초록

○ 연구결과
① 검정콩 유전자원 1,032점의 특성 및 안토시아닌 함량 분석
② 3품종 대상, 3개 지역에서 3년간 안토시아닌 함량의 환경변이 분석
③ 검정콩 안토시아닌이 함유된 기능성 식품 요구르트와 머핀 개발
④ in vitro 및 동물식이 실험을 통한 안토시아닌의 기능성 확인
⑤ NIRS를 이용한 안토시아닌의 비파괴적 분석 기술 개발

Abstract ▼

1. Genetic and environmental variations of anthocyanin contents in black soybean
Anthocyanin contents of black soybean were analyzed for development of superior breeding lines with high anthocyanin contents. Total 1,042 black soybean germplasm collected through the whole south Korea were analyzed

1. Genetic and environmental variations of anthocyanin contents in black soybean
Anthocyanin contents of black soybean were analyzed for development of superior breeding lines with high anthocyanin contents. Total 1,042 black soybean germplasm collected through the whole south Korea were analyzed with HPLC. It were detected C3G(Cyanidin-3-Glucoside), D3G(Delphinidin-3-glucoside), and Pt3G(Petunidin-3-Glucoside) as main anthocyanin pigments and each pigment contents were significantly different according to genotypes. C3G content showed the highest value in all materials and its variation was also wide, whereas D3G and Pt3G were not detected in 4 and 24 genotypes. Mean value of C3G, D3G, and Pt3G contents were 8.81, 1.78 and 0.79 mg/g, respectively. Consequently total anthocyanin content of collections were ranged 0.54~23.45mg/g and average of content was l1.67mg/g. Also total anthocyanin contents were higher in seed coat as maturation period were longer from flowering stage and seed weight were more heavy. Anthocyanin content in seed coat was different according to location, genotypes and seeding time, so it can be concluded that anthocyanin content was effected by environmetal and. genetic variation.
The correlations between color index and anthocyanin content were analyzed. L(lightness) and b(yellowness) values were correlated negatively with D3G, C3G and total anthocyanin content while a(redness) value was correlated negatively with C3G content. The correlations between climatic factors and anthocyanin content were analyzed. Anthocyanin content was correlated negatively with mean temperature and accumulated temperature whereas mean daily temperature range showed positive correlation. We could conclude that the area in which mean temperature was low and daily temperature. range was high is good for production of high anthocyanin containing black soybean.
2. Nondestructive and rapid analysis methods by NIRS to Quantify anthocyanin contents
Near infrared reflectance spectroscopy (NIRS) is a rapid and accurate analytical method for determining the composition of agricultural products and feeds. This study was conducted to measure anthocyanin contents in black colored soybean by using NIRS system. Total 300 seed coat of black colored soybean samples previously analyzed by HPLC were scanned by NIRS and over 250 samples were selected for calibration and validation equation. A calibration equation calculated by MPLS(modified partial least squares) regression technique was developed in which the coefficient of determination for anthocyanin pigment C3G, D3G and Pt3G content was 0.952, 0.936, and 0.833, respectively. Each calibration equation was applied to validation set that was performed with the remaining samples not included in the calibration set, which showed high positive correlation both in C3G and D3G content file. In case Pt3G, the prediction model was needed more accuracy because of low $R^2$ value in validation set. This results demonstrate that the developed NIRS equation can be practically used as a rapid screening method for quantification of C3G and D3G contents in black colored soybean.
3. Pharmaceutical effects and oral toxicity of anthocyanin extracts
Anthocyanin pigments in soybean seed coat were D3G(Delphinidin-3-glucoside), C3G(Cyanidin-3-glucoside) and Pt3G (Petunidin-3-glucoside), which have been known potential roles in the prevention and treatment of chronic diseases. Effects of anthocyanin to human cancer cells was analyzed by MTT assay after anthocyanin pigments treated on leukemia (Jurkat-T) and adenocarsinoma (MCF-7) cells. It showed decrement of cell numbers as anthocyanin concentration is increasing. $EC_{50}$ range of anthocyanin concentrations were 100~250 ug/mL and 100~250 ug/mL in Jurkat-T and MCF-7 cell, respectively. D3G showed higher cytotoxicity than other pigments in Jurkat- T cell whereas activity of C3G was high in MCF -7 cell. And SOD activity and inhibition of nitrogen dioxide production by anthocyanin extracts treatment were also showed the biological activity. It is believed that supplementation of human diets with soybean anthocyanin markedly reduces human cancer mortality rates. This study was also conducted to assess the toxicity of anthocyanin extracts from black soybean seed coats via oral administration in a mice feeding test. The final anthocyanin-containing products induced signs of toxicity on mortality in the 4--week feeding test. The incidence of clinical signs and changes in body and organ weight were also not observed in all anthocyanin-treated groups as compared with the control groups. In hematology analysis, RBC indices concluding MCV, MCH, and MCHC and WBC differential counting such a NEU, LYM, MONO, EOS, and BASO evidenced no significant differences between the anthocyanin treatment and control groups. The biochemical parameters in serum analysis - ALT, AST, BUN, Crea etc.- were also not significantly altered in those groups. Absolute and relative organ weights were not increased after 4 weeks of treatment with anthocyanin extracts in mice. In conclusion, anthocyanin extracts from the black soybean has biological activity without any toxic effects, which also suggests that the consumption of soy containing anthocyanin products may be a good source for health and medical supplies.
4. Development of anthocyanin-based functional foodstuff
We analyzed the anthocyanin contents of black soybean crude extracts derived using a countercurrent system and to compare the effects of stabilizers ($\beta$-cyclodextrin, maltodextrin) and sugars (sucrose, maltose) on the color deterioration of the anthocyanin. When the extraction process was kept at $100^{\circ}C$ for 120~180 min, only C3G (cyanidin-3-glucoside) was detected in the water extract. The C3G contents in the water extracts acquired at $8^{\circ}C$, $60^{\circ}C$, and $80^{\circ}C$ were 2.38 ppm, 1.73 ppm, and 1.73 ppm, respectively. Sucrose and maltose retarded color deterioration of the crude pigment extract by the countercurrent method with methanol. Finally, the additions of maltodextrin or 13-cyclodextrin did not retard thermal color deterioration of the black soybean crude pigment extract. Quality of anthocyanin extract yogurt on during storage was analyzed. To strengthen the usage of anthocyanin extract, the yogurt containing skim milk powder and anthocyanin was fermented by Streptococcus thermophilus and Lactobacillus plantarum.. Control group was yogurt containing only skim milk 15.4 % and experimental groups were replaced the part of skim milk, 0.5 %, 1 % and 1.5 % with anthocyanin extract. The amount of lactic acid were increased according to the amount of anthocyanin extract during. fermentation and the viscosity was also increased, too. The more the addition of anthocyanin extract to yogurt by Streptococcus thermophilus and Lactobacillus plantarum was increased the sensory properties, such as fragrance, texture and overall preference. This experiment suggest that it is desirable to add anthocyanin extract 0.5 % or less than 1 % to the yogurt and further research for the selection of lactic acid bacteria and for adequate proportion of skim milk powder vs. anthocyanin extract is needed. Also, quality characteristics of muffin added anthocyanin extract was analyzed. Anthocyanin extract was used in muffin production at different concentrations ranging from 0 to 50%. The lightness value of muffins decreased, however, the yellowness value increased with increasing amounts of anthocyanin extract. The bulk of the muffins was generally reduced by the addition of anthocyanin extract. At the 10% concentration, hardness and viscosity were similar to the control, and there were no changes in springiness or cohesiveness. For the sensory evaluation, parameters such as appearance, color, texture, taste, and overall acceptability increased significantly from the control at the 10% concentration of anthocyanin, but flavor increased with increasing amounts of anthocyanin extract. Based on these results and the sensory evaluation specifically, the optimal of anthocyanin added to the muffins was the 10% concentration.

