|Abstract: ||本研究目的為探討以酵素法生產之石蓴硫酸寡醣特性及其生理活性，並研究石蓴藻渣經乳酸菌發酵生產乳酸之條件。石蓴 (Ulva lactuca) 粉末經 110oC 萃取 75 min 後，以超過濾系統收集大於 30 kDa 之多醣分子，再經 95% 乙醇沉澱，凍乾製得石蓴硫酸多醣 (Ulvan)。Ulvan 之產率為 15.89% (每 100 g 石蓴粉末產得 15.89 g 之石蓴硫酸多醣)，且其總糖、還原糖、糖醛酸、硫酸基、蛋白質及總酚含量分別為 48.59%、3.05%、30.22%、21.29%、0.47% 及 0.11%。經 HPLC 分析得知 Ulvan 以 769 kDa 之多醣分子為主要醣類組成，相對比例佔 53.2%。經 FT-IR 分析得知 Ulvan 含有 O-H、C=O、C-O、及 S=O 等官能基結構。接著以 Ulvan 誘導 Pseudomonas vesicularis MA103 及 Aeromonas salmonicida MAEF108，可測得降解石蓴硫酸多醣之酵素有石蓴硫酸多醣裂解酶、澱粉酶、纖維素酶、和聚木糖酶。MA103 於誘導第三日之酵素活性分別為 1.93 U/mL、3.78 U/mL、2.72 U/mL、和 1.62 U/mL；而 MAEF108 於誘導第二日之酵素活性分別為 0.22 U/mL、0.17 U/mL、0.19 U/mL、和 3.23 U/mL。後續 Ulvan 經 MA103 與 MAEF108 之誘導粗酵素液水解後，以超過濾系統收集小於 3 kDa 之寡醣分子，石蓴硫酸寡醣 (Ulvan oligosaccharides, UOS3K) 之產率為 4.69% (每 100 g 石蓴粉末可生產 4.69 g 之石蓴硫酸寡醣)。UOS3K 之總糖、還原糖、糖醛酸、硫酸基及總酚含量分別為 7.42%、7.17%、2.54%、13.19% 及 0.73%；HPLC 圖譜顯示 UOS3K 以 800 Da 之寡醣分子為主要醣類組成，相對比例佔 82.0%；FT-IR 圖譜顯示 UOS3K 含有 O-H、C=O、C-O、及 S=O 等官能基結構。血管升壓素轉化酶 (Angiotensin I converting enzyme, ACE) 抑制活性試驗中，Ulvan 和 UOS3K 之 ACE 抑制率分別為 18.41% (50 mg/mL) 及 81.86% (12.5 mg/mL)，且 UOS3K 之 ACEI 的 IC50 值為 0.451 mg/mL。抗氧化試驗中，Ulvan 和 UOS3K 的 DPPH 清除率分別達 52.46% (10 mg/mL) 及 27.27 % (10 mg/mL)；Ulvan 和 UOS3K 之亞鐵離子螯合率分別達 4.62% (20 mg/mL) 和 97.98% (10 mg/mL)；而 Ulvan 與 UOS3K 皆無還原力之表現。抗凝血試驗中，UOS3K 與空白組相比無延遲凝集時間，但於 APTT 及 PT 試驗中可分別降低凝集程度 94% 及 78%。以抽取石蓴硫酸多醣後之石蓴藻渣誘導二株海洋細菌，MA103 於誘導第三日有較佳之石蓴硫酸多醣裂解酶、澱粉酶、和聚木糖酶活性，分別為 1.97 U/mL、3.91 U/mL、和 2.08 U/mL；MAEF108 於誘導期間石蓴硫酸多醣裂解酶、澱粉酶、纖維素酶和聚木糖酶之活性無顯著差異。最後將添加 0.5% (w/v) 酵母萃取物至石蓴藻渣多醣水解液，接種 1% (v/v) Lactobacillus plantarum BCRC12327 於 30oC 下以碳酸鈣做為中和劑，發酵 36 hr 可得乳酸濃度為 35.97 g/L 及乳酸產率為 15.65%，即每 100 g 石蓴藻渣可生產 15.65 g 乳酸。|
This study is aim to analyze characteristic and bioactivities of ulvan oligosaccharides produced by enzymatic hydrolysis and investigate conditions of lactic acid fermentation of Ulva residue. Firstly, ulvan were gained from Ulva lactuca powder that treated by hot-water (110oC/75 min), then collected by ultrafiltration system (> 30 kDa), and precipitated in 95% ethanol. The yield of ulvan is 15.89% (15.89 g of ulvan obtained from 100 g of Ulva lactuca powder), and the total sugar, reducing sugar, uronic acid, sulfate, protein and total phenols of ulvan are 48.59%, 3.05%, 30.22%, 21.29%, 0.47% and 0.11%, respectively. HPLC chromatogram is shown molecular weights (MW) of ulvan are mainly 769 kDa polysaccharides which relative ratio of fractions are 53.2%. FT-IR spectra of ulvan is presented O-H, C=O, C-O and S=O stretching. Moreover, using ulvan to induce marine bacteria Pseudomonas vesicularis MA103 and Aeromonas salmonicida MAEF108 could produce ulvan-degrading