臺灣博碩士論文加值系統

乳酸菌 (lactic acid bacteria) 是一種廣泛存在於人體、自然界與發酵食品中的菌株，一般被公認為安全之微生物，能產生多種抗菌物質，在細菌中產生的抑菌蛋白質被稱為細菌素(bacterion)，可抑制食品中的腐敗及病原菌，被認為是一種具有廣闊應用前景的天然食品防腐劑。本研究的主要目的為篩選並鑑定有較廣泛抑菌效果之乳酸菌菌株，生產及提取其細菌素，並對純化後的細菌素進行生化特性的分析。本實驗於實驗室保存菌株、臺灣南部採集海水樣品和市售自製優格中，篩選出三株具有較高抑菌效果的乳酸菌菌株，菌種鑑定後確認為 Lactiplantibacillus plantarum ( d1 )、 Companilactobacillus farciminis ( C4-67-2 )、Lacticaseibacillus rhamnosus ( H4-38 ) ，以此三株菌株作為探討對象。測試此三株菌之生長能力， d1 於24小時、 C4-67-2 於20小時、 H4-38 於20小時菌體與細菌素釋放的含量為最大量，且分別於16小時( d1 ) 和12小時( C4-67-2 和H4-38)開始出現對於大腸桿菌和金黃葡萄球菌的抑菌效果，並於24小時對金黃葡萄球菌抑制效果最佳，於32小時對大腸桿菌抑制效果最佳。本研究使用乙酸乙酯初步提取三株菌的細菌素。初步純化後的細菌素，與不同蛋白質分解酵素作用後活性降低，證實其活性物質為蛋白質。三種細菌素皆具有良好的耐熱性及耐鹽性，經 121℃ 加熱30分鐘，仍保有活性，鹽度適應範圍從1%至6%。對13株細菌有抑制作用；最低抑菌濃度( MIC )分別為158.19 μg/ml 、99.32 μg/ml 、69.59 μg/ml ，最低殺菌濃度( MBC )分別為316.38 μg/ml 、397.28 μg/ml 、278.39 μg/ml 。利用已知細菌素基因片段，透過 PCR 技術檢測，鑑定出 d1攜帶pln A、plnc8A及 plnc8B三個細菌素基因。綜合上述研究結果表明，此三株乳酸菌的細菌素具有良好的抗菌效果及耐熱、耐酸鹼性，適合用於食品工業中，它們具有可在產品中作為食品輔料的潛力，未來，可以應用於食物保存或益生菌食品的開發。

Lactic acid bacteria widely exist in the human body, nature, and fermented food; microorganisms generally recognized as safe can produce a variety of antibacterial substances. Especially antagonistic protein, which is produced by the bacteria, also called bacteriocin. It can inhibit spoilage and pathogenic bacteria in food. It is also considered as a potential natural food preservative in the future. The primary purpose of this study is to screen and identify lactic acid bacteria strains with a wide range of antibacterial effects, produce and extract their bacteriocins, and analyze their biochemical characteristics. In this study, three strains of lactic acid bacteria that were considered to be exhibited the most potent antibacterial effect were screened out from the lab-preserved strains, Seawater in southern Taiwan and yogurt, and the strains were identified as Lactiplantibacillus plantarum ( d1 )、 Companilactobacillus farciminis ( C4-67-2 )、 Lacticaseibacillus rhamnosus ( H4-38 ). We used these three strains of bacteria to be the object of this study. The growth ability of these three strains was tested; the highest bacteria and bacteriocin release content was observed during the growth of d1 for 24 hr, C4-67-2 for 20 hr, and H4-38 for 20 hr. The antibacterial effect started to appear against Escherichia coli and Staphylococcus aureus during the growth of d1 for 16 hours and the growth of C4-67-2 and H4-38 for 12 hours. The best inhibitory effect on Staphylococcus aureus was at 24 hours, and at 32 hours, it had the best inhibitory effect on Escherichia coli.Bacteriocins from three strains of bacteria were extracted from ethyl acetate.Then, the purification of bacteriocins reduced activity by different proteolytic enzymes action. It confirmed that bacteriocins be active substances in proteins. These three bacteriocins had good heat resistance, also for 30 min at 121 °C, and broad salt tolerance ranging from 1% to 6%.It is an inhibitory effect for 13 strains of bacteria, and the minimum inhibitory concentration ( MIC ) of bacteria is 158.19 μg/ml 、99.32 μg/ml 、69.59 μg/ml, and the minimum bactericidal concentration ( MBC ) of bacteria is 316.38 μg/ml 、397.28 μg/ml 、278.39 μg/ml.By using known bacteriocin gene fragments through a PCR array, it was identified that d1 carried three bacteriocin genes, plnA, plnc8A, and plnc8B.The above results show that bacteriocin-producing lactic acid bacteria have been isolated from seawater and yogurt, and bacteriocins have sound antibacterial effects, heat resistance, and salt resistance and are suitable for use in the food industry. In the future, it can be applied to food preservation or the development of probiotic food.

