표제지
Abstracts
목차
I. 서론 16
II. 연구사 20
1. Whey protein의 구조 20
1) β-lactoglobulin 20
2) α-lactalbumin 23
2. Whey protein의 가수분해 및 가수분해물의 특성 24
3. Milk protein의 calcium binding 28
III. 실험 1. Cheese whey protein의 분리, 가수분해 및 가수분해물의 특성 34
1. 서론 34
2. 재료 및 방법 36
1) 공시재료 36
2) Cheddar cheese whey의 분리 36
3) Cheddar cheese whey의 화학적 성분분석 36
4) Cheese whey protein(CWP)의 전처리, 가수분해물의 제조 및 특성 36
3. 결과 및 고찰 46
1) Cheddar cheese whey의 일반 성분 46
2) CWP의 전처리 46
3) 효소별 CWP의 가수분해 특성 51
4) 효소별 CWP 가수분해물의 용해도 68
5) 효소별 CWP 가수분해물의 RP-HPLC pattern 72
4. 적요 78
IV. 실험 2. Calcium binding protein의 분리 및 확인 80
1. 서론 80
2. 재료 및 방법 82
1) Ethanol에 의한 선택적 침전 82
2) Ion exchange chromatography에 의한 CaBP의 분리 82
3) Calcium 함량 측정 83
4) Calcium binding 능력의 측정 84
5) Preparative HPLC에 의한 분획의 정제 및 대량 분취 85
6) Calcium binding peptides의 분리 85
3. 결과 및 고찰 87
1) 효소별 CWP 가수분해물의 ion exchange chromatography 87
2) Ion exchange chromatography에 의한 효소별 분획의 특성 87
3) Calcium binding 능력 96
4) Preparative HPLC에 의한 정제 및 대량분취 99
5) Calcium binding peptides의 분리 99
4. 적요 103
V. 실험 3. Calcium binding peptides의 특성 104
1. 서론 104
2. 재료 및 방법 106
1) 분자량 분포도 측정 106
2) 유리 아미노산 조성 분석 106
3) 아미노산 염기 서열 분석 107
4) In vitro에서의 calcium binding peptides의 calcium 용해도 측정 107
3. 결과 및 고찰 110
1) 효소별 CWP 가수분해물 및 trypsin 가수분해물 분획의 분자량 분포도 110
2) Calcium binding peptide의 유리 아미노산 조성 111
3) Calcium binding peptides의 아미노산 염기 서열 113
4) In vitro에서의 calcium binding peptides의 calcium 용해도 116
4. 적요 118
VI. 참고문헌 120
III. 실험 1. Cheese whey protein의 분리, 가수분해 및 가수분해물의 특성 39
Table 1. Origin and reaction conditions of proteolytic enzymes for CWP hydrolysis. 39
Table 2. Operation conditions of RP-HPLC. 44
Table 3. General composition of Cheddar cheese whey. 46
IV. 실험 2. Calcium binding protein의 분리 및 확인 83
Table 1. Operating conditions of column chromatography. 83
Table 2. Operating conditions of ion chromatography. 84
Table 3. Operating conditions of preparative HPLC. 85
Table 4. Recovery rate of protein in heated-CWP hydrolysates with commercial enzymes by ion exchange chromatography. 94
Table 5. Recovery rate of calcium in heated-CWP hydrolysates with commercial enzymes by ion exchange chromatography. 95
V. 실험 3. Calcium binding peptides의 특성 111
Table 1. The molecular weight distribution of CWP hydrolysates by commercial enzymes and tryptic hydrolysate fractions(F-2 peak). 111
Table 2. Amino acid composition of CWP and calcium binding peptides. 113
Table 3. Peptide sequence of calcium binding peptides. 115
Table 4. Solubility of calcium after in vitro digestion of calcium binding peptides in pH 2.5 and 7.5. 117
Table 5. Solubility of calcium after in vitro digestion of calcium binding peptides in stomach and duodenal condition. 117
III. 실험 1. Cheese whey protein의 분리, 가수분해 및 가수분해물의 특성 37
Figure 1. Separation of whey from Cheddar cheese process. 37
Figure 2. SDS-PAGE patterns of CWP before hydrolysis. 47
Figure 3. SDS-PAGE patterns of tryptic hydrolysates of native CWP for 0, 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~8) at 50℃. 48
Figure 4. SDS-PAGE patterns of proteolytic hydrolysates of native CWP by sommercial enzymes for 0, 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~8) at 50℃ 50
Figure 5. Time course of hydrolysis of heated-CWP by commercial enzymes. 54
Figure 6. Time course of hydrolysis of native CWP by commercial enzymes. 55
Figure 7. Time course of NPN content in heated-CWP hydrolysates by commercial enzymes. 57
Figure 8. Time course of free SH group content in heated-CWP hydrolysates by commercial enzymes. 58
Figure 9. Time course of disulfide bond content in heated-CWP hydrolysates by commercial enzymes. 60
Figure 10. SDS-PAGE patterns of tryptic heated-CWP hydrolysates at 50℃ for 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~7). 63
Figure 11. SDS-PAGE patterns of heated-CWP hydrolysates by papain W-40 at 50℃ for 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~7). 64
Figure 12. SDS-PAGE patterns of heated-CWP hydrolysates by papain S at 50℃ for 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~7). 65
Figure 13. SDS-PAGE patterns of heated-CWP hydrolysates by neutrase 1.5 at 50℃ for 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~7). 66
Figure 14. SDS-PAGE patterns of peptic heated-CWP hydrolysates at 50℃ for 15, 30, 60, 90, 120, 180 and 240 min.(lanes 1~7). 67
Figure 15. Solubility of hydrolysates by commercial enzymes. 70
Figure 16. Solubility of hydrolysates added with CaCl₂ by commercial enzymes. 71
Figure 17. RP-HPLC chromatograms of native CWP(A) and heated-CWP(B). 74
Figure 18. RP-HPLC chromatograms of heated-CWP hydrolysates by commercial enzymes. 77
IV. 실험 2. Calcium binding protein의 분리 및 확인 88
Figure 1. Ion exchange chromatogram of heated-CWP. 88
Figure 2. Ion exchange chromatogram of tryptic heated-CWP hydrolysates. 89
Figure 3. Ion exchange chromatogram of heated-CWP hydrolysates by papain W-40. 90
Figure 4. Ion exchange chromatogram of heated-CWP hydrolysates by protease S. 91
Figure 5. Ion exchange chromatogram of heated-CWP hydrolysates by neutrase 1.5. 92
Figure 6. Ion exchange chromatogram of peptic heated-CWP hydrolysates. 93
Figure 7. SDS-PAGE patterns of tryptic hydrolysate fractions by ion exchange chromatography. 97
Figure 8. Calcium binding ability of fractions eluted with 20 mM Tris-HCl buffer containing 0, 0.25 and 0.5 M NaCl from tryptic hydrolysates. 98
Figure 9. Preparative HPLC chromatogram of tryptic hydrolysate fraction 2(peak) by ion exchange chromatography. 100
Figure 10. Calcium binding ability of fractions by preparative HPLC from tryptic hydrolysates. 101
Figure 11. SDS-PAGE patterns of 5 KDa molecular weight cut off(MWCO) treatments in peak(3, 4 and 5) by preparative HPLC. 102
V. 실험 3. Calcium binding peptides의 특성 109
Figure 1. Schematic protocol for in vitro digestion of calcium binding peptides. 109
Figure 2. RP-HPLC chromatogram of calcium binding peptides. 114