臺灣博碩士論文加值系統

本研究針對4株乳酸菌之不同部位進行體外抗氧化活性之分析，並以抗氧化活性較佳之部位探討是否具降低腸道上皮細胞氧化傷害之效果。使用之4株乳酸菌分別為Bifidobacterium longum B6、B. longum 15708、Lactobacillus bulgaricus 1006及Streptococcus thermophilus 3641，使用菌數為1010、109、108 CFU/mL，並且將其製備為活菌、熱致死菌、胞內液、熱處理胞內液及細胞壁等五個部份。抗氧化活性之評估包括總抗氧化力、清除DPPH自由基能力、清除過氧化氫能力、清除羥自由基能力及螯合亞鐵離子能力，並以細胞模式探討乳酸菌不同部分降低腸道上皮細胞 (Caco-2 cells) 氧化傷害之效果。在細胞模式中，將抗氧化活性較佳的部份與人類腸道上皮細胞進行共同培養，再以過氧化氫刺激細胞株分泌interleukin-8 (IL-8)，以觀察樣品是否有保護腸道上皮細胞降低其氧化傷害。結果顯示以乳酸菌樣品進行抗氧化活性評估及降低過氧化氫誘導Caco-2細胞株分泌IL-8的試驗中，大部分的樣品皆因為菌數之增加而有較好的抗氧化能力。當菌數為1010 CFU/mL時，同一菌株下總抗氧化能力較好的部位為熱致死菌、胞内液及熱處理胞內液，其中以B. longum B6之熱處理胞內液之總抗氧化能力最高 (291.3μM)；清除DPPH能力之結果中，4株菌中之活菌及熱致死菌清除DPPH之能力皆可達80%以上；清除過氧化氫能力以B. longum B6、B. longum 15708及S. thermophilus 3641之熱處理胞內液 (81.8、67.7及55.1%) 與L. bulgaricus 1006之熱致死菌 (88.9%) 較其它部位高；清除羥自由基能力則因菌株之不同，效果較好的部位亦有不同；螯合亞鐵離子能力較佳的部位為B. longum B6及L. bulgaricus 1006之熱致死菌 (88.1及92.5%) 與B. longum 15708及S. thermophilus 3641之熱處理胞內液 (72.3及93.5%)。於細胞試驗結果顯示B. longum B6、B. longum 15708、L. bulgaricus 1006及S. thermophilus 3641之熱致死菌、胞內液及熱處理胞內液與1 mM H2O2 處理組比較，大部分皆可降低Caco-2細胞株分泌IL-8的量 (P ＜0.05)。

Antioxidative ability of lactic acid bacteria including Bifidobacterium longum B6, B. longum 15708, Lactobacillus bulgaricus 1006 and Streptococcus thermophilus 3641 were examined in vitro. The fractions of lactic acid bacteria including intact cells, heat-killed cells, intracellular fractions, heat-treated intracellular fractions and cell wall were used for our study. The different bacterial concentration including 1010, 109, 108 CFU/mL were used. The best antioxidative ability of the fractions of lactic acid bacteria was further used for study on interleukin-8 (IL-8) secretion in H2O2-treated Caco-2 cells. The cell model can evaluate the protective effects of the fractions of lactic acid bacteria against H2O2-induced oxidative stress. Antioxidative abilities including TEAC, scavenging ability of DPPH, scavenging ability of H2O2, scavenging ability of OH• and chelating ability on ferrous ions were determined. The antioxidative ability of lactic acid bacteria depended on difference of strains and number of bacterium and expressed strain-dependent manners. Heat-treated intracellular fractions of B. longum B6 were highest TEAC (291.3μM). Scavenging ability of DPPH was more than 80% by all of these four lactic acid bacteria. Scavenging ability of H2O2 was higher than other fractions by heat-treated intracellular fractions of B. longum B6, B. longum 15708 and S. thermophilus 3641 (81.8, 67.7 and 55.1%), and heat-killed cells of L. bulgaricus 1006 (88.9%). Scavenging ability of OH• was different with difference of strains and fractions. Heat-killed cells of B. longum B6 and L. bulgaricus 1006 (88.1 and 92.5%), and heat-treated intracellular fractions of B. longum 15708 and S. thermophilus 3641 (72.3 and 93.5%) exhibited higher chelating ability on ferrous ions. Heat-killed cells, intracellular fractions and heat-treated intracellular fractions of B. longum B6, B. longum 15708, L. bulgaricus 1006 and S. thermophilus 3641 significantly decreased IL-8 secretion of Caco-2 cell.

