臺灣博碩士論文加值系統

細菌素是由核醣體所合成的抗菌胜肽，因其可作為無毒添加劑應用在食品保鮮並延長保存期限，防止食源性致病菌造成食物腐敗以及預防食物中毒的發生而引起了廣泛的關注。另一方面，可溶性咖啡是全世界最受歡迎的飲料之一，在生產過程中會伴隨著產生大量的咖啡渣，咖啡渣內含許多有用的成分，其利用性相當廣泛，是一個非常有潛力的木質纖維素原料。因此，本研究的目的為探討以咖啡渣作為原料培養植物乳桿菌以及副乾酪乳桿菌生產出細菌素的可行性。
從本研究的結果證實，經萃取過後的咖啡渣確實可做為培養植物乳桿菌以及副乾酪乳桿菌的基質，所生產出的細菌素，對於試驗革蘭氏陽性菌(仙人掌桿菌)及革蘭氏陰性菌具有抑制活性。。經由實驗設計軟體得到生產植物乳桿菌以及副乾酪乳桿菌細菌素之最佳條件為咖啡渣總醣濃度: 15 mg/ml、19.1 mg/ml，半胱胺酸含量：0.059% (w/v)、0.062% (w/v)以及pH：7.1、7.08。並且經由活性測定後植物乳桿菌以及副乾酪乳桿菌之比活性分別為1163 AU/mg以及1920 AU/mg，由兩株乳酸菌生產出之細菌素在不同溫度及pH條件下儲存均擁有很好的穩定性(pH：4-10)，最後使用Tricine-SDS-PAGE測定兩株乳酸菌所生產出細菌素之分子量大小均低於10 kDa。

Soluble coffee, being one of the world’s most popular consuming drink, produces considerable amount of spent coffee ground (SCG) along with its production. The SCG could function as a potential lignocellulosic feedstock for production of bioproducts. The objective of this study was to evaluate the production of bacteriocin by Lactobacillus plantarum BCRC 10069 and Lactobacillus paracasei LCW 23 using spent coffee ground as feedstock. Bacteriocins are ribosomally-synthesized antibacterial peptides. These compounds have attracted significant attention because of their possible applications as non-toxic additives for food preservation and prevention of food spoilage by food-borne pathogenic bacteria. The bacteriocin produced by L. plantarum and L. paracasei showed inhibitory activity against Gram-positive and Gram-negative bacteria when Escherichia coli and Bacillus cereus were tested as model targets. The factors including pH, sugar concentration, and cysteine concentration which might affected the bacteriocin production by Lactococcus plantarum and Lactobacillus paracasei were optimized using Response Surface Methodology. The optimum conditions for bacteriocin production from L. plantarum were found to be SCG concentration 15 mg/ml, Cysteine content 0.059% (w/v), and pH 7.1. For L. paracasei, the optimal bateriocin production condition was SCG; oncentration 19.1 mg/ml, Cysteine content 0.062% (w/v), and pH 7.08 . Specific activity of bacteriocin was 1163 AU/mg and 1920 AU/ml recorded after 24 h after inoculation. Based on Tricine–SDS–PAGE, the bacteriocin molecular weight produced by L. plantarum and L. paracasei were both less than 10 kDa.

摘要 i
Abstract ii
誌謝 iii
目錄 iv
表目錄 viii
圖目錄 ix
第 1 章 緒論 1
1.1 前言 1
1.2 研究目的與動機 3
第 2 章 文獻回顧 4
2.1 咖啡豆 4
2.1.1 阿拉比卡咖啡豆 5
2.1.2 羅布斯塔咖啡豆 5
2.2 咖啡廢棄物 5
2.2.1 咖啡渣 8
2.3 木質纖維素之預處理 10
2.4 還原醣之酶水解 13
2.5 初級代謝級次級代謝之比較 14
2.6 抗生物質 14
2.6.1 生物合成法 15
2.6.2 化學合成法 15
2.6.3 半化學合成法 15
2.7 細菌素 16
2.8 乳酸菌之細菌素分類 16
2.9 細菌素與抗生素之比較 17
2.10 細菌素作用機制 18
2.9.1 阻礙細胞壁合成 20
2.9.2 抑制核酸合成 20
2.9.3 抑制蛋白質合成 20
2.9.4 破壞細胞膜之功能 20
2.9.5 抑制代謝能力 21
2.11 抗微生物測試 21
2.12 聚丙烯醯胺膠體電泳(SDS Poly-acrylamide-gel-electrophoresis, SDS-PAGE) 22
2.13 實驗設計 23
第 3 章 材料與方法 25
3.1 咖啡渣水解液製備 25
3.1.1 生物質前置準備 25
3.1.2 溶劑萃取 25
3.1.3 稀硫酸水解 27
3.1.4 酶水解 27
3.2 細菌素生產 28
3.2.1 菌體保存 28
3.2.2 以MRS 培養基培養乳酸菌生產細菌素作初步測試 29
3.2.3 以咖啡渣水解液培養乳酸菌生產細菌素 30
3.2.4 細菌素純化 32
3.3 溫度穩定性測試 32
3.4 pH值穩定性測試 33
3.5 優化細菌素生產製程 33
3.6 放大細菌素生產製程 37
3.7 分析方法 39
3.7.1 HPLC 單醣分析 39
3.7.2 蛋白質測定 40
3.7.3 分子量測定 41
3.7.4 細菌素抗菌活性測試 43
3.7.5 測定細菌素之活性 45
3.7.6 測定細菌素之最低抑制濃度 46
第 4 章 結果與討論 48
4.1 初步抗菌測試 48
4.2 細菌素之pH值穩定性 50
4.3 細菌素之溫度穩定性 51
4.4 細菌素分子量測定 53
4.5 咖啡渣產量評估 53
4.6 評估以咖啡渣水解液作為培養基生產細菌素之能力 55
4.7 以咖啡渣水解液作為培養基生產細菌素之優化 57
4.8 以咖啡渣水解液作為培養基生產細菌素之放大 63
4.9 細菌素活性測定 67
4.10 細菌素之最低抑制濃度(MIC) 68
第 5 章 結論 70
參考文獻 71

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