7 day 100 800 50 50 NI 50
Original form chitosan 100 100 100 100 100 100 1 day 100 200 100 50 200 50
E. coli BCRC 10675
7 day 100 NI 100 100 NI 25 a MIC:Minimum inhibitory concentration(ppm)
b NI:No Inhibition,表示 MIC>800 ppm
86
表十二、幾丁聚醣水解產物其對 E. coli 最低殺菌濃度(MBC)
Table 12. MBC of chitosan hydrolysates for E.coli.
DD80 DD92
Indicator bacteria Degradation time
CHtri CHcel CHlys CHtri CHcel CHlys
Original form chitosan 100 100 100 50 50 50
1 day 50 100 50 50 100 50
E. coli BCRC 10314
7 day 100 NI 50 50 NI 50
Original form chitosan 200 200 200 100 100 100 1 day 100 200 100 50 200 50
E. coli BCRC 10675
7 day 100 NI 100 100 NI 25 a MBC:Minimum bactericidal concentration(ppm)
b NI:No Inhibition,表示 MBC>800 ppm
87
捌、圖
酵素誘發天數(Days)
0 1 2 3 4 5 6 7
Reducing sugar (OD 590 nm)
0.00 0.02 0.04 0.06 0.08 0.10 0.12
圖十二、以 Trichoderma viride 誘發產生幾丁質酶之活性曲線
Figure 12. Active curve(reducing sugar)of crude chitinase induced from Trichoderma viride.
Hydrolysis time (Days)
0 1 2 3 4 5 6 7
Reducing sugar (OD 590 nm)
-0.1
Figure 13. Reducing sugar of enzymatically prepared chitosan hydrolysates.
89
Hydrolysis time (hrs)
0 20 40 60 80 100 120 140 160
log Molecular weight( kDa)
1 10 100 1000
DD80-CHtri DD92-CHtri DD80-CHcel DD92-CHcel DD80-CHlys DD92-CHlys
圖十四、以酵素水解幾丁聚醣其黏度推算幾丁聚醣水解物分子量之 變化。
Figure 14. Molecular weight of enzymatically prepared chitosan hydrolysates.
圖十五、HPLC 分析幾丁聚醣水解物(DD92-CHtri)
Figure 15. HPLC profiles of chitosan hydrolysates(DD92-CHtri)
Gel filtration column:TSKgel G3000PW(7.5 mm × 300 mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
DD92-CHtri-1Day
DD92-CHtri-7Day DD92-CHtri-3Day
dextran hexamer
min
min
min
91
Figure 16. Solubility of enzymatically-prepared chitosan hydrolysates
(A)DD92(B)DD80
92
玖、附錄 表一、幾丁聚醣水解產物其對 S. aureus 抑制時間(IT)
Table 1. IT of chitosan hydrolysates for S. aureus.
DD80 DD92
Indicator bacteria Degradation time
CHtri CHcel CHlys CHtri CHcel CHlys Original form chitosan 200-8a 200-8 200-8 200-8 200-8 200-8
1 day NIb NI 400-20 200-8 NI 400-20
S. aureus BCRC 10451
7 day 800-20 NI 400-8 400-20 NI 400-32 Original form chitosan 400-10 400-10 400-10 400-34 400-34 400-34 1 day 800-10 800-4 400-10 800-20 NI 400-8
S. aureus BCRC 10780
7 day 800-4 NI 800-22 800-2 NI 400-8 Original form chitosan 200-22 200-22 200-22 200-22 200-22 200-22
1 day 200-10 400-22 400-34 400-22 800-10 400-22
S. aureus BCRC 10781
7 day 400-22 NI 400-22 800-34 NI 400-34 a IT:Inhibitory time,為 X 濃度(ppm)下,可維持在遲至其所延長之 Y 時間(hr),Xppm-Yhr b NI:No Inhibition,表示 800ppm 下不具有延長遲滯期之效果
93
表二、幾丁聚醣水解產物其對 E. coli 抑制時間(IT)
Table 2. IT of chitosan hydrolysates for E. coli.
