BIFIDUS 균주의 선발
- 한국인유래의 amylolytic BIFIDUS를 분리하고 제품발효 우수균주 선발
- 면역능력검정 system 개발
(Michigan대학, Dr. Pestka와 공동연구)
▣ Amylase 생산 BIFIDUS균을 이용한 쌀 당화/발효 시스템 개발
- 쌀 당화 시스템개발
- 쌀/사과박 발효특성구명
▣ 종균생산및 제품생산을 위한 발효공학적 기초기술확립
- 종균 생산기술 개발
- 제품발효기술개발
- 쌀/과채류 발효특성구명
▣ 선발균주의 동정, 생리 활성 특성 연구-F-6-PPK, DNA분석 등을 통한 배양, 생리, 유전학적인 동정
- Amylase 특성 연구
- 면역능력증강 균주의 선발
(Michigan대학 Dr. Pestka와 국제공동연구)
▣ 사과박 전처리 시스템 개발및 발효제품 생산 시스템 연구
- 사과박 건조기술 개발
- 쌀/건조사과박/과채류 발효시스템 개발
▣ 품질개선및 저장성 향상기술개발
- Pilot Plant규모 발효실증연구
- 종균의 혼합기술및 안정화 기술개발
- 저장성향상기술 개발
▣ 발효균주및 발효제품의 기능성제고
- Amylase활성, 쌀, 과채류 발효특성, 면역증강능력, 생리활성 종합적 고찰.
- 면역기능인자의 분리 및 발현을 통한 제품중의 면역기능증진인자 강화
▣ 제품생산공정개발
- 부산물 이용 제품개발
- 제품생산설비 선택
- 생산공정 lay-out 완성

Abstract ▼

The aim of this research is to develop a fermented rice and vegetable products using Korean-originated amylolytic Bifidobacterium strains with high immuno-enhancing activity. 47 amylolytic Bifidobacterium strains were isolated on starch-containing agar medium from the fecal samples of the various ag

The aim of this research is to develop a fermented rice and vegetable products using Korean-originated amylolytic Bifidobacterium strains with high immuno-enhancing activity. 47 amylolytic Bifidobacterium strains were isolated on starch-containing agar medium from the fecal samples of the various age groups of Korean. The amylase activities of these strains were subsequently measured and compared after liquid culture in the starch-containing medium. Based on their abilities of starch liquefaction and saccharification and specific amylase activities strians such as Int-57, JS9, HJ7, SJ8, MS1, MS5 and ZS8 were chosen, Then, fermentation by selected-Bifidobacterium strains were carried out on rice, fruit and vegetables. Amylolytic Bifidobacterium strains showed significantly higher growth than non-amylolytic strains on gelatinized rice. Among the various fruits and vegetables tested, juices of apples, peaches and carrots were shown to be good growth substrates for the Bifidobacterium.
To study the effect of the amylolytic Bifidobacterium strains on the immune function, murine macrophage cell line RAW 264.7 and immune cells isolated from the Peyer's patch immune tissue were used. For the assessment of the macrophage activation, cytokines such tumor necrosis factor(TNF-$\alpha$) and interleukin(IL)-6, nitric oxide(NO), hydrogen peroxide and phagocytic activities were measured. Lipopolysaccharide (LPS) from Salmonella typhimurium supressed the production of TNF-$\alpha$ below the Bifidobacterium-treated level when LPS and Bifidobacterium were co-incubated with RAW 264.7 cells. Among the amylolytic Bifidobacterium strains, SJ32 which showed the greatest stimulatory effect on the production of TNF-$\alpha$ also showed the highest stimulatory activities on the production of IL-6, NO and hydrogen peroxide.
The effect of the amylolytic Bifidobacterium strains which were selected for their suitability in the fermentation of rice were evaluated on their macrophage-stimulatory actions. Strains of RD35 and RD50 showed greater macrophage-stimulation in the production of TNF-$\alpha$ and IL-6. As the cell concentration increased the cytokine production increased, although the cytokine levels started to decline over cell concentration of $250{\mu}g$/mL in some strains. The strains which showed higher cytokine-stimulatating activity generally stimulated greater production of nitric oxide ever though the difference were less marked between strains. In our previous studies we showed that macrophage-stimulatory activity are co-related which the adhesion to the intestinal-epithelial cells. These results may suggest that high-adhesive strains to the epithelial cells may also retain high adhesion to the immune cells such as macrophage, T cell and B cell.
