臺灣博碩士論文加值系統

摘 要
本研究探討香杉芝之固態深層培養的條件，期望能培養出與樟芝相同具有良好生理功效之產品。香杉芝是由香杉木上所發現之新種，因外觀平伏緊貼而稱為芝，與樟芝同屬，易與樟芝混淆之藥用菇類。真菌菇類具有一次營養機能性、二次嗜好機能性及三次生理機能性，本實驗係針對香杉芝菌絲體、香杉芝燕麥及燕麥，進行一般成分、營養、物性、呈味、生理活性物質、抗氧化和抗發炎性質之分析。近來研究指出，經由香杉芝所純化出來的化合物，在人類白血球細胞具有抗氧化功效(Shen，2006)，但由於有關於香杉芝之文獻甚少，因此想深入探究。
以加水量50% 之燕麥為基礎培養基，在不同發酵條件下生長所得之香杉芝燕麥，生產品質穩定之產品，探討獲得最大量菌絲體之最適培養基組成。經探討後，於28天的培養時間，發現以第九天液態菌種接種比例10%，均質時間為0秒、於25C的培養溫度下，並分別使用碳源和氮源為 1% 葡萄糖和 0.5% 脫脂大豆粉時，可獲得最高產量的菌絲體（0.42 mg/g）。
進一步針對最適固態條件進行大量培養，結果發現，菌絲體主要由醣類（42.71%）和粗蛋白（44.21%）組成。香杉芝燕麥與燕麥在一般組成上並無太大的差異，主要差異在於組成分醣類上（63.00和70.15%）。在生理活性物質方面，腺苷含量以菌絲體最高；麥角固醇含量上，菌絲體（3.17）>香杉芝燕麥（0.42 mg/g）。在呈味成分含量（以等價鮮味值表示）方面，依序為菌絲體（3.34）>香杉芝燕麥（0.39）及燕麥（0.03 g/100 g）。
不論乙醇萃取或熱水萃取，皆是以香杉芝燕麥萃取率最高。菌絲體、香杉芝燕麥及燕麥之熱水萃取物在抗氧化力部分，於20 mg/ml時，依序為香杉芝燕麥（92.43）＞燕麥（65.12）＞菌絲體（49.26%）。在還原力部分，菌絲體、香杉芝燕麥及燕麥熱水萃取物在20 mg/ml時，分別為1.22、2.16和0.10。捕捉DPPH自由基能力方面以燕麥熱水萃取物清除能力最佳，在5 mg/ml時即可達100%。而在螯合亞鐵離子之能力上，於20 mg/ml時，依序為香杉芝燕麥（96.83）＞燕麥（67.73）＞菌絲體（46.22%）。
菌絲體、香杉芝燕麥及燕麥之乙醇萃取物在20 mg/ml時，抗氧化力分別為88.1、100和100%。在還原力上，菌絲體、香杉芝燕麥及燕麥之乙醇萃取物在10 mg/ml時，分別為0.46、1.12和0.43。在清除DPPH自由基之能力上，於20 mg/ml時，依序為香杉芝燕麥（94.20%）>菌絲體（92.85%）＞燕麥（92.68%）。在螯合亞鐵離子之能力上，在20 mg/ml 時，菌絲體、香杉芝燕麥及燕麥分別為84.04、100和92.18%。在抗氧化成分分析上，香杉芝燕麥之總酚類化合物是所有試驗樣品中含量最多的天然抗氧化成分，含量上以熱水萃取量較高。
在抗發炎部分，菌絲體、香杉芝燕麥及燕麥之熱水萃取物，在測試劑量範圍內（2.5~500 μg/ml），對小鼠巨噬細胞（RAW 264.7）皆不具有生長抑制。對於內毒素(LPS)誘導巨噬細胞生成之NO具有捕捉效果，於濃度500.0 μg/ml，清除率分別為21.34、40.84和12.45%。
綜合上述得知燕麥經過固態發酵為香杉芝燕麥後，可提高燕麥之營養、呈味和生理活性物質之含量及抗氧化及抗發炎性質。因此，本研究之結果深具開發具營養、機能及保健性之潛力產品。

Abstract
The research reported herein is designed to study the solid culture of Antrodia salmanea to produce high physicochemical properties product like Antrodia cinnamomea. Antrodia salmonea is a new specific, the fungus is associated with Cunninghamia konishii. By the appearance of calm down and as close is called the iris, and this fungus is similar to Antrodia cinnamomea. Mushrooms possess three functionalities including the first nutritional functionality, the second taste functionality and the third physiological functionality. The objectives of this study were to investigate the proximate composition, nutritional components, physicochemical properties, taste components, physiologically active components, antioxidant properties, and antinflammation effects of Antrodia salmanea mycelia, A. Salmonea colonized oat (ASCO) and oat. Recent research has pointed out, new lanostanes and naphthoquinones isolated from Antrodia salmonea and their antioxidative burst activity in human leukocytes (Shen， 2006). However, only limited information is reported in the literature about this fungus, then the objective of this research was to study Antrodia salmonea
