초록 ▼

1. 대두식품으로부터 항고혈압 펩타이드 소재의 개발
1) 여러 대두식품으로부터 항고혈압 활성의 탐색
2) 대두박 가수분해물로부터 항고혈압 펩타이드의 분리
2. 한외여과를 이용한 펩타이드의 대량생산공정 개발
3. 대두식품으로부터 항암 펩타이드 소재의 개발
1) 항암 펩타이드 검색법의 정립
2) 대두박 가수분해물로부터 항암 펩타이드의 분리
4. 대두식품으로부터 항혈전 펩타이드 소재의 개발
1) 항혈전 펩타이드 검색법의 정립
2) 대두박 가수분해물로부터 항혈전 펩타이드의 분

1. 대두식품으로부터 항고혈압 펩타이드 소재의 개발
1) 여러 대두식품으로부터 항고혈압 활성의 탐색
2) 대두박 가수분해물로부터 항고혈압 펩타이드의 분리
2. 한외여과를 이용한 펩타이드의 대량생산공정 개발
3. 대두식품으로부터 항암 펩타이드 소재의 개발
1) 항암 펩타이드 검색법의 정립
2) 대두박 가수분해물로부터 항암 펩타이드의 분리
4. 대두식품으로부터 항혈전 펩타이드 소재의 개발
1) 항혈전 펩타이드 검색법의 정립
2) 대두박 가수분해물로부터 항혈전 펩타이드의 분리
5. 유전공학기법을 이용한 고기능성 생산균주의 개발
1) 기능성 펩타이드의 대량생산을 위한 expression vector개발
2) 기능성 펩타이드의 대량생산을 위한 system개발
6. 기능성 펩타이드의 In vivo효능검정법 정립
1) 대두박 가수분해물의 항고혈압 In vivo효능검정

Abstract ▼

Recently, not only cancer but also circulatory diseases, such as hypertension, arteriosclerosis, cardiovascular disease are markedly increasing according to the change of food habit from traditional to western style. For the protection and prevention of cancer, hypertension and thrombosis, it was st

Recently, not only cancer but also circulatory diseases, such as hypertension, arteriosclerosis, cardiovascular disease are markedly increasing according to the change of food habit from traditional to western style. For the protection and prevention of cancer, hypertension and thrombosis, it was studied to find active peptides from soy foods and to develop novel functional food additives.
First, to produce anti-hypertensive peptides inhibiting angiotensin-I converting enzyme(ACE), screening of ACE inhibitory activity of various soybean foods and purification process were performed. The soybean enzrmatic hydrolyzate of soybean had much higher ACE inhibitory activity than other soybean foods such as soybean paste and soy sauce. The enzymatic hydrolyzate was purified with ultrafiltration membrane in order to remove high molecular weight polypeptides. The ACE inhibitory peptide was isolated from reverse phase, ion exchange, gel permeation column. The ACE inhibitory activity of the peptide increase 140 times during purification steps from soybean hydrolyzate. The amino acid composition of this peptides was Gly, Ser, Glu, Arg, Pro, Met, and hydrophilic amino acids(Gly, Ser, Glu, Arg) enriched in this peptides. Second, mass production of soybean hydrolyzate was made by following method : Distilled water (1,000 kg) was added to defatted soybean 15%(w/w) and heated at${90^{\circ}C}$, 10 min, and sample was hydrolyzed with Promod 278(endo-peptidase, Biocatalyst Co. Ltd.) enzyme concentration was 0.4%(w/w), at ${60^{\circ}C}$, pH 8.0, for 2 hours. Promod 279(exo-peptidase, Biocatalyst Co. Ltd.) was added after first enzyme hydrolysis with enzyme concentration 0.8%(w/w), at ${55^{\circ}C}$, pH 5.0 for 4 hours, and followed by inactivation at ${90^{\circ}C}$ for 15 min. Using this hydrolyzate, process optimizations for mass production of ACE inhibitory peptides with ultrafiltration were performed. Among the various kinds of membrane, the GR type, the hydrophobic mimbrane, had a higher average permeate flux and recovery yield of nitrogen that HEKLA type or ETNA type, the hydrophilic membranes. But their permeates showed similar ACE inhibitory activity ($IC_{50}$ = 75.3 to 78.8 ${\mu}g$/ml). As the molecular weight cut-off of membrane increased, the average permeate flux and recovery yield of nitrogen was increased, but their permeates showed similar ACE inhibitory activity. So, the GR60PP membrane, the molecular weight cut-off was 25,000 dalton, had the highest total inhibitory activity. During the ultrafiltration process, average permeate flux was increased according to the temperature increasing, but ACE inhibitory activity was decreased. Considering the next evaporation process, it was determined that optimal temperature was ${55^{\circ}C}$. As the initial nitrogen concentration for ultrafiltration process effected average permeate flux to decrease, but recovery content of nitrogen to increase, it was determined that optimal initial nitrogen concentration was 0.87 %(w/w). The final ultrafiltrate obtained from overall process was mainly consisted of low molecular weight peptides(83.8 %) lower than 2,000 dalton and had a 1.8 times purified ACE inhibitory activity.
