清酒酒粕乳酸菌發酵液抑制血管收縮素轉換酶活性之研究

Studies on ACE-inhibitory activity of lactic acid bacteria fermented sake lees

鄭淑文(Shu-Wen Cheng)

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

高血壓為心血管疾病危險因子之一。血管收縮素轉換酶(angiotensin I converting enzyme, ACE)為renin angiotension system (腎素-血管收縮素系統, RAS)中造成血壓上升的重要酵素，ACE-抑制劑 (ACE-inhibitor, ACEI)可以抑制ACE酵素，可使體內血管收縮素angiotensin-I無法變成angiotensin-II，使血管收縮減緩而達到延緩血壓上升的效果。許多研究顯示，乳酸菌可經發酵產生具有調節血壓功效之物質，本研究利用本實驗室從泡菜醱酵產物所篩選出來具乳酸菌株(Lb. acidophilus V12、Lb. casei NI、E7、E30、E33、E40、Lb. plantarum E38、E55、V29、Lb. rhamnosus B7及Lb. rhamnosusGG)對清酒酒粕進行小量(20 ml)發酵培養。培養12小時後的發酵上清液，測試酒粕發酵液抑制ACE酵素的活性。清酒酒粕的抑制能力為7.5 AU/mg，在乳酸菌株的清酒發酵液的抑制活性比較上，各清酒酒粕發酵液樣品(SLGG、SNI、SE7、SE30、SE33、SE38、SE40、SE55、SV12及SV29)，均具有抑制ACE酵素活性的能力，抑制ACE酵素活性能力為32.4 AU/mg至47.9 AU/mg，其中以SE55的抑制活性能力最好；而且這些以乳酸菌發酵的清酒酒粕發酵液，較清酒酒粕的ACE抑制能力具顯著差異。接下來再以Lb. plantarum E55對清酒酒粕進行500 ml發酵培養，並以不添加菌，只有清酒酒粕當作對照組進行比較。經12小時發酵反應後，經離心及10 kDa濾膜超濃縮過濾器(Amincon Ultrafiltration)過濾收集各濾液進行抑制ACE酵素活性的比較。清酒酒粕的ACE抑制活性(6.3 AU/mg)，經乳酸菌發酵後抑制活性明顯增加(29.4 AU/mg)，粗蛋白含量也增加。顯示清酒酒粕經乳酸菌發酵後，能增加影響抑制活性的機能性peptide含量。故將清酒發酵樣品SE55的上清液，經10 kDa濾膜超濃縮過濾器(Amincon Ultrafiltration)過濾收集濾液，調整發酵產物SE55濃度為200 mg/ml，並以HW-40膠體層析進行劃分，可得5個分劃波峰(Fraction Peaks)。經大量回收HW-40膠體層析各分劃波峰，進行機能性功效物質的抑制ACE酵素活性分析。已確認一個回收的分劃波峰具有較高的抑制ACE酵素活性能力。再經由RP-C18進行第二次的分離，已分離回收一純的分劃波峰，其ACE抑制能力為366.2±8.3 AU/mg，純化倍數為10.4，回收率為3.5%，其IC50 為 3.7±0.03 μg/mL(8.3±0.1 μM)。經由LC/MS/MS分析得知，是一個由4個胺基酸所組成之ACEI peptide。由序列結果推測的分子量為445 Dalton、pI值為3.23。

關鍵字

乳酸菌 ； 清酒酒粕 ； 血管收縮素轉換酶

並列摘要

Angiotensin I-converting enzyme (ACE) plays an important role in regulating blood pressure. In the rennin-angiotenisn system, ACE cleaves the dipeptide at the C-terminal portion of angiotensin I and produces the potent vasopressor angiotensin II. This induces the release of aldosterone that causes the retention of sodium ions by the kidney and elevates blood volume, thus increasing blood pressure. ACE-inhibitory (ACEI) can inhibit ACE activity and decrease angiotensin II be converted, thus lowering blood pressure. ACEI can lowering the blood pressure via the inhibition of the ACE activity, consequently decrease the convertion of the angiotensin II. Currently, proteins that produced through enzyme hydrolysis and/or fermented by selected lactic acid bacteria are the main sources to produce bioactive peptide for ACEI. These peptides are effective in lowering the blood pressure of moderately hypertensive humans or spontaneously hypertensive rats and are used as pharmaceuticals. In this study, nine strains isolated from local Taiwanese fermented vegetables, which could tolerate low pH conditions and high concentration of bile salts were selected for sake lees fermentation. The specific ACEI activity of LAB-fermented sake lees was determined. As the results, all LAB-fermented products could inhibit ACE activity and specific activity was ranged from 32.4 to 47.9 AU/mg. Lactobacillus plantarum strain E55 which the fermented product had the highest specific activity was selected to large scale fermentation. In a 500 mL fermention, the specific ACEI activity was 40.0 AU/mg and increased significantly (P＜0.05) compared with unfermented sake lees (7.5 AU/mg). It suggested that Lactobacillus plantarum strain E55 used during fermentation of sake lees may synthesize ACEI peptide with a potential antihypertensive effect. Then, the fermented product was purified by ToyopearlR HW-40 GL column and HPLC reverse phase C18 (ODS) column. The purified peptide had the specific ACEI activity of 366.2 AU/mg, enhanced 10.4-fold and the recovery was 3.5%. The IC50 was calculated to be 3.7±0.0 μg/mL(8.3±0.1 μM). Identification of this fraction carried out through LC/MS/MS, and knows this is a peptide composition by four animo acid. The peptide molecular weight is 445 Dalton, pI 3.23 conjecture through peptide sequences.

並列關鍵字

lactic acid bacteria ； sake lees ； Angiotensin I-converting enzyme

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