本實驗利用胃蛋白酶（pepsin）於pH 2.5、37℃下水解5％乳鐵蛋白（Lactoferrin）水溶液4小時，來製備乳鐵蛋白水解物（Lactoferrin hydrolysate），經陽離子樹脂分離後，利用高效液相層析儀分析（High Performance Liquid Chromato -graphy, HPLC）收集水解液中所含抗菌胜肽區分（Antibacterial peptide fractions）並探討其抑菌效果，結果發現水解率為7.4％之乳鐵蛋白水解液，其為濃度8.1 mg/ml之多胜肽水溶液，具抗菌活性之胜肽區分1及胜肽區分2含量分別為591.3 μg/ml及491.8 μg/ml，且胜肽區分1及胜肽區分2對L. monocytogenes之最小抑菌濃度分別為25 μg/ml及50 μg/ml。
將乳鐵蛋白水解液之稀釋液，濃度為125、250、500及1000 μg/ml，在100℃下加熱0、5、10、15、20、25、30及60分鐘，冷卻後分別測試對於兩株病原菌：革蘭氏陽性菌（Listeria monocytogenes）及革蘭氏陰性菌（Salmonella typhimurium）（菌體濃度約104 CFU/ml之對數生長期之菌體）之抑菌效果，結果發現不論是否加熱或加熱時間長短，乳鐵蛋白水解液之濃度為125 μg/ml時，其無抑菌效果，而濃度為500 μg/ml或以上時，其對兩株病源菌有顯著抑制效果。當濃度為250 μg/ml時，未加熱之水解液對於兩株病原菌也具抑制效果，但經加熱10分鐘後，其對S. typhimurium的抑菌效果會顯著降低（P < 0.05）；經加熱30分鐘後，對L. monocytogenes的抑菌效果會顯著的下降（P < 0.05）。而本實驗進一步把乳鐵蛋白水解液（500 μg/ml）加熱（100℃）0、5及10分鐘後離心並分析上澄液之乳鐵蛋白素含量；發現未加熱但經離心之上澄液所含胜肽區分1及胜肽區分2的濃度分別為571.6 μg/ml與487.3 μg/ml，為未離心之乳鐵蛋白水解液中抗菌胜肽含量的百分之97與99，可知抗菌胜肽大部分存在於乳鐵蛋白水解液之上澄液中；但加熱5分鐘的乳鐵蛋白水解液之上澄液中所含的胜肽區分1與胜肽區分2減少為原始濃度之62.8％（371.1 μg/ml）及49.9％（245.6 μg/ml），加熱10分鐘更減少為原始濃度之36.3％（214.8 μg/ml）與42.8％（210.4 μg/ml）；證明加熱時間越長，乳鐵蛋白水解液之上澄液所含抗菌胜肽濃度越低。
將乳鐵蛋白水解液進行冷凍乾燥，得到之冷凍乾燥乳鐵蛋白水解物（freeze drying lactoferrin hydrolysate, FD-LfH），本實驗添加冷凍乾燥乳鐵蛋白水解物2.03 g （FD-LfH 6.78 mg/g）及4.08 g （FD-LfH 13.56 mg/g）於300 g 絞碎豬肉中，分別為3倍（75 μg/g）與6倍（150 μg/g）胜肽區分2之最小抑菌濃度，結果發現控制組與FD-LfH 6.78 mg/g及FD-LfH 13.56 mg/g的水分含量有顯著差異（P＜0.05），但FD-LfH 6.78 mg/g與FD-LfH 13.56 mg/g組之間並無顯著差異。在儲藏實驗中之總生菌結果方面，FD-LfH 13.56 mg/g對於絞碎豬肉中之總生菌數，在儲藏時間中皆與控制組及FD-LfH 6.78 mg/g有顯著之差異（P＜0.05），而FD-LfH 6.78 mg/g亦在儲藏後的第2、4、6及8天，與控制組之總生菌數有顯著之差異（P＜0.05）。

In this experiment, lactoferrin was hydrolysis four hours by pepsin at 37℃and pH2.5 to manufacture lactoferrin hydrolysate (LfH), then separated by cation-exchanged resin and analyzed by high performance liquid chromatography to collect the peptide fractions for investigating its antibacterial activity. The hydrolysis ratio of LfH is 7.4%, and the concentration of polypeptide is 8.1 mg/ml including the antibacterial peptides-peptide1 (591.3 μg/ml) and peptide 2 (491.8 μg/ml). The minimal inhibition concentrations of peptide fraction 1 (PF 1) and peptide fraction 2 (PF 2) toward Listeria monocytogenes was 50 μg/ml and 25 μg/ml.