enzymes. Activities of ulvan lyase, amylase, cellulase, and xylanase were 1.93 U/mL, 3.78 U/mL, 2.72 U/mL, and 1.62 U/mL respectively which produced by MA103 in 3 days, and were 0.22 U/mL, 0.17 U/mL, 0.19 U/mL, and 3.23 U/mL respectively which produced by MAEF108 in 2 days. Further, ulvan were hydrolyzed by crude enzymes solution of MA103 and MAEF108, and differentiated by ultrafiltration system (< 3 kDa) to acquire ulvan oligosaccharides (UOS3K). The yield of UOS3K is 4.69% (4.69 g of ulvan gained from 100 g of Ulva lactuca powder), and the total sugar, reducing sugar, uronic acid, sulfate and total phenols of ulvan oligosaccharides are 7.42%, 7.17%, 2.54%, 13.19% and 0.73%, respectively. HPLC chromatogram is shown MW of UOS3K are mainly 800 Da oligosaccharides which relative ratio of fractions are 82.0%. FT-IR spectra of UOS3K is presented O-H, C=O, C-O and S=O stretching. Secondly, the bioactivity of ulvan or UOS3K were evaluated by angiotensin I converting enzyme (ACE) inhibitory, antioxidant activities and anticoagulant activities in vitro. In ACE inhibitory assay (ACEI), inhibition of ulvan (50 mg/mL) and UOS3K (12.5 mg/mL) were 18.41% and 81.86%. IC50 value of UOS3K on ACEI was 0.451 mg/mL (Peptide concentration). In antioxidant activity assays, the DPPH scavenging activity of ulvan (10 mg/mL) and UOS3K (10 mg/mL) were 52.46% and 27.27 %; the Fe2+ chelating ability of ulvan (20 mg/mL) and UOS3K (10 mg/mL) were 4.62% and 97.98%; both of ulvan and UOS3K were not shown the reducing power. In anticoagulant activity assays, wich are activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using rabbit plasma, UOS3K did not prolong the aggregation time compared to blank group. However, UOS3K could decrease the aggregation level to 94% and 78% in APTT and PT test. Thirdly, utilized Ulva residue which removed the ulvan from Ulva lactuca to induce MA103 and MAEF108 producing crude enzymes was investigated. Activities of ulvan lyase, amylase, and xylanase were 1.97 U/mL, 3.91 U/mL, and 2.08 U/mL respectively which produced by MA103 in 3 days, and activities of enzymes produced by MAEF108 were no significant. Additionally, hydrolyzing conditions of the Ulva residue were evaluated. Finally, Ulva residue polysaccharide hydrolysate was added 0.5% (w/v) yeast extract as nitrogen source, calcium carbonate as neutralizing agent, and then fermented by 1% (v/v) of Lactobacillus plantarum BCRC12327 at 30oC for 36 hr, the lactic acid concentration was 35.97 g/L and lactic acid yield was 15.65% (15.65 g of lactic acid gained from 100 g of Ulva residue).