臺灣博碩士論文加值系統

本研究之目的在探討龍鬚菜多醣之抗氧化活性、益菌能力及調節血脂之功效，並藉由酸、鹼和酵素方法製備龍鬚菜水解產物。實驗採用不同劑量之龍鬚菜多醣及藻體對高脂質飲食誘導雄性倉鼠進行八週調節血脂之研究。
結果顯示於不同催化脂質過氧化系統中，FeCl3/H2O2/ascorbic acid與FeCl3/H2O2/ascorbic acid，龍鬚菜多醣並無明顯之抗氧化活性。龍鬚菜溶液（1 mg/mL）在氫氧自由基清除率方面，其效果達到40%；清除超氧陰離子之能力方面，龍鬚菜多醣萃取液（0.5mg/mL）之抗氧化能力達為68.2﹪，其效果較butylated hydroxyanisole（BHA）佳。龍鬚菜之總酚類化合物含量為11.5 mg/g 乾重藻體。各種水解方法顯示，不論酸、鹼或酵素之水解，隨著時間之增加其水解產物增加有限。酵素法可獲得較佳水解產物之多樣性，主要以三醣（G3）、四醣（G4）為主。另cellulase之水解產物對Bifidobacterium bifidum有益菌效果，但略低於果寡糖及奧利多（mini-oligo），且對Clostridium perfringen之促進生長並不明顯。
龍鬚菜多醣及龍鬚菜藻體於體內降血脂功效，實驗飲食處理8週後，各組間之三酸甘油酯（TG）無顯著差異；攝食高量龍鬚菜藻體組（HA）及高量龍鬚菜多醣（HG）之總膽固醇明顯降低（p＜0.05），但各組間之HDL-C及LDL-C值並無顯著性差異，而攝食龍鬚菜多醣（HG、LG）較龍鬚菜藻體（HA、LA）更能防止脂肪肝發生。實驗結果顯示，無論是添加龍鬚菜藻體或龍鬚菜多醣其濃度與糞便中TG值及低密度脂蛋白（LDL）成正比，推論龍鬚菜可促進脂質排泄之機轉，進而達到預防倉鼠高血脂症發生之效果。

The objective of the present study was to investigate the effect of polysaccharide of Gracilaria tenuistipitata on antioxidative activity, prebiotic property and anti-hypercholesterolemia. The hydrolysates of polysaccharide, extracted from cultured Gracilaria tenuistipitata, were prepared by acidic, alkaline and enzymatic hydrolysis. Sixty five male alimentary-induced by hypercholesterol diet fed with different dosage of polysaccharide and algae of G. tenuistipitata for 8 weeks were employed.
The results indicated that no significant antioxidative activity was observed when lipid peroxidation coupling with (Fe+3/H2O2/ascorbate)and (Fe+2/H2O2)system were used under defined conditions. Forty percent of hydroxyl (OH•) free radical scavenging activity can be reached if G. tenuistipitata polysaccharide solution, up to 1 mg/ml, was employed. Better than BHA, using 0.5 mg/ml of G. tenuistipitata polysaccharide solution, 68% of superoxide scavenging effect was found. The content of total phenol was 11.48 mg/g G. tenuistipitata. The amount of hydrolysates was slightly increased by time no matter in chemical or enzymatic methodology. The better diversity of hydrolysates was obtained in enzymatic hydrolysis than those of acidic or alkaline treatments, and trimers and tetramers were the major products.
In vitro, the results of prebiotics test demonstrated that trimers and tetramers of G. tenuistipitata hydrolysates exhibited better stimulation of growth on Lactobacillus acidophilus, Bifidobacterium bifidum than that of monomer and dimmers. The prebiofic effect of cellulose hydrolysate in B. bifidum was slightly lower than those fructooligosaccharides （Taisugar Co.）and Oligos（King Car Co.）. Cellulase hydrolysate of G. tenuistipitata has little effect on growth of Clostridium perfringens. In anti-hypercholesterolemia study, the results illustrated that intake of high dosage of Gracilaria algae and polysaccharide can reduce the total cholesterol, but little significant difference was found in triglyceride after 8 weeks experimental diet period. No significant change was observed in HDL-C and LDL-C value among all test groups. The results of gross and microscopic finding indicated that Gracilaria polysaccharide was effective in preventing fatty liver. Evidence showed that the amount of TG and LDL-C excreted in feces were dependent upon the concentration of Gracilaria algae and polysaccharide and inferenced that the effect of anti-hypercholesterolemia can be improved by increase of lipid excretion.

