臺灣博碩士論文加值系統

在食品生產製程中，許多機械零件和水管因不易清潔。細菌持續繁殖並形成具有多層結構的生物膜，提供額外保護。生物膜會降低殺菌劑的有效性，從而增加微生物的風險。微氣泡（microbubble）為一項新穎技術，大幅提高了氣泡的表面/體積比，增強了清潔能力。此外， MBW能夠深入難以清潔的角落，並且可結合殺菌劑能有效提高殺菌效力。因此，本研究使用MBW結合常用殺菌劑，次氯酸鈉 (NaClO) 及二氧化氯 (ClO2) ，去除食品級不鏽鋼片 (stainless steel, SS) 與聚氯乙烯 (polyvinyl chloride, PVC) 水管上之鼠傷寒沙門氏菌 (Salmonella Typhimurium) 與金黃色葡萄球菌 (Staphylococcus aureus) 生物膜。透過結晶紫和生物膜多醣染色，確定生物膜形成的最佳條件，於SS與PVC表面形成生物膜後，以微氣泡結合次氯酸鈉 (50 ppm NaClO-MB Water, NMW) 或二氧化氯 (50 ppm ClO2-MB Water, CMW) 進行處理。於10 L水槽中，微氣泡水系統先活化5 min，之後處理2.5 min、5 min、7.5 min或10 min。對照組包括未洗、水洗、單獨微氣泡水 (MBW)、單獨次氯酸鈉水 (NaClOW) 及二氧化氯水 (ClO2W)。SS處理10 min去除效果最佳，與水洗組相比，S. Typhimurium在SS上之生物膜減少8.07 log CFU/piece，菌量降至偵測極限以下(< 1.08 log CFU/piece)，比NaClOW 增加了5.48 log CFU/piece殺菌效力。相同條件下，CMW與水洗組相比，減少5.46 log CFU/piece，與ClO2W相比，可增加2.49 log CFU/piece的殺菌效力。相同條件下，PVC上的S. Typhimurium生物膜比SS表現出更高的抵抗力。NMW 處理 10 min後，可減少6.60 log CFU/piece；與NaClOW相比，可增加殺菌效力2.85 log CFU/piece。另外，對於 SS 上的S. aureus生物膜，NMW 處理 7.5 min菌量已低於檢測限 (減少> 6.28log CFU/piece)；與NaClOW相比，增加殺菌效力3.72 log CFU/piece。在相同條件下，PVC上S. aureus生物膜的去除效果也低於SS，與水洗組相比，NMW可增加殺菌效力5.71 log CFU/piece；與NaClOW組相比，增加殺菌效力0.87 log CFU/piece。另外，掃描電子顯微鏡 (SEM) 和多醣螢光染色之觀察證實SS和PVC上生物膜之去除。水洗和MBW處理後，生物膜結構仍然完整並覆蓋大部分的表面。NaClOW洗滌僅去除生物膜的表層，大量細菌仍殘留在接觸面上。這是第一個展示MBW結合殺菌劑可有效去除食品接觸表面上生物膜的研究，結果顯示PVC上的生物膜比SS更難去除，S. Typhimurium生物膜比S. aureus生物膜更具抵抗力，此研究成果可擴大規模於工業界，具有巨大的應用潛力。

