臺灣博碩士論文加值系統

gamma-胺基丁酸 (gamma-aminobutyric acid, GABA) 為一種廣泛存在於大自然中的非蛋白質胺基酸，具有降血壓、抗憂鬱、利尿與鎮定等多種生理功能。本試驗利用由健康魚腸道所分離之生產具 GABA 乳酸菌株發酵牛乳，探討其 GABA 生產最適化條件與乳酸菌菌株之益生菌特性。10 株產 GABA 乳酸菌菌株皆不具溶血性能力，其中 FPS 2520 具有膽鹽水解能力並可通過模擬腸胃道試驗仍有高菌數存活。另外，於 Caco-2 體外吸附細胞模式中，亦具有高吸附率 (80%)。在 10 株產 GABA 乳酸菌分離株中，以分離自秋刀魚 (Pacific saury) 腸道之菌株 FPS 2520 於牛乳中具有最高 GABA 生產能力。乳酸菌菌株 FPS 2520 培養於添加 20 mM MSG、1% 紅砂糖與 0.5% 酵母萃取物之 10% 全脂牛乳中，可得到最高之 GABA 產量，其產量為 10.67 ± 1.98 mg/mL，且 ACE 抑制活性之IC50 為 0.28 ± 0.02 mg/mL。另外以 4oC 冷藏儲存 14 天後，除了乳酸菌數稍微下降以外，pH 值、可滴定酸度及 GABA 產量與 0 天發酵乳樣品相比皆無顯著差異。接著將產 GABA 乳酸菌菌株 FPS 2520 與高蛋白質分解能力乳酸菌菌株 FKR 3737 以混株方式進行發酵，與單株發酵比較，可顯著提升其 GABA 產量約得到 GABA 產量 14.74 ± 0.32 mg/mL。

gamma-Aminobutyric acid (GABA) is a well-known non-protein amino acid, which is widely distributed in animals and plants. GABA has been reported to be a major inhibitory neurotransmitter in the mammalian central nervous system and show antihypertensive activity. This aims of this research are to manufacture a GABA-enriched fermented milk by lactic acid bacteria (LAB) isolated from fish intestines and to evaluate the probiotic potential property of the GABA-producing LAB. Preliminarily, lactic acid bacteria strains were isolated from healthy fish intestine from previous study. Ten lactic acid bacteria strains of 126 LAB strains were screened based on the capacity of synthesizing GABA. And ten GABA-producing strains show that none of them exhibited hemolytic activity. Moreover, five strains exhibited partial bile salt hydrolase activity, including the strain FPS 2520. FPS 2520 also showed a high percentage of adhesion to monolayer of Caco-2 cells. Strain FPS 2520 demonstrated high survival viability to gastrointestinal conditions simulating stomach and duodenum passage. Optimal conditions of strain FPS 2520 for producing GABA in whole milk were: 10% reconstituted whole milk, initial inoculum size of 5 log CFU/mL, addition of 20 mM MSG, 1% brown sugar, and 0.5% yeast extract during fermentation at 37oC for 48 hours. The results indicated FPS 2520 show a probiotic potential property and have a great application in milk fermentation for the production of G (10.67 mg/mL), and the IC50 of ACEI-inhibitory activity is 0.28 ± 0.02 mg/mL. Besides lactic acid bacteria number slightly decreased, pH value, titratable acidity and GABA concentration didn’t have significant difference compare to control at 4oC for 14 days. Using mixed strains, GABA-producing strain FPS 2520 and proteinase-positive FKR 3737 could significantly enhance the GABA production about 14.74 mg/mL.