Third, to characterize the anticancer activities of pepetides from soybean, defatted soybean was hydrolyzed by various peptidases and the peptides were tested for the cytotoxic activity on several cell lines. Defatted soybean was hydrolyzed by 16 endopeptidases and extracted by water, ethanol, methanol, buthanol, acetone, hexene, a mixture of chloroform and methanol. Among these, ethanol fractions of bromelain-HSP and thermoase-HSP had significant cytotoxic activity on P388D1. The bromelain-HSP was further fractionized by ultrafiltration into B1-B3. Cytotoxicity assay revealed that B1 fraction had the strongest anticancer activity and it's $IC_{50}$ value was 230${\mu}g$/ml. The B1 fracion was further fractionized into 13 fractions by RP HPLC. B1-8 and B1-12 fractions showed strong anticancer acivity in cytotocic assay. Thin layer chromatography and amino acid analysis showed that these fractions contained glycopeptides and comprised high contents of Asp, Glu, Pro, and Leu. The thermoase-HSP was fractionized by XAD-2 adsoption chromatography into hydrophobic (To) and hydrophilic (Ti) fractions. To and Ti were tested for cytotoxicity on P388D1 (mouse monocyte - machrophage: ATCC HB 8065). The fraction to showed stronger in anticancer activity than Ti and it's $IC_{50}$ value was 63.37 ${\mu}g$/ml. The fraction To was further fractionized into five fractions by anion exchange chromatography. Among these, To2 fraction showed the strogest anticancer activiy and it's $IC_{50}$ value was 19.24 ${\mu}g$/ml. To2 fraction was further fractonized by reverse-phase high performnace liquid chromatography into 12 fractions. Among these, To2-9 had the strogest anticancer activity and it's $IC_{50}$ value was 30.60 ${\mu}g$/ml. The fractions obtaind by gel filtration chromatography were named as To2-9-Go, G1, G2, G3, G4, and G5. Among these frations, To2-9-G5 had relatively high anticancer activity. This fraction was confirmed in a HPLC as a single peak, and it's molecular weight was determined as 1330Da by MALDI-TOF MS.
Fourth, in order to search for antithrombotic peptides from soyprotein hydrolyzates, the inhibitory activities of soyprotein hydrolyzates and the peptide fraction on ADP-induced aggregation of washed platelets were assayed. The cold water extracts of soyprotein hydrolyzate was fractionated by gel permeation chromatography. Only one fraction showed antithrombotic activity. When the fraction was divided into many fractions by reverse phase HPLC with vydac $C_{18}$ vumn, all fractions had high antithrombotic activities. The most antithrombotic active peak at 60 minutes retention time among several active fractions was separated into two different active fraction by cation exchange HPLC, all of which appeard to be high antithrombotic activity (inhibition rate of platelet aggregation, 80%). The peptide fractions were further purified and identified by a combination of automated Edman degradation and ion-spray mass spectrometry. The sequence of peptides were Asp-Glu-Glu and Ser-Ser-Gly-Glu. The peptides were synthesized with the Fastmoc solid-phase peptide synthesis and purified by reversed-phase HPLC. The synthetic peptides had high antithrombotic activities ($IC_{50}$ value were 485 & 458 $\mu$M).