目 錄
頁次
中文摘要 I
英文摘要 III
誌謝 V
目錄 VI
圖索引 XIII
表索引 XV
第一章 前言 1
第二章 文獻回顧 3
2.1 藻類簡介 3
2.2 海藻多醣 3
2.2.1 β-葡聚醣之介紹 3
2.2.2 海藻多醣定義及特性 4
2.2.3 海藻之應用 6
2.2.4 影響海藻多醣萃取因子 10
2.2.4.1 萃取溫度 10
2.2.4.2 萃取時間 12
2.2.4.3 鹼處理 12
2.2.5 海藻多醣生理活性 12
2.2.5.1 海藻對血脂質的影響 12
2.2.5.2 海藻對免疫活性與抗腫瘤的影響 13
2.2.6 龍鬚菜多醣 14
2.2.6.1 龍鬚菜簡介 14
2.2.6.2 龍鬚菜多醣之生理活性 15
2.3 油脂氧化與抗氧化作用 16
2.3.1自由基與活性氧 16
2.3.2 多醣與抗氧化劑 20
2.3.3 天然抗氧化物質 20
2.4非消化性碳水化合物及其生理活性 22
2.4.1 膳食纖維 22
2.4.1.1 膳食纖維的定義及分類 22
2.4.1.2 膳食纖維之生理功能 23
2.4.2 寡醣與腸道菌叢 23
2.4.2.1 寡醣簡介 23
2.4.2.2 紫菜寡醣之生理活性 24
2.4.2.3 龍鬚菜寡醣之生理活性 24
2.4.2.4 海藻寡醣之應用 25
2.4.3 胃腸道菌叢與益生菌 25
2.4.3.1 胃腸道菌叢 25
2.4.3.2 益生菌 26
2.4.4 腸道菌叢發酵 28
2.4.4.1 短鏈脂肪酸 28
2.4.5 非消化性碳水化合物對脂質代謝的影響 29
第三章 材料與方法 30
3.1實驗設計 30
3.2 一般試藥 31
3.2.1 龍鬚菜多醣製備 31
3.2.1.1 龍鬚菜來源 31
3.2.1.2 龍鬚菜多醣製備 31
3.2.2 龍鬚菜多醣抗氧化性質分析 31
3.2.3 龍鬚菜多醣部分水解產物製備分析 32
3.2.4 龍鬚菜多醣部分水解產物促進益生菌生長試驗 32
3.2.4.1 菌株來源 32
3.2.4.2 菌株培養 32
3.2.5 龍鬚菜多醣及龍鬚菜藻體降血脂功效評估 32
3.2.5.1 實驗動物 32
3.2.5.2 動物飼養 33
3.2.5.3 血液生化值分析 33
3.3 儀器設備 33
3.3.1 龍鬚菜多醣製備 33
3.3.2 龍鬚菜多醣抗氧化性質分析 33
3.3.3 龍鬚菜多醣部分水解產物製備及分析 33
3.3.4 龍鬚菜多醣部分水解產物促進益生菌生長試驗 34
3.3.5 龍鬚菜多醣及龍鬚菜寡醣降血脂功效評估 34
3.4 實驗方法 34
3.4.1 龍鬚菜多醣物質之萃取 34
3.4.1.1 龍鬚菜前處理 35
3.4.1.2 龍鬚菜多醣製備 35
3.4.2 龍鬚菜多醣抗氧化性質分析 35
3.4.2.1 於不同催化脂質過氧化系統中抗氧化能力測定 35
3.4.2.1.1 氫過氧化物之測定，以硫氰酸鐵法 35
3.4.2.1.2 於FeCl3/H2O2/ascorbic acid 催化之linoleic acid脂質過氧化系統中抗氧化性 36
3.4.2.1.3 於FeCl2/H2O2 催化之linoleic acid脂質過氧化系統中抗氧化性 37
3.4.2.2 龍鬚菜多醣抗氧化特性之探討 37
3.4.2.2.1 清除氫氧自由基之能力測定 37
3.4.2.2.2 清除DPPH自由基試驗 38
3.4.2.2.3 清除超氧陰離子能力試驗 39
3.4.2.3 龍鬚菜多醣抗氧化成分分析 39
3.4.2.3.1 總酚類化合物含量之測定 40
3.4.3 龍鬚菜多醣最佳水解條件 40
3.4.3.1 龍鬚菜多醣之酵素水解 41