Fermentation of the carrot juice by Bifidobacterium were characterized. Most of the Bifidobacterium strains tested grew above $10^8$ CFU/mL. The pH reached to about 4.2 after 30h fermentation. When various supplements were added, ascorbic acid and cysteine promoted the growth of the Bifidobacterium during fermentation. Ascorbic acid may be favored for their superior sensory value to cysteine. The volatile compounds produced during fermentation are $\beta$-terpine, $\alpha$-terpine, endocarbonylactate, dl-limonene, $\beta$-farnesene, $\alpha$-bergamolene. Delta-4-carene and 2-$\beta$-pinene disappeared during fermentation.
Addition of 0.5% skim milk and ISP to the rice fermentation media brought the effect of growth acceleration from $10^7$ to $10^8$ CFU/ml, and the addition of 10% frozen apple pomace accelerated fermentation time from 24 hr. to 12 hr. After the addition of the cells of Bifidobacterium longum 35 to the rice fermentation product of Lactobacillus acidophilus KFRI 233, it was stored 8 days at $4^{\circ}C$와 $20^{\circ}C$. During the 8 days' storage, the cells of Lb. acidophilus KFRI 233 maintained at the level of $10^8$, but the cells of Bif. longum 35 decreased gradually to the $10^5$ CFU/ml.
Grinding or size reduction and preheating of rice before gelatinization facilitated the saccharification process and improved the saccharification yields. A coarse grinding of rice to the mean particle size of 453 ${\mu}m$ in an impact mill and the preheating at $60^{\circ}C$ for 45 min were appropriate for the saccharification. The optimum gelatinization condition was at $100^{\circ}C$ for 40 min. The optimum addition levels of the amylolytic enzymes for the saccharification of rice were 0.135 unit/g rice powder for $\alpha$-amylase and 3.375 unit/g rice powder for glucoamylase and the conditions were at $60^{\circ}C$ for 75 min.
The fermentation of the saccharified rice solution(SRS) was done at $37^{\circ}C$ using Bifidobacterium sp. FBD-27 for 48 hrs. A great decrease in pH from 5.39 to 3.93 was observed for first 12 hrs of the fermentation. The pH and the titratable acidity of 48hr fermented SRS was 3.58 and 0.153%, respectively. The viable number of Bifidobacterium was $4.8{\times}10^6$ CFU/$m{\ell}$ before the fermentation and showed the maximum 2.2 $\times$$10^8$ CFU/$m{\ell}$ after 36 hr and decreased graually afterward showing 1.4 $\times$$10^8$ CFU/$m{\ell}$ after 48 hr fermentation.
Drying characteristics of wet apple pomace(WAP) was investigated to optimize drying process for the yearlong supply for the raw material of the fermented products. The drying of the WAP consisted of 3 stages; constant rate drying, 1st and 2nd stages of falling rate drying. The drying rate constant of each stage of drying was calculated and its temperature dependence was estimated according to Arrhenius equation. The magnitude of activation energy of each drying stage were 21.0 kJ/mol for the initial constant rate drying, 26.2 kJ/mol for the first stage of the falling rate drying and 24.0 kJ/mol for the second stage of the falling rate drying. The optimum drying condition of the WAP was at $70^{\circ}C$ and air velocity of 0.7 m/s. Drying took 2.5 hrs under the condition.
A drying scheme of apple pomace/rice mixture(ARM) was developed. In this scheme, ground rice was used as a drying aid to increase the drying rate and thereby to reduce the drying time. The optimum mixing ratio of the ARM was 42% wet apple pomace and 58% ground rice. The drying of ARM consisted of 2 stages of falling rate drying. The activation energy of the 1st stage was 16.1 - 21.3 kJ/mol, and was 6.2 - 7.9 kJ/mol in 2nd stage. The optimum drying temperature and air velocity were $70^{\circ}C$ and 0.7 m/s, respectively. The drying time was 1.5 hrs, which 1 hr less than that of WAP alone.
