臺灣博碩士論文加值系統

硫磺菌(Laetiporus sulphureus)，為珍稀品種之一，是一種傳統藥用菇類。在治療中可作為乳腺癌、前列腺癌之輔助食物。過去研究顯示，硫磺菌多醣與其萃取物均具有許多生物活性功能，其中包括：抗氧化、抗腫瘤、增加免疫能力、降血糖等。本實驗利用液態醱酵培養硫磺菌，探討不同界面活性劑之添加以促進硫磺菌液態醱酵之多醣體產量，以期待生產高產率之胞外多醣。
在搖瓶試驗中分別添加 0.5 g L-1 之各種界面活性劑包括PEG 系列(4000、6000)、Tween 系列(20、40、80、85)及Span 系列(20、80)於搖瓶中，置於28 ℃、150 rpm之恆溫培養箱培養12天。分別測定硫磺菌液態醱酵之菌絲體及多醣體含量。篩選出對硫磺菌液態醱酵生產菌絲體及多醣體有益之界面活性劑。由實驗結果得知添加Span 80可得最大菌絲體及多醣含量為7.13 g L-1及240 mg L-1。
在搖瓶試驗中分別添加不同濃度(0、0.2、0.4、0.6、0.8、1.0、1.2 g L-1)之Span 80於搖瓶中，置於28 ℃、150 rpm之恆溫培養箱培養12天。分別測定硫磺菌液態醱酵之菌絲體及多醣含量。篩選出對硫磺菌液態醱酵生產菌絲體及多醣體之最佳濃度。由實驗結果得知在0.6 g L-1 Span 80時有最大之菌絲體及多醣含量，分別為9.33 g L-1及130 mg L-1。
利用5-L攪拌式醱酵槽改變不同濃度(0、0.2、0.4、0.6、0.8、1.0 g L-1)之Span 80培養條件醱酵培養，實驗結果顯示，攪拌式醱酵槽在0.2 g L-1可得到最大之菌絲體4.08 g L-1，在0.6 g L-1可得到最高胞外多醣體含量為213.43 mg L-1。此外，並在20-L攪拌式醱酵槽進行放大培養，由最適化培養條件0.6 g L-1培養所得之硫磺菌多醣產量為120.88 mg L-1。本研究亦針對硫磺菌醱酵產物進行分子量和蛋白質分析，在5-L醱酵槽中0.6 g L-1Span 80液態醱酵所得之胞外多醣其算術平均分子量及蛋白質含量為2.94×104和6.64 %。在20-L醱酵槽中0.6 g L-1Span 80液態醱酵所得之胞外多醣平均分子量及蛋白質含量為5.54×105和6.39 %。在5-L醱酵槽中0.6 g L-1Span 80液態醱酵所得之胞內多醣其算術平均分子量及蛋白質含量為5.30×104和7.76 %。在20-L醱酵槽中0.6 g L-1Span 80液態醱酵所得之胞內多醣平均分子量及蛋白質含量為1.03×105和6.62 %。此外，硫磺菌醱酵產物萃取液之抗氧化特性分析，硫磺菌醱酵產物萃取液包括：(硫磺菌菌絲乾燥物熱水萃取、硫磺菌菌絲乾燥物乙醇萃取、硫磺菌醱酵液乾燥物乙醇萃取、硫磺菌醱酵液胞外多醣、硫磺菌醱酵菌絲乾燥物胞內多醣)，不同濃度對硫磺菌醱酵菌絲體乾燥物萃取以及醱酵液乾燥物萃取之抗氧化特性評估，以EC50為評估依據，菌絲體乾燥物乙醇萃取、熱水萃取，胞內多醣和醱酵液乾燥物乙醇萃取及胞外多醣，其中在還原力方面以醱酵液乾燥物乙醇萃取濃度0.6 g L -1Span80，其EC50為0.06 mg mL-1；清除DPPH能力以菌絲體乾燥物熱水萃取濃度0.8 g L -1Span80，其EC50為0.09 mg mL-1；螯合亞鐵離子以醱酵液乾燥物乙醇萃取濃度0.4 g L -1Span80，其EC50為0.04 mg mL-1；清除超氧陰離子菌絲體乾燥物熱水萃取0.8 g L -1Span80，其EC50為0.22 mg mL-1。

Abstract
Laetiporus sulphureus, one of the rare species, is a traditional medicinal mushrooms. The mushroom can be used as adjuvant food during the treatment of breast cancer and prostate cancer. Previous studies indicated that polysaccharides and extracts from Laetiporus sulphureus posses various biological activities such as anti-oxidant, anti-tumor, increasing immune ability, reducing blood sugar and so on. In this study, effect of