ASCO was obtained from the solid state fermentation of cooked oat with 50% moisture content as the base under various conditions to produce high quality products, then to study the optimal conditions for solid culture of A. Salmonea to evaluate the maximum biomass. With regard to A. Salmonea for solid culture, the optimal conditions were incubation time of inoculum 8 days, inoculation rate 10%, homogenization time of inoculum 0 sec with carbon and nitrogen sources being 1% glucose and 0.5% soybean meal for 28 days at 25°C to get the highest yield (0.42 mg/g).
Furthermore, the optimal conditions for solid culture was used to get a great quantity culture. Carbohydrate (42.71%) and crude protein (44.21%) were major components found in mycelia. Generally, the proximate profiles of ASCO and oat were similar, but different with the major component carbohydrate being (63.00 and 70.15%, respectively). With regard to physiologically active components, the contents of adenosine of mycelia was highest ; the contents of ergosterol in order were mycelia (3.17 mg/g) > filtrate (0.42 mg/g). With regard to contents of taste components as expressed equivalent umami concentration, mycelia (3.34) was higher than ASCO (0.39) and oat (0.03 g/100 g).
Extraction yield of ethanolic and hot water extracts from ASCO was better than A. Salmonea mycelia and oat. The antioxidant activities of hot water extracts were in the descending order: ASCO (92.43%) > oat (65.12%) > mycelia (49.26%) at 20 mg/ml. The reducing powers of hot water extracts were 1.22, 2.16 and 0.10 at 20 mg/ml for mycelia, ASCO and oat, respectively. The hot water extracts from oat showed an excellent scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals at 5 mg/ml (100%). At 20 mg/ml, chelating abilities of the hot water extracts on ferrous ions were in the descending order : ASCO (96.83%) > oat (67.73%)> mycelia (46.22%).
For mycelia, ASCO and oat, at 20 mg/ml, antioxidant activities of ethanolic extracts were 88.1, 100 and 100% whereas at 10 mg/ml, reducing powers were 0.46, 1.12 and 0.43, respectively. At 20 mg/ml, scavenging abilities of ethanolic extracts on DPPH were in the descending order: ASCO (94.20%) > oat (92.85%) > mycelia (92.68%). For ethanolic extracts from mycelia, ASCO and oat chelated ferrous ions by 84.04, 100.00 and 92.18% at 20 mg/ml, respectively. Total phenols were the major naturally occurring antioxidant components found in all samples, and the content of hot water extracts was more then ethanolic extracts.
In the anti- inflammation activity test, the inhibition on the growth of RAW 264.7 cells was studied in a MTT test using hot water extracts from mycelia, ASCO and oat. Four samples with the increased concentrations did not exhibit the growth of RAW 264.7 cells. Effect of hot water extracts from mycelia, ASCO and oat on LPS-induced NO production in Raw 264.7 cell in the descending order : ASCO (40.84%) > mycelia (21.34%) > oat (12.45%) at 500.0 μg/ml.