Two types of products were developed; LFR/BFW, lactic acid fermented rice mixed with Bifidobacterium fermented WAP/SRS, and LFW/BFS, lactic acid fermented WAP/SR mixed with Bifidobacterium fermented SRS. The mixed products showed the best sensory properties with the mixing of 4 parts of lactic acid fermented rice and 1 part of Bifidobacterium fermented SRS. LFW/BFS had better sensory properties than LFR/BFW. The lay-out of the production processes for the developed products were proposed for 100 ml/btl $\times$ 10,000 btl/day production scale.

목차 Contents

• 표지...1
• 제출문...2
• 요약문...4
• SUMMARY...17
• 목차...28
• 제 1 장 서 론...34
• 가. 연구의 배경...34
• 나. 연구의 필요성...35
• 다. 연구개발의 목표 및 내용...40
• 제 2 장 재료 및 방법...42
• 1. 한국인 유래의 amylolytic bifidobacteria의 분리 및 선발...42
• 2. 면역능력 비피더스균주 선발...42
• 1) Macrophage cell line...43
• 2) Nitric oxide...43
• 3) Cytokine...43
• 4) H2O2 의 생성...43
• 3. 선발균주의 동정, 효소활성 및 배양학적 생리활성특성연구...43
• 4. 고농도 균체배양연구...44
• 5. 쌀 및 과채류에 대한 발효적합성 연구...44
• 6. 쌀기질의 전처리조건 설정 및 쌀 액화, 당화의 최적화...45
• 7. 사과박 건조 공정 및 건조사과박/쌀당화액 발효 시스템 개발...46
• 8. 사과박/쌀가루 혼합건조 및 발효...48
• 9. Bifidobacterium 발효음료를 이용한 호상 쌀젖산발효물의 품질개선...49
• 10. 이화학 성분분석 및 미생물 수의 측정...49
• 제 3 장 실험결과 및 고찰...52
• 제 1 절 기능성 비피도박테리아의 분리 및 선발...52
• 1. 한국인 유래의 amylolytic bifidobacteria의 선발...52
• 2. 면역능력 비피더스균주 선발실험을 위한 방법확립...54
• 3. 면역능력 비피더스균주의 선발 (in vitro macrophage activity)...56
• 가. Macrophage의 TNF-α 생산에 미치는 amylolytic Bifidobacterium의 영향...56
• 나. IL-6의 생산에 미치는 Bifidobacterium의 영향...58
• 다. Hydrogen peroxide 생산에 미치는 amylolytic Bifidobacterium의 영향...59
• 라. Nitrogen oxide(NO)의 생산에 미치는 Bifidobacterium의 영향...60
• 4. 선발균주의 동정...61
• 제 2 절 비피더스 선발균주의 배양, 생리학적 연구...65
• 1. 선발균주의 분비효소의 특성조사...65
• 2. 선발균주의 배양학적 생리연구...65
• 가. 선발 Bifidobacterium spp.의 내산성 (acid resistance)...65
• 나. Bifidobacterium spp.의 내산소실험 (oxgen resistance)...66
• 다. 선발균주의 생육인자(growth factor)에 대한 영향...67
• 3. 고농도균체배양 연구...68
• 4. Bifidobacteria의 면역증강능력연구...69
• 제 3 절 선발 균주의 쌀 및 과채류에 대한 발효적합성 연구...72
• 1. 쌀 및 과채류에 대한 발효적합성 연구...72
• 가. 쌀배지에서의 amylolytic 과 non-amylolytic bifidobacteria의 생육비교...72
• 나. Bifidobacteria에 의한 과채류의 발효...73
• 2. 발효제품화에 대한 실험...75
• 가. Bifidobacteria에 의한 쌀발효...75
• 나. 부재료첨가에 의한 bifidobacteria의 쌀발효실험...78