목차 Contents

• 제출문 ... 1
• 요 약 문 ... 2
• SUMMARY ... 8
• CONTENTS ... 14
• 목 차 ... 15
• 제 1 장 연구개발과제의 개요 ... 17
• 제 1 절 연구개발의 목적 ... 17
• 제 2 절 연구 개발의 필요성 및 범위 ... 17
• 제 2 장 국내외 기술 개발 현황 ... 20
• 제 3 장 연구개발수행 내용 및 결과 ... 23
• 제 1 절 연구 수행 방법 ... 23
• 1. 검정콩 유전자원의 안토시아닌 함량 및 환경변이 분석 ... 23
• 2. 검정콩 안토시아닌 함량의 신속ㆍ비파괴분석 기술 개발 ... 27
• 3. 검정콩 안토시아닌 효능의 in vitro 분석 ... 27
• 4. 동물실험을 통한 안토시아닌 독성 평가 ... 30
• 5. 추출조건과 첨가물에 따른 안토시아닌 함량과 색소 안정성 평가 ... 36
• 6. 안토시아닌 첨가 발효유 개발 ... 41
• 7. 안토시아닌 활용 머핀 개발 ... 44
• 제 2 절 연구 수행 결과 및 고찰 ... 49
• 1. 검정콩 유전자원의 안토시아닌 함량 및 환경변이 분석 ... 49
• 가. 검정콩 유전자읜 안토시아닌 함량 평가 ... 49
• 나. 검정콩 안토시아닌 함량의 환경 변이 평가 ... 62
• 2. 검정콩 안토시아닌 함량의 신속.비파괴분석 기술 개발 ... 75
• 가. 검정콩 종피를 이용한 안토시아닌 함량의 NIRS 검량식 작성 ... 75
• 나. 검정콩 종실을 이용한 총 안토시아닌 함량의 NIRS 검량식 작성 ... 81
• 3. 검정콩 안토시아닌 효능의 in vitro 분석 ... 88
• 가. Superoxide dismutase(SOD) assay ... 88
• 나. Nitric Oxide(NO) assay ... 88
• 다. 항균력 효과 분석 ... 92
• 라. 항암성 평가 ... 92
• 4. 동물실험을 통한 안토시아닌 독성 평가 ... 98
• 5. 추출조건과 침가물에 따른 안토시아닌 함량과 색소 안정성 평가 ... 109
• 가. 검정콩 안토시아닌 추출 ... 109
• 나. 안토시아닌 색소의 안정성 ... 113
• 6. 안토시아닌 첨가 발효유 개발 ... 121
• 7. 안토시아닌 활용 머핀 개발 ... 132
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 146
• 제 1 절 목표 달성도 ... 146
• 제 2 절 관련분야에의 기여도 ... 147
• 제 5 장 연구 개발 성과 및 성과활용 계획 ... 148
• 제 1 절 연구개발 성과 ... 148
• 1. 계량적 성과 ... 148
• 2. 성과 내용 ... 148
• 제 2 절 연구개발의 활용 계획 ... 150
• 제 6 장 연구개발과정에서 수집한 해외과학기술 정보 ... 150
• 제 7 장 참 고 문 헌 ... 151
• 끝페이지 ... 157