目 錄
表次 ix
圖次 xii
前言 1
文獻整理 2
一、香杉芝之介紹 2
二、食藥用菇之機能性 2
三、固態發酵 4
四、食藥用菇之生理活性物質 8
五、菇類之呈味特性 10
六、抗氧化性質 19
七、發炎反應與相關因子 22
材料與方法 26
一、實驗材料 26
二、實驗方法 26
（一）培養條件探討 26
1. 試藥 26
2. 菌種保存 26
3. 菌種活化及接種原製備 26
4. 液態發酵 27
5. 固態發酵 27
6. 樣品粉末製備 29
（二）一般成分分析 29
1. 試藥 29
2. 水分 29
3. 灰分 30
4. 粗脂質 30
5. 粗纖維 30
6. 粗蛋白 30
7. 還原糖 31
（三）品質評估 31
1. 試藥 31
2. 物理性質分析 31
3. 生理活性物質分析 32
（四）呈味特性成分分析 34
1. 試藥 34
2. 可溶性糖 34
3. 游離胺基酸 35
4. 核苷酸 36
（五）抗氧化性質分析 37
1. 試藥 37
2. 樣品製備 37
3. 抗氧化力 38
4. 還原力 38
5. 捕捉1,1-二苯基-2-苦味肼基團（DPPH）能力 38
6. 清除羥自由基之能力 39
7. 螯合亞鐵離子之能力 39
8. 抗氧化成分分析 39
（六）抗發炎試驗 41
1. 試藥 41
2. 細胞株 41
3. 熱水萃取物之製備 41
4. 巨噬細胞存活率之測定 42
（七）統計分析 43
結果與討論 44
一、培養條件之探討 44
二、一般成分分析 57
（一）水分 57
（二）碳水化合物 57
（三）粗灰分 59
（四）粗脂質 59
（五）粗纖維 59
（六）粗蛋白 60
三、品質評估 60
（一）物理性質分析 60
（二）生理活性物質分析 68
四、呈味特性成分分析 74
（一）可溶性糖 74
（二）游離胺基酸 79
（三）核苷酸 80
五、抗氧化性質分析 86
（一）熱水和乙醇萃取物之萃取率 86
（二）熱水萃取物之抗氧化性質 88
1. 熱水萃取物之抗氧化力 88
2. 熱水萃取物之還原力 88
3. 熱水萃取物之捕捉1,1-二苯基-2-苦味肼基團能力 93
4. 熱水萃取物之清除羥自由基能力 96
5. 熱水萃取物之螯合亞鐵離子能力 99
6. 熱水萃取物抗氧化性質之EC50 102
7. 熱水萃取物之抗氧化成分分析 102
（三）乙醇萃取物之抗氧化性質 107
1. 乙醇萃取物之抗氧化力 107
2. 乙醇萃取物之還原力 110
3. 乙醇萃取物之捕捉1,1-二苯基-2-苦味肼基團能力 110
4. 乙醇萃取物之螯合亞鐵離子能力 110
5. 乙醇萃取物抗氧化性質之EC50 117
6. 乙醇萃取物之抗氧化成分分析 117
六、發炎反應之評估 121
（一）對小鼠巨噬細胞（RAW 264.7）生長之影響 121
（二）LPS誘導巨噬細胞(RAW 264.7)生成NO之影響 124
結論 130
參考文獻 131

表 次
附表1、L-胺基酸之呈味特性 13
附表2、胺基酸之相對甜度 14
附表3、鮮味胺基酸及鮮味核苷酸之個別相對鮮味強度 15
附表4、菇類子實體的等價鮮味值 16
附表5、菇類子實體的等價鮮味值(Continued) 17
附表6、菇類菌絲體之等價鮮味值 18
表1、液態發酵期間香杉芝生物質、pH值和殘糖量之變化 45
表2、香杉芝固態發酵期間，生物質和pH值之變化 47
表3、不同培養條件所得香杉芝燕麥之生物質及最終pH值 51
表4、香杉芝菌絲、香杉芝燕麥和燕麥之一般組成 58
表5、香杉芝固態發酵期間，生物質和pH值之色澤分析 61
表6、不同培養條件所得香杉芝燕麥之色澤分析 63
表7、香杉芝菌絲體、香杉芝燕麥和燕麥之色澤分析 67
表8、不同溫度下香杉芝菌絲、香杉芝燕麥和燕麥之溶解度 69
表9、不同溫度下香杉芝菌絲、香杉芝燕麥和燕麥之膨潤力 70
表10、不同溫度下香杉芝菌絲、香杉芝燕麥和燕麥之吸水指標 71
表11、香杉芝菌絲、香杉芝燕麥和燕麥之麥角固醇、腺苷和粗三
帖含量 73
表12、香杉芝菌絲、香杉芝燕麥和燕麥多醣分子量分布 77
表13、香杉芝菌絲、香杉芝燕麥和燕麥之可溶性糖類組成 78
表14、香杉芝菌絲、香杉芝燕麥和燕麥之游離胺基酸組成 81
表15、不同溫度下香杉芝菌絲、香杉芝燕麥和燕麥之游離胺基
酸之呈味特性 82
表16、香杉芝菌絲、香杉芝燕麥和燕麥之核苷酸組成 83
表17、香杉芝菌絲、香杉芝燕麥和燕麥之等價鮮味值 85