• 다. Bifidobacteria에 의한 쌀/사과박발효...81
• 라. Bifidobacteria에 의한 양파발효...82
• 마. Bifidobacteria에 의한 당근발효...82
• 제 4 절 Bifidobacterium 이용 발효제품의 생산...83
• 1. 냉동사과박 첨가 비피더스 쌀 발효제품의 제조...83
• 2. Bifidobacterium에 의한 당근 발효식품의 제조...86
• 가. 과일과 채소의 종류에 따른 발효...86
• 나. 부원료 첨가에 따른 당근발효...87
• 다. Bifidobacterium 배양에 의한 관능의 변화...88
• 라. Lactobacillus acidophilus와 Str. thermophilus와의 혼합배양...89
• 마. 당근 발효중의 휘발 향기 성분의 변화...90
• 제 5 절 쌀 당화시스템 및 Bifidobacterium 발효 시스템 개발...93
• 1. Bifidobacterium 발효를 위한 쌀 당화 시스템 개발...93
• 가. 당화를 위한 쌀의 분쇄조건 확립...93
• 나. 최적당화를 위한 호화 전 예비가온 조건...94
• 다. 최적당화를 위한 호화 조건...94
• 라. 쌀 당화액 제조를 위한 당화 조건 확립...96
• 마. Bifidobacterium발효 쌀당화액의 특성...96
• 2. 쌀당화액의 비피더스 발효특성 규명...97
• 가. Bifidobacterium의 최적 발효조건을 위한 혐기적 조건 확립...98
• 나. 환원제를 첨가한 Bifidobacterium발효 쌀당화액의 특성...98
• 다. 쌀당화액 발효에 적합한 균주 선발...100
• 3. 사과박/쌀당화액 혼합물의 Bifidobacterium 발효특성...101
• 가. 사과박/쌀당화액 혼합물의 Bifidobacterium 발효를 위한 환원제 선발...101
• 나. 사과박/쌀당화액 Bifidobacterium발효를 위한 균주 선발 및 혼합 비율 결정...102
• 제 6 절 사과박 건조 공정 및 건조사과박/쌀당화액 발효 시스템 개발...103
• 1. 사과박 건조기술 개발...103
• 가. 사과박 건조특성...103
• 나. 사과박 건조조건 결정...106
• 2. 건조사과박/쌀당화액 Bifidobacterium 발효 시스템 개발...107
• 가. 쌀당화액 제조 공정 개발...107
• 나. 건조사과박/쌀당화액 혼합물의 발효...108
• 다. 생사과박과 건조사과박의 발효특성 비교...111
• 라. Bifidobacterium 발효의 최적화...113
• 제 7 절 사과박/쌀가루의 혼합건조에 의한 건조효율 향상 및 건조물의 발효특성...114
• 1. 사과박/쌀가루 혼합물 (apple-pomace/rice-flour mixture; ARM)의 건조기술 개발...114
• 가. ARM의 WAP (wet apple pomace) 첨가 비율 결정...114
• 나. ARM의 건조양상...116
• 다. ARM 최적 건조조건 결정...118
• 2. 사과박/쌀가루 건조물 (dried apple-pomace/rice-flour mixture; DARM)의 발효 특성...120
• 3. 사과박/쌀가루 건조물 (DARM)을 이용한 비피더스 발효음료(Bifidobacterium fermented dried ARM; BFD) 제조 물질수지...121
• 제 8 절 Bifidobacterium 발효음료를 이용한 호상 쌀젖산발효물의 품질개선...124
• 1. 쌀 젖산 발효...124
• 2. Bifidobacterium 발효...124
• 3. 쌀젖산발효물과 비피더스발효물과의 혼합...126
• 가. 쌀젖산발효물 (LFR)과 Bifidobacterium발효한 WAP/SRS(BFW)의 혼합제품 (LFR/BFW)...127
• 나. 젖산발효한 사과박/쌀당화물 (LFW)과 Bifidobacterium 발효한 쌀당화액 (BFS)과의 혼합제품 (LFW/BFS)...128
• 4. 혼합 제품의 제조를 위한 물질수지...130
• 가. Bifidobacterium발효한 WAP/SRS (BFW)의 쌀젖산발효물(LFR)과의 혼합제품 (LFR/BFW)의 물질수지...130
• 나. Bifidobacterium발효한 쌀당화액 (BFS)과 젖산발효한 사과박/쌀당화물(LFW)의 혼합제품 (LFW/BFS)의 물질수지...131
• 제 9 절 개발제품의 생산공정 lay-out...131
• 제 4 장 참고문헌...137