臺灣博碩士論文加值系統

　茯苓（Wolfiporia cocos）屬於食藥兼用之真菌菇類。主要生物活性成份為三帖類及茯苓多醣體（pachymaran）。藥理研究證實茯苓的有效成分可對人體有抗腫瘤、調節免疫、利尿、抗發炎、抗氧化、免疫調節、抗腫瘤、鎮靜、利尿、止吐、抗氧化、抑制發炎、腎臟炎、糖尿病等多種功能。近年來，雖有許多研究者集中在茯苓醱酵產物中活性成分、功能以及在功能食品中的開發應用。本實驗應用單因子法，探討茯苓在5升醱酵槽液態培養生產胞外多醣體之培養條件(包括pH、溫度及通氣量)最佳化，其中在5升醱酵槽培養，最佳化培養條件為pH 3.0、溫度28℃及0.9vvm，並放大至20升醱酵槽培養，以期生產高產率之胞外多醣，其中在第7天時可得最大菌絲量為5.614 mgmL-1，在第7天時可得多醣產量0.236 mgmL-1。再利用凝膠過濾管柱層析法分離純化茯苓菌粗多醣體，可得分區多醣 (EPS-A、 EPS-B 、EPS-C、 EPS-D)。利用茯苓菌醱酵液所得之分區多醣與20升醱酵槽生產醱酵胞外多醣，分析其抗氧化活性在共軛雙烯方面以EPS-C為較佳，其EC50為4.03 mgmL-1; DPPH方面以EPS-A為較佳，其EC50為0.04 mgmL-1，螯合亞鐵方面以20升為較佳，其EC50為0.15mgmL-1，還原力方面以EPS-D為較佳，其EC50為0.018 mgmL-1，超氧陰方面以EPS-C為較佳，其EC50為0.25 mgmL-1。
此外，分析分區多醣與20升醱酵槽生產醱酵胞外多醣之蛋白質含量結果顯示，EPS-A為31.46%，EPS-B為32.16%、EPS-C為31.81% 、EPS-D為31.46%、20升為34.2%。利用分子篩析管柱(GPC)分析EPS-A、 EPS-B 、EPS-C、 EPS-D、20升醱酵槽, EPS-A為4.13×106Da，EPS-B 為2.48×104Da、EPS-C為2.48×104Da 、EPS-D為2.81×105 Da、20升醱酵槽為7.23×103Da。
關鍵字：茯苓、抗氧化特性、多醣體、液態培養

Poria cocos (Schw.) Wolf is an important oriental edible and medical fungus. The main active component isolated from Poria cocos is triterpenes and pachymaran. Pharmacology studies have proven that its active ingredients possess anti-inflammatory, anti-tumor, anti-emetic, ant-inflammation, anti-nephritis, anti-diabetes, anti-aging, immunity, sedative, diuretic and antioxidant properties. Recently, although many researchers focus on activity components and function of fermentation product as well as development in health-food, little literature was reported concerning effects of culture conditions on production and antioxidant activity of polysaccharides of Poria cocos by submerged culture. Thus, the project was proposed to investigate the optimization of culture conditions (including pH, temperature and aeration rate) on the production of polysaccharide in submerged culture of Poria cocos by one-factor-at-a-time design methodology and their antioxidant properties. The results showed that the optimal operation conditions are pH 3.0, 36℃and 0.6 vvm in 5 L stirred tank bioreactor submerged cultures. Besides, In 20 L stirred tank bioreactor culture on day 7, the optimal operation conditions also can obtain the maximum biomass and exopolysaccharide (EPS) production with 5.614 gL-1 and 236.32 mgL-1, respectively. The crude EPS from submerged cultures of Poria cocos was purified by using Sepharose CL-4B column chromatography with four EPS peaks (EPS-A, EPS-B, EPS-C and EPS-D) eluted. Antioxidant activity results point out that EPS-C has a high antioxidant activities and scavenging effects on superoxide anions. Corresponding EC50 values are 4.03 mgmL-1 and 0.25 mgmL-1. The high EC50 values of scavenging effects on 1,1-diphenol-2-picrylhydrazyl radicals of EPS-A are 0.04 mgmL-1. EPS-A showed high reducing powers with EC50 values of 0.018 mgmL-1. Crude EPS from 20 L stirred tank bioreactor culture by Poria cocos. Besides, the protein/polysaccharide ratios of EPS-A, EPS-B, EPS-C, and EPS-D are 31.46%, 32.16%, 31.81%, and 31.46% (w/w), respectively. The protein/polysaccharide ratio of crude EPS from 20 L stirred tank bioreactor culture by Poria cocos is 34.2% (w/w). Molecular weight analyses of EPS-A, EPS-B, EPS-C, and EPS-D by using Molecular Sieve chromatography tell that molecular weights of four fractions are 4.13×106Da for EPS-A, 2.481×104Da for EPS-B, 2.48×104Da for EPS-C, and 2.81×105 Da for EPS-D. In addition, molecular weight of crude EPS from 20 L stirred tank bioreactor culture by Poria cocos is 7.23×103 Da.
Keywords: Poria cocos; antioxidant properties; polysaccharide; submerged cultur

