台灣鯛為目前世界上廣為養殖的一種經濟魚類，其生長速度快、高環境適應性以及高換肉率等的特性已經讓台灣鯛成為了世界分布最為廣泛的經濟魚種之一。本實驗的主要目的為利用目前已被研究的芥末萃取物中之異硫氰酸丙烯脂 (allyl isothiocyanate, AITC) 作為一個台灣鯛魚片的前置殺菌步驟，取代常用的次氯酸鈉水溶液 (漂白水) ，以達到除去魚腥味，防止細菌生長以及延長保值期的效果。本次使用的菌株為李斯特菌，近年來已被認定為輕度加工水產品的主要汙染菌，加上此菌耐冷、抗低 pH 值的特性而難以被抑制。實驗以不同濃度的 AITC 與正控制組漂白水進行比較兩者抗菌機制，殺菌能力，並推估經處理後，最多可延長多久的保值期。實驗分為三個部分，第一部分為探討 AITC 對於李斯特菌純菌株的抑菌能力， AITC 的 pH 約在 5 左右，而氧化還原電位則位於 450-480 mv 的區間，最低抑菌濃度為 250 ppm。藉由光學顯微鏡觀察到經過 AITC 處理的李斯特菌在經過 1 小時候即產生大量菌體凝聚死亡現象。在培養24 小時，不論低濃度或高濃度皆無產生李斯特菌生長，而控制組為 106 菌數，然而在經過 48 小時培養後， 250 ppm AITC 的組別則產生了回復生長的情形，達到與控制組相同的菌數。第二部分分別探討經由 AITC 處理後，李斯特菌之 DNA 損失率、鉀離子外漏之影響、超氧化物歧化酶 (superoxide dismutase, SOD) 以及 TTC-dehydrogenase 之相對活性。DNA流失率以 500 ppm AITC 的組別流失最多，與控制組相比，達到損失率超過 100% 的效果，隨著劑量降低，流失率下降，50 ppm 組別最低。而經過不同濃度所處理的氯化三苯四唑 (2, 3, 5-Triphenyl tetrazolium chloride, TTC) 含量，經過處理 24 小時後，500、300、250 ppm 顯示可將 TTC 相對活性降低至小於 100%，而三組之間並無顯差，而濃度低於 250 ppm 者，則無明顯差異。K+ 離子的損失代表菌體細胞膜的穩定性被破壞，在經過不同劑量之 AITC 分別處理 30 分鐘以及 24 小時之後，兩者皆無顯著差異。第三部分是不同殺菌劑濃度處理對台灣鯛魚片在不同溫度儲藏下之探討，pH 值在2°C、25°C以及37°C 皆是先降後升，然而經過 AITC 處理的變化程度較小。而在 TBARS 方面， AITC 顯示出在 2°C時具有抑制魚肉氧化的效用，而於其他較高溫度，則會加速氧化進行；在經過不同的 AITC 處理後，各個溫層之腐敗指標如TVB-N 以及 TMA 皆顯示出良好的抑制效果，其中又以 2°C 下的效果最為明顯，在儲藏到達 15 天時，尚未達到腐敗值。經由保存性試驗可知， AITC 在不同溫度以及不同溫層下皆可有效抑制李斯特菌的生長，其中又以 1000 ppm 的 AITC 效果最為顯著。結果顯示， AITC 相較於漂白水下，具有對人體無毒性，且具有良好的保存效果，為未來極具潛力的一個新興抗菌產品。

Tilapia (Oreochromis niloticus) is one of the most popular aquaculture species in Taiwan. Tilapia has relatively high nutritional value and are rich in protein and enzymes. Recent changes in consumer life style have increased the demand for safe, high-quality fish products. In this study, allyl isothiocyanate (AITC), which is known as an effective antibacterial agents widely used in agriculture, could have more benefit than bleaching liquid for tilapia fillet sanitization. In terms of antibacterial mechanism, intracellular potassium leakage, DNA leakage, dehydrogenase activity were analyzed to evaluate antimicrobial mechanism of AITC. The result shows that Listeria monocytogenes treated with different concentration of AITC (500, 1000 ppm) has dose effect in DNA leakage and dehydrogenase activity. Up to 137% of DNA leakage and 40.38% of dehydrogenase relative activity after treated with AITC. No significant effect in intracellular potassium leakage. AITC can reduce the drop of pH value during storage at 2°C for 9 day. The total plate count (TPC) in pure culture treated with 250 ppm AITC for 30 minutes had reduce to no detectable survivors by a direct plating procedure (ND) after cultivated at 37°C. In terms of shelf-life evaluation for tilapia fillets with or without L. monocytogenes inoculum and treated with AITC, different storage temperatures at 2°C, 25°C and 37°C and time periods were analyzed. When storage at 2°C, sample which treated with AITC reduce the surviving cell number of L. monocytogenes up to 2.3 log CFU/mL. Be synonymous with the 2°C group, 25°C and 37°C groups show the same tendency. This study demonstrated that AITC as an excellent and safe antibacterial agent for tilapia fillets contaminated with L. monocytogenes.