supplementation of surfactants in submerged cultures on biomass and exopolysaccharide (EPS) production of Laetiporus sulphureus was investigated to promote biomass and exopolysaccharide formation. In the shake flask experiments, various surfactants (0.5 g L-1), including PEG series (4000, 6000), Tween series (20, 40, 80, 85) and Span series (20, 80), were added into basal medium in shake flasks , and cultivated at 28 ℃, 150 rpm for 12 days. The results indicated that Span 80 showed the maximum biomass and exopolysaccharoide production with 7.29 g L-1 and 0.178 g L-1, respectively. To find the optimal Span 80 concentration different span 80 concentration (0 , 0.2 , 0.4 , 0.6 , 0.8 ,1.0 , 1.2 g L-1) where cultured in the shake flask at 28 ℃ , 150 rpm for 12 days. The results revealed that the maximum biomass (9.33 g L-1) and exopolysaccharide production (0.13 g L-1) was found to be at 0.6 g L-1 of Span 80. For a 5-L stirred tank fermenter test, experimental results showed that the maximum mycelium (4.08 g L-1) was obtained at 0.2 g L-1 Span 80 and the maximum exopolysaccharide production was 213.43 mg L-1 at 0.6 g L-1 Span 80. In addition, the average EPS molecular weights and protein content in EPS at 0.6 g L-1 Span 80 in 5-L stirred tank fermenter were 2.94×104 and 6.64 %. The average EPS molecular weights and protein content in EPS at 0.6 g L-1 Span 80 in 20-L stirred tank fermenter were 5.54×104 and 6.39 %. Moreover, the average IPS molecular weights and protein content in IPS at 0.6 g L-1 Span 80 in 5-L stirred tank fermenter were 5.30×104 and 7.76 %. Tthe average IPS molecular weights and protein content in IPS at 0.6 g L-1 Span 80 in 20-L stirred tank fermenter were 1.03×105 and 6.62 %. The lowest EC50 values are described as follows: for antioxidant activity. For reducing power, EC50 values of ethanol extract from broth by L. sulphureus submerged culture at 0.6 g L-1 Span 80 are 0.06 mg mL-1. For scavenging DPPH effects, EC50 