目錄
摘要 I
Abstract II
誌謝 IV
目錄 V
圖目錄 IX
表目錄 XI
第一章緒論 1
1.1研究動機與背景 1
1.2研究目的 3
第二章文獻回顧 4
2.1菇類的介紹和機能性 4
2.1.1菇類的應用 5
2.1.2菇類食品生理活性 7
2.1.3菇類多醣的生理活性 10
2.2茯苓介紹 11
2.2.1茯苓的成分組成 13
2.2.2多醣體介紹 14
2.2.3多醣體之分類 15
2.2.4多醣體之作用 15
2.2.5多醣體及其抗腫瘤機制 17
2.2.6多醣體之機能性 17
2.3醱酵深層培養技術介紹 19
2.3.1深層醱酵培養的定義及培養基的成份和功能 19
2.3.1.1 碳源 19
2.3.1.2 氮源 20
2.3.1.3 碳氮比( C/N ) 21
2.4.1.4 無機鹽和微量元素 21
2.3.1.4 其他添加物 22
2.3.2影響深層醱酵培養的其他物理化學因子 23
2.3.3 培養基滅菌 30
2.4多醣體之分離與純化 30
2.4.1 透析法 30
2.4.2 酒精沉澱法 30
2.4.3層析法 30
2.4.4膠體過濾管柱層析 32
2.5自由基與抗氧化介紹 33
2.5.1 自由基如何產生 33
2.5.2 自由基對細胞造成的傷害 33
2.5.3 抗氧化物質(Antioxidant) 34
2.5.4 抗氧化性質分析 35
第三章實驗方法 36
3.1實驗架構 36
3.1實驗藥品與器材 37
3.1.1實驗藥品 37
3.1.2菌種 39
3.1.3儀器設備 39
3.1.4 實驗裝置 41
3.2實驗方法 46
3.2.1 菌株保存 46
3.2.2 培養基成分 46
本章節分別介紹菌接種於固態平面培養、種瓶培養、液態培養和醱酵槽培養需要營養源之組成成份。 46
3.2.2.1 固態平面培養基 46
3.2.2.2 液態培養基 46
3.2.2.3 醱酵槽培養 46
3.2.3 操作條件 48
3.2.3.1固態培養基 48
3.2.3.2種瓶液態培養基 48
3.2.3.3 5升醱酵槽液態培養 48
3.3分析方法 50
3.3.1 醱酵產物分析流程 50
3.3.2 菌體乾重測定 51
3.3.3 粗多醣含量測定 51
3.3.4 殘糖分析 (DNS method) 52
3.3.5醱酵液之胞外多醣的製備 53
3.3.5.1醱酵產物 53
3.3.5.2醱酵液之胞外多醣的製備 53
3.3.6 管柱層析與分離純化 54
3.3.7 茯苓菌胞外多醣分區純化蛋白質含量分析 54
3.3.8 茯苓菌醱酵胞外多醣分子量分析 55
3.3.9茯苓菌醱酵物萃取之抗氧化性質分析 56
第四章結果與討論 59
4.1 茯苓菌絲型態 59
4.1.1固態培養 59
4.1.2種瓶培養 60
4.1.3攪拌式醱酵槽 61
4.2攪拌式醱酵槽實驗 62
4.2.1不同pH值對茯苓菌菌絲生長及生產胞外多醣之效應 62
4.2.1.1不同pH值對茯苓菌菌絲生長之影響 62
4.2.1.2不同pH值對茯苓菌胞外多醣產量之影響 63
4.2.1.3不同pH值對μ、Qx、Yx/s之影響 67
4.2.1.4不同PH值對Qp、Yp/x、Yp/s之影響 67
4.2.2不同溫度對茯苓菌菌絲生長及生產胞外多醣之效應 68
4.2.2.1不同溫度對茯苓菌菌絲生長之影響 68
4.2.2.2不同溫度對茯苓菌胞外多醣產量之影響 69
4.2.2.3不同溫度對μ、Qx、Yx/s之影響 72
4.2.2.4不同溫度對Qp、Yp/x、Yp/s之影響 72
4.2.3不同通氣量對茯苓菌菌絲生長及生產胞外多醣之效應 73
4.2.3.1不同通氣量對茯苓菌菌絲生長之影響 73
4.2.3.2不同通氣量對茯苓菌胞外多醣產量之影響 74
4.2.3.3不同通氣量對μ、Qx、Yx/s之影響 78
4.2.3.4不同通氣量對Qp、Yp/x、Yp/s之影響 78
4.2.4.5最適化20升醱酵槽培養條件放大生產對茯苓俊菌絲生長及生產胞外多醣之影響 79
4.3 茯苓菌粗多醣體分離純化結果 81
4.3.1茯苓菌胞外粗多醣體純化之分布 81
4.3.2茯苓分區多醣與20升醱酵槽生產醱酵胞外多醣之分子量及蛋白質含量分析 82
4.3.3茯苓菌胞外分區多醣與20升醱酵槽生產醱酵胞外多醣之分子量組成分析 83
4.4茯苓分區多醣與20升醱酵槽生產醱酵胞外多醣抗氧化特性分析 85
4.4.1茯苓分區多醣與20升醱酵槽生產醱酵胞外多醣之還原能力 85
4.4.2茯苓分區多醣與20升醱酵槽生產醱酵胞外多醣之清除1,1-diphenyl-2-picry- lhydr- azyl(DPPH)自由基能力 87
4.4.3茯苓分區多醣與20升醱酵槽生產醱酵胞外多醣之抗氧化能力 89
4.4.4茯苓菌分區多醣與20升醱酵槽生產醱酵胞外多醣之螯合亞鐵離子能力 90
4.4.5茯苓菌分區多醣與20升醱酵槽生產醱酵胞外多醣之清除超氧陰離子能力 92
4.5茯苓菌分區多醣與20升醱酵槽生產醱酵胞外多醣之EC 50值 94
第五章結論與建議 96
5.1結論 96
5.2 建議 98
第六章參考文獻 99
作者簡介 105

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