臺灣博碩士論文加值系統

酒，自古以來一直陪伴著人類的文明前進，酒在華人的文化中佔有重要的地位，在應酬上不可或缺的就是酒，而相關研究指出，長期過度攝入酒精，容易對個人健康、家庭甚至危害他人安全。亞洲人又因基因缺陷，相當一部分的人體內代謝乙醛 ( 酒精代謝之中間物 ) 的酵素活性較差，也就是乙醛去氫酶 ( Acetaldehyde dehydrogenase，ALDH ) 的基因型突變，導致酵素活性大幅降低，乙醛更容易在體內堆積，造成一系列身體損傷。
實驗室先前由自然醱酵酸白菜醱酵汁中篩選出能夠有效代謝酒精的菌株YW108，但在進一步的人體實驗後，發現YW108確實能幫助人體代謝酒精，但代謝乙醛的速率並沒有如預期般的好，YW108在人體中反而會提高乙醛產生的速率，加速堆積，更快進入酒醉狀態。因此本實驗第一個目的就是利用酵素法分析YW108酒精代謝活性部位，再利用基因質體與蛋白質體學分析找出可能提升乙醛代謝能力的酵素，添加到YW108產品中，提升代謝乙醛能力。
酵素法首先發現乙醛誘導後的YW108胞內可溶蛋白部分具有能將乙醛代謝成乙酸的活性，後用蛋白質體學分析發現到經乙醛誘導後的YW108確實會表現多種酒精代謝相關酵素，其中兩種具有代謝乙醛能力，我們將這兩種酵素定義為目標蛋白質，未來則可利用本實驗建立的基因資料庫進行進一步的純化表現實驗，更加針對性的提升YW108產品代謝乙醛的能力。
本實驗另一個目的則是利用Phenotype MicroArrays ( PM ) 進行YW108生長源分析，目的是找出YW108最適培養條件。過去實驗室培養YW108都以MRS當作主要培養基進行培養，利用PM後得到大量YW108對各式營養源的嗜好性，這些數據在未來YW108進入工業規模生產可以用來改良培養基以提升YW108的產量。

Wine has been accompanying the progress of human civilization since ancient times. In the east, wine plays an important role in the Chinese culture, especially at sociality. According to the report, long-term excessive intake of alcohol is likely to endanger individual health and even harm others. Moreover, about1/4 of Asia people suffer from lower activity of acetaldehyde ( Intermediate of alcohol metabolism ) metabolism due to the gene mutation of acetaldehyde dehydrogenase ( ALDH ). This genetic mutation is the main reason of the significant decrease in ALDH activity and that causes the accumulate of acetaldehyde in the body, and therefore causing a series of physical damage.
Previously, our lab screened a probiotic strain from fermented sour cabbage liquid. The strain was characterized and designated as Lactobacillus brevis YW108 which have ability to metabolize alcohol. However, in human trial YW108 does help human body to metabolize alcohol, but the rate of acetaldehyde metabolism is not as good as expected. Thus for people who suffering from ALDH mutation, L. brevis YW108 accelerate accumulation of acetaldehyde, and get into a drunken state more quickly.
In the first part of this study, we analyze alcohol and acetaldehyde metabolic active fraction of YW108 by enzyme method. Then, genomic and proteomic analysis were used to identify enzymes that may enhance the metabolism of acetaldehyde We found that the intracellular soluble protein fraction of YW108 induced by acetaldehyde has the activity of metabolizing acetaldehyde by enzymeatic analysis. Using proteomic analysis, it was found that YW108 induced by acetaldehyde does exhibit a variety of enzymes related to alcohol metabolism, two of which have the ability to metabolize acetaldehyde. We define these two enzymes as target proteins which can seen as candidate for acetaldehyde reduction product development.
In another part of this study, we used Phenotype MicroArrays ( PM ) for YW108 growth source analysis to find the optimal culture conditions. In the future, we can use these data to improve the medium to increase the yield of YW108 when it enters industrial scale production.

壹、 前言大綱 1
一、 飲酒習慣與健康影響 1
二、 人體體內酒精吸收代謝 1
(一) 影響酒醉的因素 2
三、 酒精對人體的影響 3
(一) 乙醇對人體健康的影響 3
(二) 乙醛對人體健康的影響 3
(三) 酒精中毒 4
四、 人體內酒精代謝與相關酵素 5
(一) 人類之乙醇脫氫酶 6
(二) CYP2E1 7
(三) 人類之乙醛脫氫酶 7
(四) ALDH 相關SNP 7
1、 ADH1B 8
2、 ADH1C 8
3、 ALDH2 8
(五) 微生物酒精代謝相關研究 9
1、 乳酸菌(lactic acid bacteria, LAB)介紹 9
2、 釀酒酵母Saccharomyces cerevisiae 10
3、 Streptococcus 10
4、 Lactobacillus brevis YW108 11
(六) 破菌系統 11
1、 機械法 11
2、 非機械法 13
(七) 菌體基因分析與蛋白質鑑定 14
1、 基因組體學( Genomics ) 14
2、 蛋白質體學 16
(八) YW108生長源分析 18
貳、 研究源起與實驗目的 19
參、 實驗架構 20
肆、 材料與方法 21
一、 菌種 21
二、 實驗中的儀器、藥品及試劑 21
三、 YW108基因體與蛋白質體研究分析 21
(一) YW108全基因鑑定 21
(二) YW108蛋白質體分析 22
(三) Phenotype MicroArrays(PM) 26
(四) 酵素活性產生部位分析 27
(五) YW108顯微鏡觀察與平板計數 27
(六) SNP檢驗 27
伍、 結果與討論 29
一、 具代謝酒精能力之乳酸菌株YW108特性分析 29
(一) YW108在乙醛與酒精誘導下的生長狀況及顯微鏡觀察 29
(二) Lactobacillus brevis YW108 Phenotype MicroArrays Assay 29
(三) 酵素法分析YW108活性部位 31
(四) YW108蛋白質與基因質體分析 33
(五) SNP分析 36
陸、 結論 38
附錄一、表格 39
壹、 附錄二、圖片 81
貳、 附錄三、藥品與儀器 129
一、 培養基 129
二、 DNA抽取藥品 129
三、 破菌用藥品 129
四、 PM所需藥品 129
五、 酵素法用藥品 130
六、 蛋白質濃度測定 130
七、 儀器 131
壹拾、 參考文獻 132

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