臺灣博碩士論文加值系統

病原性大腸桿菌 (Escherichia coli, E. coli)、金黃色葡萄球菌 (Staphylococcus aureus, S. aureus) 和沙門氏腸炎桿菌 (Salmonella enterica serovar Enteritidis, Sal. Enteritidis) 是常見引起食物中毒的食品病原菌。全球的研究中發現，由食物中分離出的病原菌對抗生素耐受性有持續增加的趨勢，而乳酸桿菌具有產生有機酸和細菌素的特定蛋白質或胜肽之能力，可抑制多種病原菌的生長。本研究自發酵芥菜、醃菜和其他植物發酵產品中分離出 50 株乳酸菌，研究其對抗 E. coli、S. aureus 和具抗藥性 Sal. Enteritidis 的抗菌活性，並利用聚合酶鏈反應 (PCR) 方法鑑定菌種及 plantaricin 基因。結果顯示乳酸菌分離株具有抑制 S. aureus、E. coli 及 Sal. Enteritidis 生長之能力，其中B0055、B0106、B0115 和 B0126 利用 PCR 方法檢測出含有 plnABCD、plnEFI、plnJ/ K 和 plnG/ V 的基因，且經由 16S rDNA 序列鑑定之結果為 Lactobacillus plantarm (L. plantarm)，推測所產生抑菌活性物質可能為蛋白質或胜肽類之細菌素。後續研究以 B0106 之發酵上清液 (spent culture supernatants, SCS) 進行穩定性試驗，結果顯示 B0106 之 SCS 對熱及酵素具有穩定性。B0106 菌體對人類結腸腺癌 (Caco-2) 細胞模擬腸道上皮細胞組織具有良好的吸附能力，而 B0106 菌體及其 SCS 亦能有效降低 Sal. Enteritidis SE07吸附於 Caco-2 細胞。另外本研究亦利用豆腐製作過程中所產生之廢水 (簡稱豆腐水，TFW) 作為乳酸菌的培養基，並探討乳酸菌發酵後之 TFW 上清液抗氧化及抗菌的活性。結果顯示 L. plantarm B0106 培養於 TFW 中，經 20 小時培養可獲得菌數高達9 log CFU/ ml，且具有抑制抗耐甲氧西林金黄色葡萄球菌 (methicillin-resistant S. aureus, BCRC 15211) 生長之能力，可於 6 個小時達到 90％ 以上之抑制效果。本乳酸菌分離株含有大部分的 plantaricin 細菌素基因，且對於病原菌及具抗藥性之病原菌有著高度的抑制活性，L. plantarm B0106 亦能利用 TFW 中的營養物質達到 9 log CFU/ ml菌量，其發酵液亦具有良好的抗氧化活性及GABA含量。在未來有潛力發展成為抗菌劑來降低致病菌的感染發生，亦可減少抗藥性菌株的產生，且在保健食品及化妝品上亦具應用潛力。

Pathogenic Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Salmonella enterica serovar Enteritidis (Sal. Enteritidis) are food pathogens that cause severe food poisoning. In the global studies, it has been found that pathogens isolated from food have a tendency to continue to increase resistance to antibiotics, whereas lactic acid bacteria (LAB) has the ability to produce specific proteins or peptides that can inhibit the growth of many pathogens. In this study, 50 strains of LAB were isolated from fermented mustard, pickle and other plant fermentation products, and their antibacterial activities against E. coli, S. aureus and Sal. Enteritidis were studied. The strains with antibacterial activity were determined by polymerase chain reaction (PCR) to identify strains and plantaricin genes. The results showed that the strains of LAB in this study were identified as Lactobacillus plantarm. Among of the tested LAB, B0055, B0106, B0115 and B0126 were detected positively in plnABCD, plnEFI, plnJ/ K and plnG/ V gene by PCR, suggesting that the antibacterial activity of the substance may be protein or peptides of plantaricin. B0106 spend culture supernatants (SCS) was stable to heat and enzyme. B0106 and its spend culture supernatants (SCS) could effectively reduce the adsorption of Sal.Enteritidis SE07 on human colonic adenocarcinoma cells (Caco-2). In addition, the waste water of tofu (TFW) was tested as the culture medium of LAB and the antioxidant, antimicrobial activity of the fermented TFW were also studied. The results showed that L. plantarm B0106 inoculated in TFW could grow up to 9 log CFU / ml after 20 hours and had the ability to inhibit the growth of methicillin-resistant S. aureus (BCRC 15211) to 90% after 6 hours. In this study, LAB strains contained most of the plantaricin genes and with inhibitory activity against pathogens. The TFW culture of L. plantarm B0106 has a good antioxidant activity and GABA content. In the future, it will be possible to use an antibacterial agent to reduce the incidence of pathogenic bacteria and reduce the emergence of drug-resistant strains, and application in health food and cosmetics has unlimited potential.