目錄
摘要.....................................................................i
Abstract................................................................ii
致謝....................................................................iv
目錄.....................................................................v
圖目錄..................................................................ix
表目錄...................................................................X
一、前言.................................................................1
1-1 乳酸菌...............................................................1
1.1.1 乳酸菌的定義及特性..................................................1
1.1.2 乳酸菌的分類........................................................1
1.1.3 乳酸菌對人體保健的功效...............................................3
1.1.4 乳酸菌的抗菌作用.....................................................6
1-2 細菌素................................................................8
1.2.1 細菌素的定義及起源...................................................8
1.2.2 細菌素的基本特性.....................................................8
1.2.3 由乳酸菌生產的細菌素.................................................9
1.2.4 乳酸菌細菌素的分類與抗菌機制..........................................9
1.2.5 乳酸菌細菌素的應用..................................................12
1-3 食源性病原菌..........................................................15
1.3.1 金黃葡萄球菌( Staphylococcus aureus )...............................15
1.3.2大腸桿菌( Escherichia coli )........................................16
1-4 研究動機及目的........................................................16
二、 材料與方法...........................................................17
2-1 實驗架構圖............................................................17
2-2 實驗材料..............................................................18
2.2.1實驗藥品.............................................................18
2.2.2儀器設備.............................................................20
2.2.3培養基配製...........................................................21
2.2.4本研究使用之菌株.....................................................22
2.2.5本研究使用之細胞株...................................................23
2-3 乳酸菌菌株篩選........................................................24
2-4 以洋菜膠孔洞擴散法初步篩選具抑菌能力之菌株...............................24
2-5 菌種鑑定..............................................................25
2-6 菌株保存與純化........................................................26
2-7 乳酸菌的抗菌能力......................................................27
2-8 乳酸菌生長特性........................................................27
2.8.1乳酸菌生長曲線.......................................................27
2.8.2乳酸菌 pH 值變化.....................................................27
2.8.3乳酸菌對革蘭氏陰性菌及陽性菌的抗菌活性.................................28
2-9 細菌素的萃取與部分純化.................................................28
2-10 乙酸乙酯萃取活性確認.................................................28
2-11 部分純化細菌素回溶於 PBS 之活性確認...................................29
2-12 蛋白質含量測定.......................................................29
2-13 細菌素生化特性.......................................................29
2.13.1 溫度對細菌素抑菌活性的影響..........................................29
2.13.2 不同酵素對細菌素抑菌活性的影響......................................30
2.13.3 pH 值環境對細菌素抑菌活性的影響.....................................30
2.13.4 鹽度對細菌素抑菌活性的影響..........................................30
2.13.5 有機溶劑對細菌素抑菌活性的影響......................................30
2.13.6 界面活性劑對細菌素抑菌活性的影響....................................31
2-14 細菌素最小抑菌濃度及最小殺菌濃度......................................31
2.14.1 最小抑菌濃度( Minimal inhibition concentration )..................31
2.14.2 最低殺菌濃度( Minimal bactericidal concentration )................32
2-15 細胞實驗............................................................32
2.15.1 細胞解凍與培養....................................................32
2.15.2 細胞繼代..........................................................33
2.15.3 細胞毒殺活性測試 ( MTT assay )....................................33
2-16 攜帶細菌素基因之確認.................................................33
2.16.1 細菌素基因篩選....................................................33
2.16.2 PCR 產物純化......................................................34
2.16.3 核酸接合反應( Ligation )..........................................35
2.16.4 熱休克轉型反應( Heat shock transformation ).......................35
2.16.5 Colony PCR.......................................................35
2.16.6 序列比對..........................................................36
三、 實驗結果.............................................................37
3-1 菌株篩選..............................................................37
3-2 菌種鑑定..............................................................37
3-3 乳酸菌之抑菌能力及抑菌譜範圍............................................38
3-4 乳酸菌生長特性.........................................................38
3-5 初步純化細菌素及回溶活性確認............................................40
3-6 蛋白質含量測定.........................................................41
3-7 溫度對細菌素抑菌活性的影響..............................................41
3-8 不同酵素對細菌素抑菌活性的影響...........................................42
3-9 細菌素對pH 酸鹼值的耐受性...............................................42
3-10 鹽度對細菌素抑菌活性的影響…............................................43
3-11 有機溶劑對細菌素抑菌活性的影響..........................................43
3-12 界面活性劑對細菌素抑菌活性的影響........................................44
3-13 細菌素之 MIC 、MBC....................................................44
3-14 細胞存活率............................................................45
3-15 細菌素基因選殖及序列比對...............................................45
四、 討論..................................................................46
參考文獻...................................................................48

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