DD80 DD92
Indicator bacteria Degradation time
CHtri CHcel CHlys CHtri CHcel CHlys
Original form chitosan 50-10 50-10 50-10 25-48 25-48 25-48 1 day 25-10 50-22 25-10 25-22 50-22 25-10
E. coli BCRC 10314
7 day 50-22 400-22 25-22 25-10 400-10 25-10 Original form chitosan 50-2 50-2 50-2 50-10 50-10 50-10 1 day 50-10 100-10 50-10 25-4 100-10 25-2
E. coli BCRC 10675
7 day 100-48 NI 50-4 50-10 NI 25-48 a IT:Inhibitory time,為 X 濃度(ppm)下,可維持在遲至其所延長之 Y 時間(hr),Xppm-Yhr b NI:No Inhibition,表示 400ppm 下不具有延長遲滯期之效果。
圖一、HPLC 分析幾丁聚醣水解物(DD80-CHtri)
Figure 1. HPLC profiles of chitosan hydrolysates(DD80-CHtri) Gel filtration column:TSKgel G3000PW(7.5 mm × 300 mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
DD80-CHtri-7Day DD80-CHtri-3Day DD80-CHtri-1Day
dextran hexamer
min min min
95
圖二、HPLC 分析幾丁聚醣水解物(DD92-CHlys)
Figure 2. HPLC profiles of chitosan hydrolysates(DD92-CHlys) Gel filtration column:TSKgel G3000PW(7.5 mm × 300
mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
DD92-CHlys-7Day DD92-CHlys-3Day DD92-CHlys-1Day
dextran hexamer
min min
min
圖三、HPLC 分析幾丁聚醣水解物(DD80-CHlys)
Figure 3. HPLC profiles of chitosan hydrolysates(DD80-CHlys) Gel filtration column:TSKgel G3000PW(7.5 mm × 300
mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
min dextran hexamer
DD80-CHlys-1Day
DD80-CHlys-7Day DD80-CHlys-3Day
min min
97
圖四、HPLC 分析幾丁聚醣水解物(DD80-CHcel)
Figure 4. HPLC profiles of chitosan hydrolysates(DD80-CHcel) Gel filtration column:TSKgel G3000PW(7.5 mm × 300 mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
dextran hexamer
DD80-CHcel-1Day
DD80-CHcel-3Day
DD80-CHcel-7Day
min min min
圖五、HPLC 分析幾丁聚醣水解物(DD92-CHcel)
Figure 5. HPLC profiles of chitosan hydrolysates(DD92-CHcel) Gel filtration column:TSKgel G3000PW(7.5 mm × 300 mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
DD92-CHcel-1Day
DD92-CHcel-3Day
DD92-CHcel-7Day
min
min min dextran hexamer
99
圖六、HPLC 分析幾丁寡醣寡聚物標準品
Figure 6. HPLC profiles of chitooligosaccharides standard.
Gel filtration column:TSKgel G3000PW(7.5 mm × 300 mm;TOSOH Co.), flow rate 1.0 ml / min, mobile phase 100 mM acetic buffer (pH 4.0)
dextran hexamer
monomer
trimer
min
圖七、HPLC 分析幾丁聚醣水解物(DD92-CHtri)
Figure 7. HPLC profiles of chitosan hydrolysates(DD92-CHtri)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
DD92-CHtri-1Day
DD92-CHtri-7Day DD92-CHtri-3Day
min
min
min hexamer monomer
101
圖八、HPLC 分析幾丁聚醣水解物(DD80-CHtri)
Figure 8. HPLC profiles of chitosan hydrolysates(DD80-CHtri)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
DD80-CHtri-7Day DD80-CHtri-3Day DD80-CHtri-1Day
min
min
min hexamer monomer
圖九、HPLC 分析幾丁聚醣水解物(DD92-CHlys)
Figure 9. HPLC profiles of chitosan hydrolysates(DD92-CHlys)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
DD92-CHlys-7Day DD92-CHlys-3Day DD92-CHlys-1Day
monomer
min
min
min
103
圖十、HPLC 分析幾丁聚醣水解物(DD80-CHlys)
Figure 10. HPLC profiles of chitosan hydrolysates(DD80-CHlys)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
monomer
DD80-CHlys-7Day DD80-CHlys-3Day DD80-CHlys-1Day
min
min
min
圖十一、HPLC 分析幾丁聚醣水解物(DD92-CHcel)
Figure 11. HPLC profiles of chitosan hydrolysates(DD92-CHcel)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
DD92-CHcel-7Day DD92-CHcel-3Day DD92-CHcel-1Day
min min min hexamer monomer
105
圖十二、HPLC 分析幾丁聚醣水解物(DD80-CHcel)
Figure 12. HPLC profiles of chitosan hydrolysates(DD80-CHcel)
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
DD80-CHcel-7Day DD80-CHcel-3Day DD80-CHcel-1Day
min min min hexamer monomer
圖十三、HPLC 分析幾丁寡醣寡聚物標準品
Figure 13. HPLC profiles of chitooligosaccharides standard.
Column:Lichrospher 100 NH2(250 × 4 mm;Merck), flow rate 1.0 ml / min, mobile phase 60% acetonitrile
hexamer monomer
min
107
對照組 實驗組 空白組 對照組 實驗組 空白組
圖七、96 well micro-plate 最低抑制濃度測試示意圖。上面為針對 S. aureus,下面為針對 E. coli。
400ppm 400ppm
100ppm~400ppm 100ppm~400ppm
400ppm