Fifth, the tripeptide His-His-Leu, derived from Soybean paste is identified as the angiotensin converting enzyme inhibitor which lowers blood pressure. To overproduce this antihypertensive peptide in E. coli, we constructed oligoncleotide cassette encoding three times repeated His-His-Leu, and subcloned into maltose-binding-protein fusion vector pMAL-c2 and pMAL-p2 and resulting in pMAL-c2-$HHL_9$ and pMAL-p2-$HHL_9$, respectively. The fusion protein, His-His-Leu fused to maltose-binding-protein(MBP) was overexpressed in protease-dificient E.coli transformants bearing pMAL-c2-$HHL_9$ and pMAL-p2-$HHL_9$ by 1mM IPTG induction and purified by amylose affinity chromatography and preparative SDS-PAGE. Factor Xa and α-chymotrypsin was used to isolate the tripeptide His-His-Leu from the MBP-HHL fusion protein. The digested fusion peptides including recombinant tripeptide HHL was fractionationed by gel filtration chromatography. The tripeptide HHL that has same molecular weight with synthetized HHL was identified by MALDI mass spectroscopy.
Sixth, to develop a novel functional food for preventing hypertension or atherosclersis, soybean peptide was investigated for its antihypertensive or antiatherosclerotic activities In vivo. Effects of soybean hydrolysates UF-peptide prepared by ultrafiltration and soybean peptide (soypep279) by modification of the UF-peptide were evaluated their action on blood pressure, selected biochemical changes in spontaneously hypertensive rats (SHR). Male SHR were divided into three groups and fed diets with UF-peptide or soypep279 for 6-8 weeks. Development of hypertension was determined by tail cuff method during the experimental period. After the experimental period, angiotensin I converting enzyme (ACE) activity and vascular tissue, and lipids level in serum or plasma were measured. Spontaneous elevation of blood pressure was suppressed by the UF-peptide supplementation after two weeks of feeding. After five weeks of feeding the development of hypertension was significantly inhibited in the UF-peptide administered groups compared to control. There were no histological changes in various organs in SHR fed UF-peptide or soypep279. In addition, the UF-peptide and soypep279 were effective for lowering serum/plasma lipids level such as triglyceride and total cholesterol, and LDL-cholesterol as well as lowering atherosclerotic index, indicating that the soybean peptide exerts antiatherosclerotic activity, as well as antihypertensive activity In vivo. The active site of the peptide derived from Korean soybean fermented paste for inhibiting ACE activity In vivo has been shown to be His-His Leu (HHL). In order to confirm whether the HHL exerts antihypertensive activity In vivo, we prepared synthetic HHL and invsestigated its antihypertensive effect In vivo on SHR. HHL was injected into vein femoralis in SHR and then the change of systolic blood pressure in artery of vervis was measured for 100 min by using poly-graph 1000 (Letical Scientific Intrument). Synthetic peptide HHL resulted in significant decrease of blood pressure in SHR. After 20 min of the third injection of HHL, the blood pressure was significantly decreased compared to control (p<0.01).