In the food manufacturer plants, many machine parts and water pipelines are difficult to clean. Thus, bacteria multiply and gradually form biofilm, which has a multi-layer structure and provide additional protection. Biofilm forming reduces the effectiveness of disinfectants and consequently increases the microbial risk. Microbubble (MB), a novel technique, greatly increase the ratio of surface/volume of bubbles and enhance cleaning ability. Additionally, studies showed MB could reach those hard-to-clean areas and the combination of MB and disinfectants increased efficacy of disinfectants. Therefore, this study used MB combining with the commonly used disinfectants, sodium hypochlorite (NaClO) and chlorine dioxide (ClO2), to remove the biofilm of Salmonella Typhimurium and Staphylococcus aureus on the surface of stainless steel (SS) and polyvinyl chloride (PVC), which are regularly used materials for machinery and water pipelines. The optimal conditions of biofilm formation were confirmed by the stain of crystal violet and polysaccharide first, then applied onto the surface of SS and PVC. MB combining with NaClO or ClO2 (both at 50 ppm) was activated for 5 min to fulfill a 10-L tank, then the SS or PVC pieces containing biofilm were treated for 2.5, 5, 7.5, or 10 min. Control groups included unwashed, water washing, MB water only (MBW), NaClO water only (NaClOW), and ClO2 water only (ClO2W). The highest reductions were obtained by the treatment of NMW on SS for 10 min. Bacterial population of S. Typhimurium were below the detection limit (1.08 log CFU/piece), which achieved reductions of 8.07 and 5.48 log CFU/piece using water washing and NaClOW as the baselines, respectively. Using the same parameters, CMW achieved lower reductions, which were 5.46 and 2.29 log CFU/piece using water washing and ClO2W as the baselines, respectively. The S. Typhimurium biofilm on PVC showed higher resistance than on SS. After 10-min treatment of NMW, reductions were 6.60 and 2.85 log CFU/piece using water washing and NaClOW as the baselines, respectively. For S. aureus on SS, 7.5-min treatment of NMW achieved a bacterial count below the detection limit. Thus, the reductions were 6.28 and 3.72 log CFU/piece using water washing and NaClOW as the baselines, respectively. The reductions of S. aureus on PVC were also lower than on SS under the same condition, which were 5.71 and 0.87 log CFU/piece using water washing and NaClOW as the baselines, respectively. Observation of scanning electronic microscope (SEM) and polysaccharide staining confirmed NMW treatment achieved the most complete removal of biofilm on SS and PVC. In contrast, biofilm remains still covered most part of SS and PVC pieces after water washing and MBW treatment. The washing of NaClOW eliminated the upper layers of biofilm but bacteria colonies still remained on the surface. This is the first study using the combinations of MB and disinfectants to remove biofilm on food contact surfaces. Results demonstrated NMW treatment was effective, both bacterial biofilms were more difficult to remove on PVC than SS. This technique demonstrated a great potential to apply in industry because of using common disinfectants and easily enlarging to industrial scale.

中文摘要 I
Abstract III
致謝 V
目錄 VII
圖目錄 XI
表目錄 XIII
壹、前言 1
貳、文獻回顧 8
2.1生物膜之介紹 8
2.1.1生物膜之形成階段 9
2.1.2生物膜形成之影響因子 12
2.1.3食品接觸面上的生物膜 14
2.1.4生物膜之危害 15
2.1.5生物膜-食品致病菌 15
2.2沙門氏菌 (Salmonella spp.) 16
2.2.1菌種特性 16
2.2.2菌種分類、介紹 17
2.2.3感染來源及傳染途徑 18
2.2.4中毒症狀 19
2.2.5沙門氏菌食品中毒事件 19
2.3金黃色葡萄球菌 (Staphylococcus aureus) 20
2.3.1菌種特性 20
2.3.2菌種分類、介紹 20
2.3.3感染來源及傳染途徑 21
2.3.4中毒症狀 21
2.3.5金黃色葡萄球菌食品中毒事件 21
2.4殺菌劑 22
2.4.1次氯酸鈉 (sodium hypochlorite, NaClO) 22
2.4.2二氧化氯 (Chlorine dioxide, ClO2) 23
2.4.3食品接觸面氯系洗潔劑使用濃度及食品之殘氯規範 25
2.5微氣泡 25
2.5.1微氣泡的產生 26
2.5.2微氣泡之特性 29
2.5.3微氣泡在食品工業之清潔應用 30
叁、材料與方法 32
3.1實驗架構 32
3.2實驗材料 33
3.2.1常壓微氣泡水系統 33
3.2.2食品接觸面材料 34
3.2.3實驗菌株 34
3.2.4實驗藥品 34
3.3實驗方法 36
3.3.1確定生物膜形成條件 36
3.3.2殺菌試驗 37
3.3.3統計分析 42
肆、結果與討論 43
4.1確定生物膜形成條件 43
4.1.1 Salmonella. Typhimurium 43

4.1.2 Staphylococcus aureus 47

4.2微氣泡結合氯系殺菌劑對食品接觸面上生物膜之殺菌效力 52
4.2.1不同處理對接觸面上S. Typhimurium生物膜之殺菌結果 52
4.2.2不同處理對接觸面上S. aureus生物膜之殺菌結果 61
4.3微氣泡結合殺菌劑對食品接觸面上生物膜之處理水中理化性質 71
4.3.1酸鹼值 (pH值) 71
4.3.2氧化還原電位 (ORP值) 71
4.3.3溫度 (Temp) 72
4.3.4有效氯 (Available chlorine) 72
4.4接觸面上的生物膜-多醣染色 81
4.4.1 S. Typhimurium 81

4.4.2 S. aureus 85

4.5接觸面上的生物膜-掃描式電子顯微鏡 (SEM) 88
4.5.1 S. Typhimurium 88

4.5.2 S. aureus 91

伍、結論 94
協助研究單位 96
參考文獻 97

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