目錄
壹、 前言 1
貳、 文獻回顧 3
一、 台灣鯛之生理、生態以及肉品利用 3
(一) 台灣鯛簡介 3
(二) 世界分布情形 4
(三) 台灣鯛之肉品利用狀況 5
(四) 食品安全疑慮 5
二、 李斯特菌之食品中毒 6
(一) 生理特性 6
(二) 分布、汙染途徑以及潛伏期 6
(三) 致病機制 6
(四) 感染案例 7
(五) 預防方法 7
三、 異硫氰酸丙烯酯脂 (AITC) 化學性質以及利用 8
(一) 基本特性以及來源 8
(二) 抗菌機制以及影響因子 8
(三) 實際應用範圍 9
(四) 毒性以及安全性 9
四、 魚類的鮮度以及判斷指標 10
(一) 好氧性總生菌數 10
(二) 揮發性鹽基態氮 10
(三) TMA 11
(四) TBARS 11
(五) pH 值 11
參、 實驗設計 13
(一) AITC 於 pure culture 上殺菌能力之探討 13
(二) AITC 殺菌能力之探討 14
(三) AITC 於台灣鯛魚片上肢殺菌能力探討 15
肆、 材料與方法 16
一、 實驗材料 16
(一) 實驗菌株 16
(二) 菌株培養藥品 16
(三) 菌體分析樣品 16
(四) 魚體萃取藥品 17
(五) 魚體鮮度分析藥品 17
(六) 蛋白質萃取藥品 17
(七) 蛋白質定量 17
二、 分析方法 17
(一) 菌株保存 17
(二) 菌株活化以及培養 17
(三) AITC 最低抑菌濃度試驗 18
(四) AITC 以及漂白水物化性質測試 18
(五) 生長曲線測定 18
(六) AITC 及漂白水殺菌能力之測定 18
(七) AITC 對於李斯特菌 DNA 外漏之影響 19
(八) 測量 AITC 對李斯特菌 TTC-dehydrogenase 相對活性 19
(九) AITC 對李斯特菌鉀離子外漏之影響 19
(十) AITC 對超氧化物岐化酶之抑制效果 20
(十一) 光學顯微鏡鏡檢 20
(十二) AITC 對於台灣鯛魚片之殺菌及保鮮效果 20
三、 統計分析 22
伍、 結果與討論 23
一、 AITC 於單純培養下對李斯特菌之殺菌能力 23
(一) AITC 物化性質測試 23
(二) 最低抑制濃度 23
(三) AITC 處理李斯特菌生長曲線圖 23
(四) AITC 與之殺菌能力 25
(五) AITC 處理李斯特菌顯微鏡鏡檢 26
二、 AITC 對於李斯特菌之抑菌機制探討 27
(一) 李斯特菌經AITC 處理後 DNA 損失比率 27
(二) 李斯特菌經AITC 處理後TTC-dehydrogenase 相對活性 27
(三) 李斯特菌經 AITC 處理後鉀離子流失比率 28
(四) 李斯特菌經 AITC 處理後 SOD 相對活性 29
三、 以 AITC 處理台灣鯛魚片後之化學性質變化 31
(一) pH 值 31
(二) 滴漏損失 31
(三) AITC 抗李斯特菌能力 32
(四) 總生菌數 33
(五) 總揮發性鹽基態氮 33
(六) TMA 34
(七) TBARS 36
(八) 官能檢測 37
陸、 結論 39
柒、 參考文獻 40
捌、 圖表 48

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