Our results demonstrated that soybean hydrolysates exerted antihypertensive and antiatherosclerotic activities In vivo, which were associated with the inhibitory activity of ACE and the lowering activity of lipids levels in serum or plasma. Soybean peptide could be useful to develop a novel functional food additive for preventing hypertension or atherosclerosis.

목차 Contents

• 표지...1
• 제 출 문...2
• 요약문...3
• Summary...12
• 목 차...19
• 제 1 장 서론...32
• 제 1 절 국내.외 연구동향...32
• 제 2 절 국내.외 기술수준...41
• 제 3 절 원료 수급현황...43
• 제 4 절 산업계 현황...45
• 참고문헌...46
• 제 2 장 대두식품으로부터 항고혈압 펩타이드소재의 개발...49
• 제 1 절 대두식품으로부터 항고혈압 펩타이드의 분리 및 정제...49
• 1. 연구수행방법...49
• 가. 여러 대두식품으로부터 항고혈압 활성의 탐색...49
• 나. 혈압강하 펩타이드의 추출...49
• 다. 1차 HPLC 분취...49
• 라. 2차 HPLC 분취...50
• 마. 3차 HPLC 분취...50
• 바. 4차 chromatography...50
• 사. 5차 chromatography...50
• 아. 6차 chromatography...51
• 자. Total Nitrogen(TN) 분석...51
• 차. ACE 저해활성 측정...51
• 카. 총 아미노산 분석...51
• 2. 연구수행결과...52
• 가. 여러 대두식품으로부터 항고혈압 활성의 탐색...52
• 나. 혈압강하 펩타이드의 추출...52
• 다. 1차 chromatography...52
• 라. 2차 chromatography...53
• 마. 3차 chromatography...53
• 바. 4차 chromatography...53
• 사. 5차 chromatography...54
• 아. 6차 chromatography...54
• 자. Chromatography 정제도에 따른 ACE 저해활성 비교...54
• 차. 각 column chromatography의 step별 아미노산 분석...55
• 제 2 절 한외여과를 이용한 펩타이드의 대량생산 공정개발...56
• 1. 연구수행방법...56
• 가. 탈지대두박 가수분해물의 산업적 제조...56
• 나. 한외여과를 이용한 펩타이드의 대량생산 공정개발...56
• 다. 산업적 분리공정의 최적화...57
• 2. 연구수행결과...57
• 가. 최적 막의 종류 선정...57
• 나. 최적 운전온도 선정...58
• 다. 최적 시료농도 선정...59
• 라. 최적 운전압력 선정...59
• 마. 산업적 분리공정의 최적화...59
• 제 3 장 대두식품으로부터 항암펩타이드의 분리 및 정제...61
• 1. 연구수행 방법...61
• 가. 대두단백 가수분해물의 제조...61
• 나. 펩타이드의 용매 추출...61
• 다. 펩타이드 용매 추출물의 항암활성 분석...61
• 라. 함암 펩타이드의 정제...63
• 2. 연구수행결과...63
• 가. Brome1ain/EtOH 항암 펩타이드의 정제...63
• 1) Fractionation by ultrafiltration...63
• 2) Brome1ain/EtOH B1 분획의 역상 HPLC 수행...63
• 3) Bromelain/EtOH Bl-8 빛 Bl-12 분획의 아미노산 분석...64
• 4) Bromelain/EtOH Bl-8 빛 Bl-12 분획의 박충 크로마토그래피...64
• 5) Bromelain/EtOH 항암 펩타이드의 특성...65
• 나. Thermoase/EtOH 추출물 항암 펩타이드의 정제...65
• 1) XAD-2 흡착 크로마토그래피에 의한 소수성 펩타이드의 분획...65
• 2) Thermoase/EtOH 소수성 펩타이드 분획의 항암활성 분석...65
• 3) Thermoase에 의 한 가수분해도 측정...66
• 4) Thermoase To 소수성 펩타이드 분획의 이온교환 크로마토그래피...66
• 5) Thermoase/EtOH To 세부분획들의 염 제거...68
• 6) Thermoase/EtOH To 세부분획들의 항암활성 분석...68
• 7) Thp.rmoase/EtOH T02 분획의 역상 HPLC 수행...69
• 8) Thermoase/EtOH T02 역상 HPLC 세부분획들의 항암활성 검정...69
• 9) Thermoase/EtOH To-9 분획에 대한 gel filtration chromatography...70
• 10) Thermoase/EtOH T02-9-G 분획에 대한 항암활성...70
• 11) Re-RP HPLC 수행...71
• 12) Re-RP HPLC peak 의 분자량 확인...71
• 제 4 장 대두식품으로부터 항혈전 펩타이드 소재의 분리 및 정제...72
• 1. 연구수행 방법...72
• 가. 대두단백가수분해물의 항혈전 검정 (in vitro)...72
• 1) 항혈전 assay (in vitro)...72
• 2) 혈소판 분리...72
• 3) 혈소판 웅집 실험...72