values of ethanol extract from broth by L. sulphureus submerged culture at 0.4 g L-1 Span 80 are 0.09 mg mL-1. For chelating ability, EC50 values of ethanol extract from broth by L. sulphureus submerged culture at 0.4 g L-1 Span 80 are 0.04 mg mL-1. For scavenging superoxide radical ability, EC50 values of hot water extract from mycelia by L. sulphureus submerged culture at 0.8 g L-1 Span 80 are 0.22 mg mL-1.

目錄
摘 要 I
Abstract III
誌謝 V
目錄 VI
表目錄 X
圖目錄 XI
第一章 緒論 1
1.1研究動機與背景 1
1.2研究目的 2
第二章 文獻回顧 3
2.1菇類的介紹和機能性 3
2.1.1 菇類的應用 4
2.1.2 菇類食品生理活性 6
2.1.3 菇類多醣的生理活性 9
2.2 硫磺菌介紹 10
2.3 多醣體介紹 13
2.3.1 多醣體之分類 14
2.3.2 多醣體之作用 14
2.3.3 多醣體及其抗腫瘤機制 15
2.3.4 多醣體之機能性 16
2.4 醱酵深層培養技術介紹 18
2.4.1深層醱酵培養的定義及培養基的成份和功能 18
2.4.2影響深層醱酵培養的其他物理化學因子 22
2.4.3 培養基滅菌 27
2.5 多醣體之分離與純化 28
2.5.1 透析法 28
2.5.2 酒精沉澱法 28
2.5.3 冷凍離心法 28
2.6 自由基與抗氧化介紹 28
2.6.1 自由基如何產生 29
2.6.2 自由基對細胞造成的傷害 29
2.6.3 抗氧化物質(Antioxidant) 29
2.6.4 抗氧化性質分析 30
2.7界面活性劑 31
第三章 材料與方法 33
3.1實驗架構 33
3.2 實驗材料 34
3.2.1 實驗菌株 34
3.2.2 實驗藥品 34
3.2.3 實驗設備與儀器 37
3.2.4 實驗裝置 38
3.3 實驗方法 45
3.3.1 菌株保存 45
3.3.2 培養基成分 45
3.3.3 操作條件 47
3.3.4 單因子液態搖瓶實驗 48
3.4 分析方法 50
3.4.1 醱酵產物分析流程 50
3.4.2 菌體乾重測定 51
3.4.3 粗多醣含量測定 51
3.4.4 殘糖分析 (DNS method) 52
3.4.5 硫磺菌醱酵物之製備 53
3.4.6 硫磺菌醱酵胞外多醣及胞內多醣分子量分析 55
3.4.7 硫磺菌醱酵物萃取之抗氧化性質分析 56
第四章 結果與討論 58
4.1 硫磺菌菌絲型態 58
4.1.1 固態培養 58
4.1.2種瓶培養 59
4.1.3搖瓶液態培養 59
4.1.4攪拌式醱酵槽 60
4.2液態搖瓶培養實驗 61
4.2.1不同界面活性劑對硫磺液態醱酵之生物質量(Biomass)及胞外多醣含量 61
4.2.2 不同濃度界面活性劑Span 80對硫磺菌菌絲生長(Biomass)及生產胞外多醣(EPS)之影響 62
4.3攪拌式醱酵槽實驗 65
4.3.1不同濃度界面活性劑Span 80對硫磺菌菌絲生長(Biomass)及生產胞外多醣(EPS)之影響 65
4.3.2 20L醱酵槽放大液態培養硫磺菌生產菌絲及胞外多醣 70
4.4 硫磺菌醱酵物萃取之抗氧化特性分析 73
4.4.1 硫磺菌醱酵物萃取之還原能力 73
4.4.2 硫磺菌醱酵物萃取之清除 1,1-diphenyl-2-picry- lhydr- azyl (DPPH)自由基能力 77
4.4.3 硫磺菌醱酵物萃取之螯合亞鐵離子能力 81
4.4.4 硫磺菌醱酵物萃取之清除超氧陰離子能力 85
4.5硫磺菌硫磺菌醱酵物萃取之EC50值 89
4.5.1 不同濃度對硫磺菌硫磺菌醱酵物萃取之EC50值 89
4.6 硫磺菌醱酵胞外多醣、胞內多醣分子量含量分析 94
4.7硫磺菌多醣體與蛋白質含量影響 95
第五章 結論與建議 96
5.1 結論 96
5.2 建議 98
參考文獻 99
作者簡介 110




表目錄
表2-1 菇類機能性 4
表2-2 菇類的藥效成分 8
表2-3 Tween 系列界面活性劑 32
表2-4 Span 系列界面活性劑 32