3.4.3.1.1 agarase 水解 41
3.4.3.1.2 cellulase 水解 41
3.4.3.2 龍鬚菜多醣之酸水解 41
3.4.3.2.1 HCl水解 42
3.4.3.2.2 甲酸水解 42
3.4.3.3 龍鬚菜多醣之複合式水解-鹽酸或甲酸處理-酵素之水解液 42
3.4.3.4 龍鬚菜多醣之鹼水解 42
3.4.4 水解產物分析 42
3.4.5 龍鬚菜部分水解產物促進益生菌生長試驗 43
3.4.5.1. Lactobacillus acidophilus 43
3.4.5.2 Bifidobacterium bifidum 43
3.4.5.3 Clostridium perfringens 43
3.4.6 龍鬚菜多醣及龍鬚菜藻體降血之功效評估 44
3.4.6.1 實驗動物品種及飼養條件 44
3.4.6.2 分組及實驗設計 45
3.4.6.3 犧牲、解剖與臟器收集 46
3.4.6.4 血液收集 47
3.4.6.4.1 血漿三酸甘油酯 47
3.4.6.4.2 血漿總膽固醇 48
3.4.6.4.3 血漿高密度脂蛋白-膽固醇 48
3.4.6.4.4 血漿低密度脂蛋白-膽固醇 49
3.4.6.4.5 肝功能生化指數 49
3.4.6.5 肝臟脂質分析 49
3.4.6.6 糞便脂質分析 50
3.4.6.7 盲腸內容物短鏈脂肪酸分析 50
3.4.7 培養基配製 52
3.4.7.1 MRS broth medium 52
3.4.7.2 MRS agar medium 53
3.4.7.3 Dulbecco’s modified Eagles medium 54
3.4.8 溶液配製 54
3.4.8.1 無菌生理食鹽水 54
3.4.8.2 中性福馬林緩衝溶液 54
3.4.8.3 醋酸緩衝溶液 55
3.4.8.4 磷酸緩衝生理食鹽水 55
3.4.8.5 Linoleic acid emulsion 55
3.5 統計分析 56
第四章 結果與討論 57
4.1 龍鬚菜多醣抗氧化性質分析 57
4.1.1 於不同催化脂質過氧化系統中抗氧化能力測定 57
4.1.2 龍鬚菜多醣抗氧化特性之探討 58
4.1.3 龍鬚菜多醣抗氧化成分分析 60
4.2 龍鬚菜多醣最佳水解條件 60
4.2.1 水解條件之最適化 61
4.2.2 水解產物分析 61
4.3 龍鬚菜寡糖對於有益菌及有害菌生長之影響 63
4.3.1 以HCl水解龍鬚菜多醣對Lactobacillus acidophilus之影響 64
4.3.2 Agarase水解龍鬚菜多醣對Lactobacillus acidophilus之影響 64
4.3.3 甲酸水解龍鬚菜多醣對Lactobacillus acidophilus生長之影響 65
4.4 龍鬚菜多醣及龍鬚菜藻體降血脂功效評估 66
4.4.1 動物實驗設計 66
4.4.2 攝食量變化 66
4.4.3 體重變化 67
4.4.4 血脂質分析 67
4.4.5 肝功能分析 69
4.4.6 器官重量 69
4.4.6.1 肝臟 70
4.4.6.2 盲腸 70
4.4.7 外觀觀察及組織切片 71
4.4.7.1 外觀紀錄 71
4.4.7.2 切片紀錄 71
4.4.8 肝臟及糞便中之脂質 72
4.4.8.1 肝中脂質 72
4.4.8.2 糞便之脂質 73
4.4.9 盲腸短鏈脂肪酸分析 73
第五章 結論 100
第六章 參考文獻 102
作者簡介 120

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