• 나. 대두단백 가수분해물의 항혈전 펩타이드의 분리...73
• 다. 분리된 획분의 동정...74
• 라. 동정 한 펩타이드의 합성 및 분리...74
• 마. 합성 펩타이드의 항혈전 검정 (in vitro와 in vivo)...74
• 2. 연구수행결과...75
• 가 대두단백가수분해물의 항혈전 검정 (in vitro)...75
• 나. 대두단백가수분해물의 항혈전 펩타이드의 분리...75
• 1) Gel permeation chromatography에 의한 분리...75
• 2) Semi-preparative reverse phase HPLC에 의한 분리...75
• 3) 새로운 protocol 에 의한 분획...76
• 4) Gel permeation chromatography에 의한 충분한획분의 확보...76
• 5) 새로운 조건에서의 reverse phase HPLC에 의한 분리...77
• 6) Cation exchange HPLC에 의한 분리 및 reverse phase HPLC에 의한 desalting...77
• 다. 분리된 획분의 동정...78
• 1) LC-MS 에 의한 동정...78
• 2) Sequencer에 의 한 동정...78
• 라 동정한 펩타이드의 합성 및 분리...78
• 1) 동정한 펩타이드의 합성...78
• 마. 합성 peptide들의 항혈전 검정 (in vitro, in vivo)...79
• 제 5 장 유전공학기법을 이용한 고기능성 펩타이드의 대량생산균주의 개발...80
• 1.연구수행방법...80
• 가. 혈압강하 펩타이드 (ACE inhibitory peptide)말현 vector 개발...80
• 1) 혈압강하펩타이드 유전자의 합성...80
• 2) multiple copy의 HHL 유전자 cassette 제작...80
• 3) Cloning vector와 HHL 유전자의 Ligation...80
• 4) E.coli 로의 Transformation...82
• 5) PCR을 통한 HHL 유전자의 확인...82
• 6) DNA Sequencing 을 통한 HHL 유전자의 확인...82
• 나. 혈압강하 펩타이드 (ACE inhibitory peptide)의 발현 시스템 구축 및 대량생산 균주 개발...83
• 1) overexpression vector pMAL-p2 , pMAL-c2 로의 subc10ning...83
• 2) 유전공학기법을 이용한 혈압강하 펩타이드 생산 균주의 확보...83
• 3) IPTG induction 을 통한 혈압강하 펩타이드 생산균주의 확인...83
• 다. 혈압강하 펩타이드의 대량생산...84
• 1) IPTG induction &Sonication...84
• 2) Amylose affinity chromatography...84
• 3) Preparative SOS-PAGE...85
• 4) Factor Xa 처리...85
• 5) Chymotrypsin 처리...85
• 6) HPLC를 통한 HHL의 정제...86
• 7) MALOI{Matrix-associated laser desorption/ionization) mass-spectroscopy를 이용한 HHL의 확인...86
• 2.연구수행결과...86
• 가. 혈압강하 펩타이드발현 vector 개발...86
• 1) 혈압강하펩타이드 유전자의 합성...86
• 2) multiple copy의 HHL 유전자 cassette 제작...87
• 3) Cloning vector 와 HHL 유전자의 Ligation...87
• 4) E. coli 로의 Transformation...87
• 5) PCR을 통한 H바 유전자의 확인...87
• 6) DNA Sequencing 을 통한 HHL 유전자의 확인...88
• 나. 혈압강하 펩타이드의 발현 시스템 구축 및 대량생산 균주 개발...88
• 1) overexpression vector pMAL-p2. pMAL-c2 로의 subcloning...88
• 2) 유전공학기법을 이용한 혈압강하 펩타이드 생산 균주의 확보...89
• 3) IPTG induction 을 통한 혈압강하 펩타이드 생산균주의 확인...89
• 다. 혈압강하 펩타이드의 대량생산...89
• 1) Amylose affinity chromatography...89
• 2) preparative SOS-PAGE...90
• 3) Factor Xa 처리...90
• 4) Chymotrypsin 처리...90
• 5) Mass spectroscopy를 통한 tripeptide His-His-Leu의 확인...90
• 제 6 장 기능성 펩타이드 식품소재의 개발...92
• 1. 연구수행방법...92
• 가. 실험동물 및 사료조제...92
• 나. UF-peptide 제조...92
• 다. HHL 펩타이드 합성...93
• 라. 비관혈식 혈압측정...93
• 마. 관혈식 혈압측정...93
• 바. 생화학적 검사 및 조직증 ACE 조효소액 조제...93
• 사. HMG-CoA reductase 효소원 조제 및 활성측정...94
• 아. 장기증량 측정 및 병리조직학적 검사...95
• 자. 통계처리...95
• 2. 연구수행결과...95
• 가. 대두가수분해물에서 분러한 UF-peptide가 In vivo에서 자발성 고혈압 흰쥐의 혈압 강하에 미치는 영향...95
• 나. 합성 웹타이드 His-His-Leu에 의한 자발생 고혈압 흰쥐의 혈압 강하효과...98
• 다. 분무건조한 대두가수분해물 SoyPep279 식이투여가 자발성 고혈압 흰쥐의 혈중지질수준 빛 혈압강하에 미치는 영향...99
• 제 7 장 기능성 펩타이드 식품소재의 개발...103
• 1. 연구수행방법...103
• 가. 기능성 펩타이드 분말 (SoyPep279)의 제조...103
• 나. 기능성 펩타이드 분말 (SoyPep279)의 특성분석...103
• 2. 연구수행결과...103
• 가. 기능성 펩타이드 분말 (SoyPep279)의 제조...103
• 나. 혈압강하 펩타이드 분말 ( SoyPep279)의 특성...104
• 참고문헌...105