The experiment to investigate that antibacterial activity of two pathogenic bacteria: Listeria monocytogenes (gram positive bacteria) and Salmonella typhimurium (gram negative bacteria) from LfH was diluted to 125, 250, 500 and 1000 μg/ml, and heated for 0, 5, 10, 15, 20, 25, 30 and 60 minutes at 100℃. The results were non-antibacterial activity in 125μg/ml LfH without heated or not, inversely, LfH had significantly antibacterial activity at 500 μg/ml. LfH at 250 μg/ml still have antibacterial activity, but the antibacterial activity of S. typhimurium from LfH heated for 10 minutes was significantly degraded (P < 0.05). And the antibacterial activity of L. monocytogenes from LfH heated for 30 minutes was significantly degraded (P < 0.05).
LFH was heated at 100℃ for 0, 5 and 10 minutes, than collected the suspending liquid of centrifuged LfH to analyse the concentration of antibacterial peptide fractions. The concentration of PF 1 and PF 2 in unheated and centrifuged LfH is 571.6 μg/ml and 487.3 μg/ml, and that was 97% and 99% of original consistence respectively. The result was meant that the most of antibacterial peptides existed in the suspending liquid of LfH. The concentration of PF 1 and PF 2 in LfH heated for 5 minutes is 62.8% (371.1 μg/ml) and 49.9% (245.6 μg/ml) of original consistence, and the concentration of PF 1 and PF 2 in LfH heated for 10 minutes is 36.3% (214.8 μg/ml) and 42.8% (210.4 μg/ml) of original consistence. So these were proved that the longer heating time and the lower concentration of antibacterial peptides in suspending liquid of LfH.
The final purposes of this study were to investigate the microbial and composition properties of non-vacuum-package ground pork loin added freeze drying lactoferrin hydrolysate (FD-LfH) to 6.78 mg/g (FD-LfH 6.78 mg/g) or 13.56 mg/g (FD-LfH 13.56 mg/ml), then stored at 2℃ for 0, 2, 4, 6 and 8 days. The moisture of control is significantly higher than FD-LfH 6.78 mg/g and FD-LfH 13.56 mg/g (P < 0.05), but the moisture of FD-LfH 6.78 mg/g and FD-LfH 13.56 mg/g were non-significantly different. The total plate counts were lower in FD-LfH 13.56 mg/g than other treatments during storage (P < 0.05), and the total plate counts in FD-LfH 6.78 mg/g also lower than control in the 2nd, 4th, 6th and 8th day (P < 0.05).