目錄
壹、前言 1
貳、文獻整理 2
一、-胺基丁酸 (-aminobutyric acid, GABA) 2
1. -胺基丁酸簡介 2
2. -胺基丁酸之生化代謝途徑 2
3. GABA 生產方式 3
4. -胺基丁酸生理功能 4
5. Glutamate decarboxylase (GAD) 9
二、血管收縮素轉化酶 11
1. 血管收縮素轉化酶簡介 11
2. 血管收縮素轉化酶抑制胜肽的生理活性 11
三、益生菌及乳酸菌之介紹 12
1. 益生菌簡介 12
2. 乳酸菌之定義、分類與特性 12
3. 乳酸菌之營養保健功效 13
4. 乳酸菌之腸胃道耐受性 15
5. 乳酸菌與 GABA 之連結 16
四、發酵方法 18
2. 饋料批式發酵 18
3. 連續式發酵 19
4. 固定化發酵 19
參、實驗設計 21
肆、實驗材料與方法 22
一、材料 22
1. 原料 22
2. 實驗菌株 22
3. 化學藥品與培養基 23
二、儀器設備 25
三、實驗方法 26
1. 菌株保存 26
2. 菌株活化 26
3. 以牛乳為發酵基質生產 GABA 之最適條件 26
4. 混株發酵製備含 -胺基丁酸之發酵乳 28
5. 乳酸菌於模擬腸胃道耐受性試驗 29
6. 分析方法 30
7. 貯存安定性試驗 34
8. Caco-2 細胞吸附試驗 34
9. 統計分析 37
伍、結果與討論 38
1. 源自魚腸道之乳酸菌分離株於牛乳中產 GABA 潛力菌株之篩選 38
2. 全脂牛乳生產 GABA 最適條件探討 39
3. 混合菌株發酵 44
4. 乳酸菌在模擬腸胃道耐受性試驗 45
5. 乳酸菌菌株溶血性試驗 46
6. 膽鹽水解能力分析 47
7. Caco-2 細胞吸附性試驗 47
陸、結論 49
柒、參考文獻 50
捌、圖表 72
圖目錄
圖一、GABA 標準品 (A)、FPS 2520 發酵乳 (B) 之 HPLC 分析圖譜。 72
圖二、以 HPLC 分析的 GABA 標準曲線。 73
圖三、十株自魚腸道分離之乳酸菌株在含 1% MSG 之 10% 全脂牛乳中於 37oC 培養 48 小時所得菌數 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量。 74
圖四、十株乳酸菌株培養於含 1% MSG 之 10% 脫脂牛乳中所得菌數 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 75
圖五、含 1% MSG 之全脂牛乳濃度對 FPS 2520 生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之變化。 76
圖六、含 1% MSG 之 10% 全脂牛乳接種不同接種量對 FPS 2520生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之變化。 77
圖七、含不同 MSG 濃度之 10% 全脂牛乳對 FPS 2520 生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之變化。 78
圖八、含 20 mM MSG 之 10% 全脂牛乳添加 1% 不同碳源對 FPS 2520生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 79
圖九、含 60 mM MSG 之 10% 全脂牛乳添加 1% 不同碳源對 FPS 2520生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 80
圖十、含 0.5% 不同種類氮源與 20 mM MSG 及 1% 紅糖之 10% 全脂牛乳對 FPS 2520生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之變化。 81
圖十一、含 0.5% 不同種類氮源與 60 mM MSG 及 1% 紅糖之 10% 全脂牛乳對 FPS 2520生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 82
圖十二、含 1% 紅糖及 0.5% 酵母萃取物之 10% 全脂牛乳中添加不同濃度 MSG 對 FPS 2520 生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 83
圖十三、FPS 2520 培養於含不同濃度 PLP 與 20 mM MSG、1% 黑糖及 0.5% 酵母萃取物 之 10% 全脂牛乳對其生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 84
圖十四、FPS 2520 於含 20 mM MSG、1% 黑糖和 0.5% 酵母萃取物之 10% 全脂牛乳中，培養於不同溫度對 FPS 2520 生長 (A)、pH 值 (B)、可滴定酸度 (C) 與 GABA (D) 含量之影響。 85
圖十五、FPS 2520 培養於 37oC 之牛乳中，其 GABA 產量、乳酸菌生長、pH 值與可滴定酸度之變化。 86
圖十六、FPS 2520 發酵乳於 4oC 冷藏過程中其 GABA 產量、生長、pH 值、與可滴定酸度之變化。 87
圖十七、以 Folin Ciocalteu method 分析的 L-tyrosine 標準曲線。 88
圖十八、十株乳酸菌分離株及三株標準菌株於 37oC 在 Lactobacilli MRS broth 培養 48 小時所產之蛋白酶活性。 89
圖十九、乳酸菌 FPS 2520 劃線培養於 Blood agar 之溶血情形。 90
圖二十、(A) 乳酸菌分離株 FPS 2520 與 (B) Lactobacillus rhamnosus GG 吸附於 Caco-2 cell 單層膜上之倒立光學顯微鏡照相圖 (400 倍放大倍率)。 91

表目錄
表一、FPS 2520 發酵乳於 4oC 儲存期間胜肽含量、ACE 抑制活性及其 IC50 之變化。 92
表二、單株及混株發酵對乳酸菌菌數生長、pH 值、可滴定酸度及 GABA 含量之影響。 93
表三、以乳酸菌發酵製備含 GABA 之發酵乳之情形。 94
表四、FPS 2520 於含 0.3% (w/v) 胃蛋白酶 (pepsin) 之模擬胃液 (pH 3.0) 中三小時之不同時間點存活菌數及其存活率。 95
表五、FPS 2520 於含 0.3% (w/v) 胃蛋白酶 (pepsin) 之模擬胃液 (pH 2.0) 中九十分鐘之不同時間點存活菌數及其存活率。 96
表六、FPS 2520 於含 0.1% (w/v) 胰酶 (pancreatin) 和 0.3% 膽鹽之模擬腸液 (pH 8.0) 中四小時之不同時間點存活菌數及其存活率。 97
表七、FPS 2520 於連續模擬腸胃道試驗 (pH 3.0 添加 3 g/L 胃蛋白酶之模擬胃液與 pH 8.0 添加 0.3% 膽鹽及 1 g/L 胰酶) 之菌量變化及其存活率。 98
表八、10 株乳酸菌分離菌株之膽鹽水解能力及溶血活性之測定。 99
表九、各試驗乳酸菌菌株對 Caco-2 cell 單層膜之吸附率。 100

附錄目錄
附錄一、GABA的中性分子結構 (a) 及兩性離子結構 (b)。 101
附錄二、GABA shunt 代謝路徑圖。 102
附錄三、GABA 受體結構。 103
附錄四、血管收縮素轉化酶之整體蛋白質構型。 104
附錄五、人體腸胃道菌相分布圖。 105
附錄六、一級胺與 OPA 之衍生化反應式。 106
附錄七、不同之維生素B6 構型。 107
附錄八、腸胃道消化概況。 108
附錄九、膽鹽結合方式及 BSH 之作用機制。 109

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