臺灣博碩士論文加值系統

乳酸菌（Lactic acid bacteria, LAB）一般為公認安全菌種（Generally recognized as safe, GRAS），LAB作為益生菌必須通過人體消化系統，且有效定殖於腸道中，能維持腸道機能、降低膽固醇、免疫調節等對人體發揮其健康效益。本實驗室自短尾大目鯛（Priacanthus marcracanthus）魚體腸道內，成功分離出一株γ-胺基丁酸（γ-Aminobutyric acid, GABA）高產率與耐酸、耐膽鹽特性之乳酸菌菌株，經16S rRNA鑑定命名為Lactobacillus brevis RK03。故本研究首先分析L.brevis RK03潛在之益生菌特性，並探討不同乾燥條件對其儲存安定性之影響，L.brevis RK03對九種抗生素敏感試驗之結果顯示，其對氨苄青黴素（Ampicillin）、氯黴素（Chloramphenicol）、紅黴素（Erythromycin）、克林黴素（Clindamycin）、福利平（Rifampicin）與四環素（Tetracycline）具有敏感性即不具有抗藥性基因；抑制病原菌試驗方面，其對大腸桿菌（Escherichia coli）、沙門氏菌（Salmonella enterica sp.）、金黃色葡萄球菌（Staphylococcus aureus sp.）、李斯特菌（Listeria monocytogenes）與腸炎弧菌（Vibrio parahaemolyticus）皆具有抑菌活性；菌體之絮凝能力（Autoaggregation）試驗經24小時結果為42.25%；表面疏水性(Surface hydrophobicity)為46.98%；體外吸附HT-29細胞吸附率為66.79%，熱安定性方面，添加5%乳清蛋白保護劑組，於50℃加熱1小時存活率達87%。冷凍乾燥技術最佳菌體活性以添加混合保護劑20%麥芽糊精及5%乳清蛋白組，其菌數達11.41 ± 0.47 log CFU/g，並經12小時連續式腸胃道模擬試驗存活率為72.8%。流動床造粒最佳菌活性條件為入風溫度60℃、排風速1500rpm、噴霧流速5mL/min、載體麥芽糊精、進樣口中段、菌液添加5%乳清蛋白，其菌數達9.13 ± 0.08 log CFU/g，並經12小時連續式腸胃道模擬試驗存活率為61.6%。L. brevis RK03依上述兩種最佳乾燥條件所製備之粉末，經加速儲藏試驗，套用阿瑞尼斯方程式(Arrhenius equation)皆成線性關係，依據此線性關係進行儲藏期間之預估值與實際值比較之結果，預估值與實際值無顯著性差異，於4℃及25℃ 儲存兩個月皆具有良好穩定性，其菌數約8.17-9.19 log CFU/g。綜合上述結果，L. brevis RK03為一株具有作為益生菌之潛力菌株，並且，經由冷凍乾燥與流動床造粒技術可成功製備具機能性之粉末化菌體。