目 次
中文摘要 i
Abstract ii
第一章 前言 1
第二章 文獻整理 3
一、乳酸菌 3
二、乳酸菌作為益生菌所具備之特性 3
三、乳酸菌的益生功能 3
(一) 維持腸道內菌相平衡 4
(二) 減緩乳糖不耐症 4
(三) 降低膽固醇 4
(四) 預防癌症 5
(五) 調節免疫系統 5
(六) 降低血壓 6
四、活性氧與氧化傷害 6
(一) 自由基與活性氧 6
(二) 活性氧對生物體的傷害 6
(三) 氧化壓力 7
(四) 抗氧化防禦系統 8
五、發炎反應與趨化激素 9
六、乳酸菌的抗氧化能力 10
七、實驗目的 10
第三章 材料與方法 18
一、 實驗材料 18
(一) 試驗菌株 18
(二) 乳酸菌培養基 18
(三) 細胞株 18
(四) 細胞株培養基 18
(五) 試藥 18
(六) 培養基及緩衝液配方 20
(七) 商業套組 21
(八) 試驗儀器 21
二、 實驗方法 22
(一) 菌株活化與保存 22
(二) 乳酸菌菌數測定 22
(三) 乳酸菌之樣品製備 22
(四) 乳酸菌之抗氧化活性分析 24
1. 總抗氧化力測定 24
2. 清除DPPH自由基能力測定 24
3. 清除過氧化氫能力測定 25
4. 清除羥自由基能力測定 26
5. 螯合亞鐵離子能力測定 27
(五) 細胞培養 27
1. 細胞解凍與培養 27
2. 細胞繼代培養 27
3. 細胞保存 28
4. 細胞計數 28
(六)細胞存活率分析 28
(七)過氧化氫誘導 IL-8 分泌試驗 29
(八)IL-8 之測試 29
(九) 蛋白質濃度測定 30
(十) 蛋白質電泳 31
(十一) 統計分析 31
第四章 結果與討論 32
一、乳酸菌之抗氧化活性分析 32
(一) 總抗氧化力 32
(二) 清除 DPPH 自由基能力 33
(三) 清除過氧化氫能力 34
(四) 清除羥自由基能力 35
(五) 螯合亞鐵離子能力 36
二、細胞存活率分析 37
三、乳酸菌對過氧化氫誘導 Caco-2 分泌 IL-8 之影響 38
四、乳酸菌胞內液及熱處理胞內液之蛋白質濃度測定及蛋白質電泳 40
第五章 結論 76
第六章 參考文獻 77
附錄 88

表目錄
表一、評估益生菌應具備的特性 12
表二、常見的自由基 13
表3-1、B. longum B6之總抗氧化力 42
表3-2、B. longum 15708之總抗氧化力 43
表3-3、L. bulgaricus 1006之總抗氧化力 44
表3-4、S. thermophilus 3641之總抗氧化力 45
表4-1、B. longum B6對DPPH自由基之清除能力 46
表4-2、B. longum 15708對DPPH自由基之清除能力 47
表4-3、L. bulgaricus 1006對DPPH自由基之清除能力 48
表4-4、S. thermophilus 3641對DPPH自由基之清除能力 49
表5-1、B. longum B6清除過氧化氫之能力 50
表5-2、B. longum 15708清除過氧化氫之能力 51
表5-3、L. bulgaricus 1006清除過氧化氫之能力 52
表5-4、S. thermophilus 3641清除過氧化氫之能力 53
表6-1、B. longum B6清除羥自由基之能力 54
表6-2、B. longum 15708清除羥自由基之能力 55
表6-3、L. bulgaricus 1006清除羥自由基之能力 56
表6-4、S. thermophilus 3641清除羥自由基之能力 57
表7-1、B. longum B6對亞鐵離子螯合能力 58
表7-2、B. longum 15708螯合能力 59
表7-3、L. bulgaricus 1006對亞鐵離子螯合能力 60
表7-4、S. thermophilus 3641對亞鐵離子螯合能力 61
表八、乳酸菌胞內液及熱處理胞內液之蛋白質濃度 62

圖目錄
圖一、人類腸道菌相的分布 14
圖二、三重態氧形成各種反應性氧分子之過程 15
圖三、體內造成氧化壓力的原因 16
圗四、趨化激素之生物功能 17
圖5-1、H2O2及GSH不同部位對Caco-2存活率之影響 63
圖5-2、B. longum B6不同部位對Caco-2存活率之影響 64
圖5-3、B. longum 15708不同部位對Caco-2存活率之影響 65
圖5-4、L. bulgaricus 1006不同部位對Caco-2存活率之影響 66
圖5-5、S. thermophilus 3641不同部位對Caco-2存活率之影響 67
圖6-1、B. longum B6對過氧化氫誘導Caco-2 細胞株分泌IL-8細胞激素之影響 68
圖6-2、B. longum 15708對過氧化氫誘導Caco-2 細胞株分泌IL-8細胞激素之影響 69
圖6-3、L. bulgaricus 1006對過氧化氫誘導Caco-2 細胞株分泌IL-8細胞激素之影響 70
圖6-4、S. thermophilus 3641對過氧化氫誘導Caco-2 細胞株分泌IL-8細胞激素之影響 71
圖7-1、100℃加熱處理對B. longum B6胞內液 (2×109 CFU/mL) 蛋白質濃度之影響 72
圖7-2、100℃加熱處理對B. longum 15708胞內液 (2×109 CFU/mL) 蛋白質濃度之影響 73
圖7-3、100℃加熱處理對L. bulgaricus 1006胞內液 (2×109 CFU/mL) 蛋白質濃度之影響 74
圖7-4、100℃加熱處理對S. thermophilus 3641胞內液 (2×109 CFU/mL) 蛋白質濃度之影響 75

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