乳酸菌發酵清酒酒粕產物對肝細胞CYP2E1活性及清除ROS之探討

Effect of Lactic Acid Bacteria-fermented Sake Lees on Activating CYP2E1 Activity and Scavenging ROS in HepG2 Cell

許瀞文(Ching-Wen Hsu)

指導教授 ： 顏聰榮

大同大學/工程學院/生物工程學系所/碩士(2011年)

摘要

人體內的酒精代謝路徑為酒精經過酒精脫氫酶(alcohol dehydrogenase, ADH)或是由細胞色素P450 (cytochrome P450 2E1, CYP2E1)進行代謝生成乙醛，乙醛由乙醛脫氫酶(aldehyde dehydrogenase, ALDH)代謝生成乙酸然後排出體外。CYP2E1在代謝酒精過程中，會產生活性氧自由基(reactive oxygen species, ROS)。慢性酒精中毒是因人體內酒精濃度過高，產生大量的CYP2E1和ROS，造成肝臟細胞的傷害。本研究選用三株實驗室自行篩選的益生性乳酸菌Lactobacillus casei E7、Lactobacillus acidophilus V12、Lactobacillus casei N1及Lactobacillus rhamnosus GG進行清酒酒粕發酵(取自台灣菸酒公司林口酒廠)並以未發酵之酒粕當作對照組。發酵產物(SLGG、SE7、SV12及SN1)進行抗氧化能力確認並探討發酵產物對於酒精代謝酵素的影響。在抗氧化效果上，分別進行清除DPPH自由基、還原力、oxygen radical antioxidant capacity (ORAC)和清除細胞ROS等測試。結果發現發酵產物SE7及SV12具有較好的抗氧化能力，相當於同濃度之BHA。清除細胞ROS上，皆與陽性對照組維生素C (0.25 mg/ml)有相似的消除細胞內活性氧的能力。在酒精代謝酵素活性測試上，分別進行發酵產物對ADH、CYP2E1及ALDH酵素活性影響的測試。在0.75 mg/ml發酵產物濃度下，SE7及SV12可以增加CYP2E1酵素活性且為控制組(未加酒粕發酵產物)的2及1.5倍(p<0.05)。也可以增加ALDH酵素活性且為控制組的1.2倍及1.1倍，但皆不能影響ADH活性。結果顯示，酒粕發酵產物中含有影響CYP2E1酵素活性及抗氧化能力的成分物質。將發酵液與proteinase K反應後發現CYP2E1活性未提升，因此確認提升酵素活性之成分為蛋白質。用Sephacryl 100HR進行純化，並將管數回收跑native和 SDS-PAGE確認純度和分子量，再進行活性測試，結果發現有效蛋白之分子量為30 kDa。乳酸菌清酒酒粕發酵液可用來增加酒精代謝效率，降低酒精濃度，增加代謝所產生的ROS亦由酒粕發酵液本身所消除，故可減少慢性酒精中毒所帶來的肝臟傷害，達到保肝的功效。

關鍵字

乳酸菌 ； 細胞色素P450 ； 抗氧化 ； 酒粕

並列摘要

In the hepatocyte, ethanol is metabolized to acetaldehyde by alcohol dehydrogenase (ADH) or cytochrome P4502E1 (CYP2E1). Then, acetaldehyde is metabolized to acetic acid by aldehyde dehydrogenase (ALDH). CYP2E1 plays a significant role in regenerating reactive oxygen species (ROS) as well as oxidative stress. Chronic alcoholism induces the pressure of alcohol, and it can increase the level of CYP2E1, which in turn results in the generation of more ROS, exacerbating the potential for cell and tissue damage. In this study, lactic acid bacteria-fermented sake lees were used to test their antioxidant ability and affects on CYP2E1, ADH and ALDH activity. Sake lees fermented by Lactobacillus casei E7 and Lactobacillus acidophilus V12 have the highest antioxidant abilities. Also, the CYP2E1 activities from these two LAB-fermented sake lees culture filtrates are increased by 2.0 folds and 1.5 folds, respectively. ALDH activities are increased by 1.2 folds and 1.1 folds, respectively. However, there is no affects on the activity of ADH. Using Sephacryl 100HR to purify protein and run a native-PAGE and SDS-PAGE to check its purity and molecular weight. The molecular weight of purified protein is about 30 kDa and its isoelectric focusing point is close to 5.85. In conclusion,SE7-fermented sake lees culture filtrate not only increase CYP2E1 activity but also have the antioxidant ability. This helps alleviate the cell damages caused by chronic alcoholism.

並列關鍵字

antioxidant ； sake lees ； lactic acid bacteria ； CYP2E1

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