Lactic acid bacteria is generally recognized as safe. As a probiotic, LAB must pass through the human digestive system and effectively colonize the intestines. It can maintain intestinal function, lower cholesterol, and regulate immune function. Research strain was isolated from fish intestines of Priacanthus macracanthus. It was identified by 16s rRNA, and named as Lactobacillus brevis RK03. It can produce high yield of γ-aminobutyric acid and also has potential probiotic characteristics with acid and bile resistant. The aim of this study was to analyze the potential probiotic characteristics and effect of different drying conditions on the shelf life of L.brevis RK03. The probiotic characteristics of L.brevis RK03 result showed that sensitive and non-drug resistant gene to ampicillin, chloramphenicol, erythromycin, clindamycin, rifampicin and tetracycline. Inhibit pathogenic bacteria, antagonizes Escherichia coli, Salmonella sp., Staphylococcus sp., Listeria monocytogenes and Vibrio parahaemolyticus. Autoaggregation is 42.25% colonization ability after 24 hours. Cell surface hydrophobicity is 46.98% potential adsorption. Adsorption rate of HT-29 cells is 66.79%. It was heated at 50 ° C for 1 hour to add 5% (w/v) whey protein the survival rate of sample which 87%.The optimal bacterial activity of the vacuum freeze drying was added to the mixed protective agent 20% maltodextrin and 5% whey protein group, the cell counts reached 11.41 ± 0.47 log CFU / g, and the survival rate after 12 hours of continuous gastrointestinal simulation test 72.8%. The optimum conditions for the granulation of the fluidized bed were the inlet air temperature of 60 ° C, the exhaust air velocity of 1500 rpm, the spray flow rate of 5 mL/min, the carrier maltodextrin, the middle of the injection port and the addition of 5% whey protein to the bacterial liquid. The cell counts reached 9.13. ± 0.08 log CFU/g, and the survival rate was 61.6% after 12 hours of continuous gastrointestinal simulation test. L. brevis RK03 has good stability after storage for about two months at 4 ℃and 25 ℃ after two drying techniques, and the cell counts is about 8.17-9.19 log CFU/g. The estimated values obtained by the Arnes formula are consistent with the experimental results. In summary, L. brevis RK03 is a potential strain of probiotics, and functional pulverized cells can be successfully prepared by freeze drying and fluid bed granulation techniques.

摘要 I
Abstract III
致謝 V
目錄 V
圖目錄 XI
表目錄 XIII
附圖目錄 XV
附表目錄 XVI
壹、前言 1
貳、文獻整理 3
一、益生菌（Probiotic） 3
（一）益生菌特性 3
（二）益生菌功效 4
二、乳酸菌（Lactic acid bacteria LAB） 7
（一）乳酸菌定義與特性 7
（二）乳酸菌分類 8
（三）乳酸菌生長代謝 8
（四）乳酸菌之應用 10
（五）短乳桿菌 11
三、冷凍乾燥技術（Freeze drying） 14
（一）冷凍乾燥原理 14
（二）冷凍乾燥之應用 15
（三）冷凍乾燥優缺點 15
四、流動床造粒（Fluid bed granulation） 16
（一）流動床造粒機制 17
（二）流動床造粒之種類 18
五、保護劑對菌體乾燥之影響 22
六、阿瑞尼斯方程式（Arrhenius equation） 24
（一）應用於加速儲藏試驗 25
參、實驗架構 26
肆、材料與方法 27
一、實驗材料 27
（一）實驗菌株與細胞來源 27
（二）培養基 28
（三）實驗藥品 28
（四）儀器設備 29
二、實驗方法 30
（一）菌株之保存 30
（二）菌株之活化 31
（三）益生菌特性分析 31
（四）熱安定性試驗 35
（五）冷凍乾燥 35
（六）流動床造粒乾燥 36
（七）模擬腸胃道試驗 37
（八）掃描式電子顯微鏡 38
（九）菌體粉末之特性分析 38
（十）儲藏性試驗 40
（十一）菌落數測定 41
（十二）統計分析 42
伍、結果與討論 43
一、培養時間對L.brevis RK03菌體生長之影響 43
二、L.brevis RK03益生菌特性之分析 43
三、L.brevis RK03熱安定性之探討 46
四、冷凍乾燥對L.brevis RK03活性之探討 47
（一）添加單一保護劑對L.brevis RK03菌體存活之影響 47
（二）添加混合保護劑對L.brevis RK03菌體存活之影響 48
五、流動床乾燥對L.brevis RK03活性之影響 49
（一）不同入風溫度對菌數之影響 49
（二）不同載體對菌數之影響 50
（三）不同進樣口對菌數之影響 50
（四）添加保護劑對菌數之影響 51
六、體外模擬腸胃道試驗 51
（一）冷凍乾燥處理之L.brevis RK03菌體 51
（二）流動床乾燥處理之L.brevis RK03菌體 53
七、L. brevis RK03菌體粉末之物理特性分析 55
八、L.brevis RK03最適乾燥條件之儲藏性 59
（一）加速儲存實驗 59
（二）一般儲存試驗 60
陸、結論 95
柒、參考文獻 97

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