封面內頁 頁次
目錄……………………………………………………………………… I
圖次…………………………………………………………………….. V
表次…………………………………………………………………… VII
中文摘要…………………………………………………………….. VIII
英文摘要……………………………………………………………….. X
壹、前言………………………………………………………………… 1
貳、文獻回顧…………………………………………………………… 3
一、乳鐵蛋白……………………………………………………………. 3
（一）乳鐵蛋白之發現…………………………………………...……. 3
（二）乳鐵蛋白之結構與特性………………………………………… 3
（三）乳鐵蛋白之功能………………………………………………… 6
二、乳鐵蛋白水解物…………………………………………………… 9
（一）乳鐵蛋白水解物之製備………………………………………… 9
（二）乳鐵蛋白水解物之抑菌活性…………………………………… 9
（三）乳鐵蛋白水解物之殺菌活性………………………………….. 13
（四）乳鐵蛋白水解物之抗菌胜肽………………………………….. 15
1. 乳鐵蛋白素…………………………………………………….. 16
1.1 乳鐵蛋白素之結構與特性………………………………. 18
1.2 乳鐵蛋白素之功能………………………………………. 18
1.2.1 抗菌活性…………………………………………….. 20
1.2.1.1 乳鐵蛋白素之抗菌機制………………………… 22
1.2.1.2 乳鐵蛋白素之抗菌中心………………………… 27
1.2.1.3 乳鐵蛋白素抗菌活性之影響因子……………… 28
(1) 其他抗生素之影響………………………………….. 28
(2) 異構體的差異……………………………………….. 30
(3) 不同溫度的影響…………………………………….. 30
(4) 不同酸鹼度的影響………………………………….. 30
(5) 不同離子濃度的影響……………………………….. 31
1.2.2 抗真菌活性………………………………………….. 32
1.2.3 抗病毒活性………………………………………….. 32
1.2.4 抗腫瘤活性………………………………………….. 33
1.2.5 免疫系統反應……………………………………….. 34
1.3 乳鐵蛋白素之生物安全性………………………………. 34
1.4 乳鐵蛋白素之應用………………………………………. 35
2. Lactoferrampin………………………………………………… 35
2.1 Lactoferrampin之結構與特性…………………………... 35
2.2 Lactoferrampin之功能…………………………………... 35
2.2.1 抗菌活性…………………………………………….. 36
2.2.2 抗真菌活性………………………………………….. 36
2.3 Lactoferrampin之生物安全性…....................................... 37
（五）乳鐵蛋白水解物之應用.............................................................. 37
参、材料與方法……………………………………………………….. 39
一、實驗材料….……………………………………………………….. 39
二、實驗儀器…………………………………………………………... 41
三、實驗設計…………………………………………………………... 43
四、實驗方法………………………………………………………….. 43
（一）水解乳鐵蛋白………………………………………………….. 43
（二）多胜肽濃度測定……………………………………………..… 46
（三）水解率測定…………………………………………………….. 48
（四）陽離子交換樹脂分離………………………………………….. 50
（五）高效液相層析之分析與製備………………………………….. 51
（六）乳鐵蛋白水解液之加熱處理………………………………….. 55
（七）抗菌活性測試………………………………………………….. 55
（八）絞碎豬肉之製備……………………………………………….. 60
（九）水分…………………………………………………………….. 62
（十）總生菌試驗…………………………………………………….. 62
（十一）統計分析…………………………………………………….. 63
肆、結果與討論……………………………………………………….. 64
一、乳鐵蛋白水解液之分析………………………………………….. 64
（一）陽離子樹脂分離……………………………………………….. 64
（二）高效液相層析之分析………………………………………….. 64
