|Abstract: ||本研究之目的在於探討 Agarase 基因 AS-IIIb 及 PV-Ib 利用穿梭 載體對乳酸菌進行轉殖，並以乳酸菌發酵含青海菜寡醣之脫脂牛乳 ，且探討青海菜寡醣對發酵產物之抗氧化、抗凝血活性、美白活性的影 響。乳酸菌 Ped. pentosaceus MFS、MFL、Lb. bulgaricus BCRC 10696、Lb. plantarum BCRC 10069 以 mutanolysin 處理後之原生質體(Protoplast) 形成效率，分別為 46.3%、3.4%、5.6%、及 9.9%。原生質體經 2.0 kV 電穿孔處理後還有 7.3 log CFU/mL以上之菌體存活。 轉殖之部分以 RF-clonimg 技術分別將pDG148-Stu 和 pVA838 載體對 Agarase AS-IIIb 與 PV-1b 基因進行構築，但未成功獲得 Agarase 基因 重組菌株。後續將載體 pDG148-Stu 以限制酶 Stu、T4 polymerase 及 dTTP 反應處理，也尚未獲得到轉殖株。青海菜寡醣 (Monostroma nitidum oligosaccharides, MnOS) 區分為分子質量 (A) 3-5 kDa 區分液含 (Degree of polymerization, DP) DP 6、12、和 30，且有最高之還原醣量及硫酸根含量 (0.28 mg/mL 和 0.68 mg/mg) 與 (B) 1-3 kDa 區分液含 DP 6。含 0.1%、0.05%、和 0.02% MnOS 脫脂牛乳發酵後，結果顯示 Lb. Plantarum BCRC 10069 (2%) + 12250 (2%) 發酵 16 小時後 pH 值可降至 4.6 以下，可滴定酸度 0.67-0.68%、乳酸菌數達 9 log CFU/mL 以上，Ped. pentosaceus MFL + MFS 則發酵 24 小時後達 pH 4.6 以下，可滴定酸度 0.60-0.70，乳酸菌數達 8.5 log CFU/mL 以上。發酵乳製 MnOS-GAI-0.1% 有最好之 DPPH 清除力 21.73%，而 MnOS-GBII- 0.1% 有最高之螯合亞鐵離子能力為 72.56%。4oC 儲藏試驗部分，於 14 天後，pH 值隨時間增加持續下至 4.47-4.07，而可滴定酸度則會上升至 0.76%-0.89%，抗氧化能力隨儲藏時間增加而降低。抗凝血活性部分，牛乳發酵產物 MnOS-GAI-100 g/mL 於兔血 APTT 結果與控制組相比，凝血時間延後 20 秒，凝血程度也降低了 32.4%，而 PT 的部分，可降低凝血程度 2.7%，於人血 APTT 與 PT 測試之結果，則降低了 8.7% 及 7.5% 之凝血程度。取抗氧化效果較好之發酵樣品進行細胞試 驗及美白測試之結果，皆有使老鼠纖維母細胞 NIH-3T3 及人類纖維母 細胞 CCD-966SK 增長之效果，細胞存活率可達 124-128% 及 122- 138%。於酪胺酸酶活性抑制測試中抑制力隨濃度提高而增加，抑制能力 為 35-37%，而後對人類黑色素腫瘤細胞 A375 測試，有抑制細胞生長 之效果，最低存活率為 46-87%。實驗結果雖無法成功獲得洋菜酶重組 乳酸菌株，但原生質體之處理及電穿孔條件可做為相關實驗之參考，而 添加青海菜之牛乳發酵產物具有做為健康食品及美白成分之潛力。|
The object of this study was clone the Agarase gene AS-IIIb and PV-Ib into the lactic acid bacteria (LAB) with shuttle vector. And investigate LAB ferment Monostroma nitidum oligosaccharides (MnOS) contained milk on the effect of MnOS of their antioxidant properties, anticoagulant properties, and whitening properties. The results showed that the effectively of protoplast formation of Ped. pentosaceus MFS, MFL, Lb. bulgaricus BCRC 10696, and Lb. plantarum BCRC 10069 treated with mutanolysin were 46.3%, 3.4%, 5.6%, and 9.9% respectively. There was more than 7.3 log CFU/mL LAB kept alive after the protoplast electroporated by 2.0 kV voltage. The constructions of Agarase AS-IIb and PV-IB genes and vectors pVA838 and pDG148-Stu by restriction-free (RF) cloning were not completed. The constructed gene of pDG148-Stu vector were treated with restricted enzyme Stu, T4 polymerase, and dTTP but the clone was not observed yet. MnOS was separated by ultrafiltration to obtain (A) 3-5 kDa fraction contained degree of polymerization (DP) 6, 12, and 30 with the highest reducing sugar content and sulftate content (0.28 mg/mL and 0.68 mg/mg) and (B) 1-3 kDa fraction contained DP 6. In the fermented experiment of 0.10%, 0.05%, and 0.02% MnOS contained milks, Lb. plantarum BCRC 10069 + 12250 group took 16 hr to reached the pH < 4.6, titratable acidity were 0.67-0.68%, and LAB counts were above 9 log CFU/mL. Ped. pentosaceus MFL + MFS group took 24 hr to reached the pH < pH 4.6, titratable acidity were 0.60-0.70%, and LAB counts were above 8.5 log CFU/mL. The MnOS-GAI-0.1% cultured milk had the best scavenging DPPH free radicals ability 21.73% and MnOS-GBII-0.1% had the best chelating Fe2+ ability 72.56%. The pH value of two MnOS cultured milks were fell to 4.47-4.07, and titratable acidity were increased to 0.76%-0.89% during 4oC for 14 days. The abilities of antioxidation were decreased as storage time increased. The APTT anticoagulants activity test of rabbit plasma showed the MnOS-GAI-100 g/mL group compared with control which could delay the coagulant time for 20 sec, and decrease the coagulation effect for 32.4%. The rabbit plasma PT anticoagulants activity test result showed it could decrease the coagulation effect for 2.7%, and on human plasma it decreased 8.7% and 7.5% coagulation effect for APTT and PT. The MnOS cultured milks with better antioxidative ability could increase the cell viability of NIH-3T3 and CCD-966SK of 124-128% and 122-138%. The inhibition of tyrosinase was increased with increasing the concentration of MnOS cultured milk, and the inhibition were 35-37%. The MnOS cultured milks could inhibited the A375 cell, and the cell viability were 46-87%. The experiment results did not obtain the recombined clone, but the result of protoplast formation and electroporation conditions could be consulted. The MnOS cultured milk had the potential to be developed as health food product and used as whitening ingredient in cosmetic product.