表3- 1 實驗藥品全目錄 34
表3- 2實驗儀器及設備全目錄 37
表3-3 實驗裝置全目錄 39
表3-4 硫磺菌固態培養基之組成 46
表3-5 硫磺菌種瓶培養基之組 46
表3-6 硫磺菌液態培養基之組成 47
表3-7 硫磺菌醱酵槽液態培養基之組成 47
表4- 1 不同濃度操作於(5L)攪拌式醱酵槽培養之結果 69
表4-2 最適化於(20L)攪拌式醱酵槽培養之結果 71
表4-3不同濃度對硫磺菌醱酵產物還原化能力EC50値 90
表4-4不同濃度對硫磺菌醱酵產物清除DPPH自由基能力EC50値 91
表4-5不同濃度對硫磺菌醱酵產物螯合亞鐵離子能力EC50値 92
表4-6不同濃度對硫磺菌醱酵產物清除超氧陰離子能力EC50値 93


圖目錄
圖2-1為硫磺菌之子實體型態 11
圖2-2 胞外多醣合成流程圖 13
圖2-3 β-葡聚糖結晶結構 15
圖2-4 由β-葡聚醣投藥產生之寄主免疫反應 17
圖2-5 各種常見之醱酵槽(Chisti, 1989) 23
圖3-1實驗架構 33
圖3-2 攪拌式生物反應設備( 5L ) 39
圖3-3 攪拌式生物反應設備( 20L ) 40
圖3-4 高壓滅菌釜 40
圖3-5 無菌操作台 41
圖3-6 菌絲均勻機 41
圖3-7 微生物培養箱 42
圖3-8 分光光度計 42
圖3-9 減壓濃縮機 43
圖3-10 高效率液相層析設備 43
圖3-11 冷凍離心機 44
圖3-13 超低溫冷凍櫃 44
圖3- 14 醱酵產物分析流程圖 50
圖3- 15 多醣檢量線 51
圖3- 16 殘糖檢量線 52
圖3- 17 胞內多醣製備之實驗流圖 54
圖3- 18 多醣分子量檢量線 55
圖4-1 硫磺菌固態培養生長圖 58
圖4-2 硫磺菌錐形瓶種瓶培養生長圖 59
圖4-3 硫磺菌錐形瓶培養生長圖 59
圖4-4 磺菌在攪拌式醱酵槽中生長之型態 60
圖4-5不同的界面活性劑對硫磺菌菌絲體含量之影響 61
圖4-6不同的界面活性劑對硫磺菌胞外多醣產量影響 62
圖4-7不同濃度Span 80對硫磺菌菌體產量影響 63
圖4-8不同濃度Span 80對硫磺菌胞外多醣產量影響 64
圖4-10硫磺菌醱酵槽生長曲線圖(0.2 g L-1 Span 80) 66
圖4-11硫磺菌醱酵槽生長曲線圖(0.4 g L-1 Span 80) 67
圖4-12硫磺菌醱酵槽生長曲線圖(0.6 g L-1 Span 80) 67
圖4-13硫磺菌醱酵槽生長曲線圖(0.8 g L-1 Span 80) 68
圖4-14硫磺菌醱酵槽生長曲線圖(1.0 g L-1 Span 80) 68
圖4-15硫磺菌於20L醱酵槽生長曲線圖(0.6 g L-1 Span 80) 71
圖4-16硫磺菌醱酵液胞外多醣之還原力 74
圖4-17硫磺菌醱酵菌絲體乾燥物乙醇萃取之還原力 74
圖4-18硫磺菌醱酵液乾燥物乙醇萃取之還原力 75
圖4-19硫磺菌醱酵菌絲體乾燥物熱水萃取之還原力 75
圖4-20硫磺菌醱酵菌絲體胞內多醣之還原力 76
圖4-21 20L硫磺菌醱酵產物之還原力 76
圖4-22硫磺菌醱酵液胞外多醣之清除DPPH自由基之能力 78
圖4-23硫磺菌醱酵菌絲體乾燥物乙醇萃取之清除DPPH自由基之能力 78
圖4-24硫磺菌醱酵液乾燥物乙醇萃取之清除DPPH自由基之能力 79
圖4-25硫磺菌醱酵菌絲體乾燥物熱水萃取之清除DPPH自由基之能力 79
圖4-26硫磺菌醱酵菌絲體胞內多醣之清除DPPH自由基之能力 80
圖4-27 20L硫磺菌醱酵產物之清除DPPH自由基之能力 80
圖4-28硫磺菌醱酵液胞外多醣之螯合亞鐵離子能力 82
圖4-29硫磺菌醱酵菌絲體乾燥物乙醇萃取之螯合亞鐵離子能力 82
圖4-30硫磺菌醱酵液乾燥物乙醇萃取之螯合亞鐵離子能力 83
圖4-31硫磺菌醱酵菌絲體乾燥物熱水萃取之螯合亞鐵離子能力 83
圖4-32硫磺菌醱酵菌絲體胞內多醣之螯合亞鐵離子能力 84
圖4-33 20L硫磺菌醱酵產物之螯合亞鐵離子能力 84
圖4-34硫磺菌醱酵液胞外多醣之捕捉超氧陰離子 86
圖4-35硫磺菌醱酵菌絲體乾燥物乙醇萃取之捕捉超氧陰離子 86
圖4-36硫磺菌醱酵液乾燥物乙醇萃取之捕捉超氧陰離子 87
圖4-37硫磺菌醱酵菌絲體乾燥物熱水萃取之捕捉超氧陰離子 87
圖4-38硫磺菌發酵菌絲體胞內多醣之捕捉超氧陰離子 88
圖4-39 20L硫磺菌發酵產物之捕捉超氧陰離子 88

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