摘要....................................................i
Abstract..............................................iii
第一章 前言..............................................1
第二章 文獻回顧..........................................3
2.1. 食品傳染性病原菌..................................3
2.1.1. 金黃色葡萄球菌 (S. aureus)....................5
2.1.2. 大腸桿菌 (E. coli)...........................5
2.1.3. 沙門氏菌 (Salmonella spp.)...................6
2.2. 乳酸菌...........................................6
2.2.1. 乳酸菌的分類..................................7
2.2.2. 植生型乳酸菌..................................9
2.2.3. 乳酸菌在食品工業上的應用......................10
2.2.4. 細菌素之介紹.................................11
2.2.5. 乳酸菌對人體之保健功效........................16
2.3. 豆腐製作過程之廢棄物介紹..........................24
第三章 從發酵蔬菜中分離的 Lactobacillus plantarum 之抗菌活性
和 plantaricin 基因的探討........................26
3.1. 摘要............................................26
3.2. 前言............................................26
3.3. 材料與方法.......................................28
3.3.1. 實驗材料....................................28
3.3.2. 實驗方法....................................32
3.4. 結果與討論.......................................35
3.4.1. 乳酸菌之抗菌活性.............................35
3.4.2. 乳酸菌分離株之鑑定...........................37
3.4.3. plantaricin 基因的增幅與偵測.................40
3.5. 結論............................................44
第四章 乳酸菌對沙門氏菌之抗菌活性及抑制其吸附細胞之能力.....45
4.1. 摘要............................................45
4.2. 前言............................................45
4.3. 材料與方法.......................................47
4.3.1. 實驗材料....................................47
4.3.2. 實驗方法....................................51
4.4. 結果與討論......................................56
4.4.1. Sal. Enteritidis VSE01 與 SE07 抗生素敏感性試
驗..........................................56
4.4.2. 乳酸菌發酵上清液對 Sal. Enteritidis 的抗菌活
性..........................................58
4.4.3. 乳酸菌菌種鑑定及 plantaricin 基因偵測.........60
4.4.4. 乳酸菌 SCS 經加熱和酵素處理對抑菌活性之影響....63
4.4.5. 乳酸菌吸附 Caco-2 細胞能力試驗................66
4.4.6. B0106 及其 SCS 防止 Sal. Enteritidis 吸附 Caco-
2 細胞之試驗.................................66
4.4.7. B0106-SCS 對 Sal. Enteritidis 生長情形之影響.69
4.5. 結論............................................71
第五章 豆腐廢水特性及其應用於乳酸菌培養之評估..............72
5.1. 摘要............................................72
5.2. 前言............................................72
5.3. 材料與方法.......................................75
5.3.1. 實驗材料....................................75
5.3.2. 實驗方法....................................77
5.4. 結果與討論.......................................81
5.4.1. TFW 及發酵後之蛋白質、胜肽及 GABA 含量變化.....81
5.4.2. 乳酸菌在 TFW 中之生長曲線及添加糖類或蛋白質對乳酸
菌生長之影響.................................84
5.4.3. TFW 發酵後之抗氧化活性分析....................87
5.4.4. 乳酸菌發酵 TFW 抑制病原菌 MRSA 之能力.........89
5.5. 結論............................................91
第六章 參考文獻.........................................92
附錄一................................................111
附錄二................................................112
附錄三................................................113
附錄四................................................114

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