（三）胜肽區分之最小抑菌濃度測試……………………………….. 68
二、乳鐵蛋白水解液之耐熱性……………………………………..…. 71
（一）乳鐵蛋白水解液加熱後之抑菌效果………………………….. 71
（二）乳鐵蛋白水解液加熱上澄液之抑菌效果…………………….. 78
（三）乳鐵蛋白水解液加熱上澄液之胜肽含量…………………….. 81
三、凍乾乳鐵蛋白水解物對絞碎豬肉之影響………………………... 84
（一）凍乾乳鐵蛋白水解物對絞碎豬肉中水份之影響………….…. 84
（二）凍乾乳鐵蛋白水解物對絞碎豬肉中總生菌之影響………….. 84
伍、結論……………………………………………………………….. 89
陸、參考文獻………………………………………………………….. 91
柒、附錄……………………………………………………………….102


圖次 頁次
圖一、、(A)牛與(B)人乳鐵蛋白之立體結構圖……………………… 4
圖二、牛乳鐵蛋白N球葉之鐵離子結合位置圖………………… …… 5
圖三、(A) 革蘭氏陽性菌及 (B) 革蘭氏陰性菌之細胞膜結構……… 8
圖四、牛與人乳鐵蛋白素之初級結構圖……………………………... 17
圖五、（A）牛及（B）人乳鐵蛋白素於水溶液中之二級結構
與電荷分佈圖……………………………………………………….… 19
圗六、(A) 抗菌胜肽對細胞膜作用的特性(B) 細胞膜穿孔模型…… 25
圖七、乳鐵蛋白素17-31中胺基酸逐一由丙胺酸替代後，對
E coli所表現的最小抑菌濃度………………………………………… 29
圖八、乳鐵蛋白水解液之抗菌胜肽分析試驗設計…………………... 44
圖九、乳鐵蛋白水解液抗菌活性之耐熱性實驗設計………………... 45
圖十、高濃度多胜肽水溶液之牛血清蛋白標準曲線………………... 47
圖十一、低濃度多胜肽水溶液之牛血清蛋白標準曲線………….….. 48
圖十二、抗菌胜肽區分1之標準曲線….………………………….…. 54
圖十三、抗菌胜肽區分2之標準曲線……….……………………….. 55
圖十四、Listeria monocytogenes之(A)生長曲線及(B)對數生長期….. 58
圖十五、Salmonella typhimurium之(A)生長曲線及(B)對數生長期… 59
圖十六、冷凍乾燥乳鐵蛋白水解物於絞碎豬里肌肉中之
抗菌活性試驗設計……………………………………………………. 61
圖十七、乳鐵蛋白水解液之陽離子交換樹脂層析圖………………... 65
圖十八、乳鐵蛋白水解液經陽離子樹脂分離後之高效液相層析圖... 66
圖十九、乳鐵蛋白水解液中抗菌胜肽區分之高效液相層析圖..…..... 67
圖二十、胜肽區分1對於L. monocytogenes之抑菌效果…………..… 69
圖二十一、胜肽區分2對於L. monocytogenes之抑菌效果………….. 70
圖二十二、乳鐵蛋白水解液對S. typhimurium抑菌活性之影響…….. 73
圖二十三、乳鐵蛋白水解液對L. monocytogenes抑菌活性之影響 …74
圖二十四、加熱乳鐵蛋白水解液（100℃, 250 μg/ml）對
L. monocy-togenes抑菌活性之影響…………………………………... 75
圖二十五、加熱乳鐵蛋白水解液（100℃, 250 μg/ml）對
S. typhimurium抑菌活性之影響……………………………………… 76
圖二十六、加熱（100℃）乳鐵蛋白水解液之上澄液對
L. monocytogenes抑菌活性之影響……………..…………………….. 79
圖二十七、加熱（100℃）乳鐵蛋白水解液之上澄液對
S. typhimurium抑菌活性之影響……………...………………………. 80
圖二十八、加熱（100℃）乳鐵蛋白水解液上澄液之
陽離子樹脂層析圖………………………………………………..…... 82
圖二十九、冷凍乾燥乳鐵蛋白水解物對絞碎豬肉於儲藏時間
總生菌數之影響………………………………………………………. 85


表次 頁次
表一、乳鐵蛋白之酵素水解物對Escherichia coli O111生長
菌數之影響……………………………………………………………. 10
表二、牛乳鐵蛋白與胃蛋白酶水解牛乳鐵蛋白針對不同菌株
之抑菌活性……………………………………………………………. 11
表三、乳鐵蛋白水解物經不同pH值處理及一般滅菌加熱後對Escherichia coli O111的抑菌活性……………………………………. 14
表四、乳鐵蛋白素對於各種微生物的抑菌活性…………………….. 21
表五、不同動物間的乳鐵蛋白素之胺基酸序列與抑菌效果………... 23表六、加熱（100℃）乳鐵蛋白水解液上澄液的抗菌胜肽濃度…… 83

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