表18、香杉芝菌絲、香杉芝燕麥和燕麥乙醇和熱水萃取物之萃取率 87
表19、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物之抗氧化力 89
表20、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物之還原力 91

表21、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物清除1,1-二苯
基-2-苦味肼基團自由基之能力 94
表22、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物清除羥自由
基之能力 97
表23、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物對亞鐵離子
之螯合能力 100
表24、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物抗氧化性質
之EC50 103
表25、香杉芝菌絲、香杉芝燕麥和燕麥粉末抗氧化性質之
EC50（熱水） 104
表26、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物中抗壞血酸、
β-胡蘿蔔素、生育酚和總多酚類之含量 106
表27、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物之抗氧化力 108
表28、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物之還原力 111
表29、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物清除1,1-二
苯基-2-苦味肼基團自由基之能力 113
表30、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物對亞鐵離子
之螯合能力 115
表31、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物抗氧化性質
之EC50 118
表32、香杉芝菌絲、香杉芝燕麥和燕麥粉末抗氧化性質之
EC50（乙醇） 119
表33、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物中抗壞血酸、
β-胡蘿蔔素、生育酚和總多酚類之含量 120
表34、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物之總酚類和
其抗氧化能力之相關係數 122
表35、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物之總酚類和
其抗氧化能力之相關係數 123
表36、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物對巨噬細胞
（RAW 264.7）生長之影響 125
表37、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物對LPS
誘導巨噬細胞（RAW 264.7）生成NO之影響 128

圖 次
圖1、液態發酵期間香杉芝生物質和pH值之變化 46
圖2、香杉芝燕麥固態發酵期間，生物質和pH值之變化 48
圖3、香杉芝固態發酵期間菌絲生長情形 49
圖4、不同（A）溫度（B）初始pH值（C）氮源（D）碳源（E）
接種原均質時間（F）接種量（G）培養空間對培養28天之
香杉芝燕麥生物質和最終pH之影響 55
圖5、高效液相層析之糊精標準曲線 75
圖6、(A) 香杉芝菌絲、(B) 香杉芝燕麥和 (C) 燕麥之 PolySep-G
FC-P 4000 and PolySep-GFC-P 5000層析圖 76
圖7、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物之抗氧化力 90
圖8、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物之還原力 92
圖9、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物清除1,1-二苯基-
2-苦味肼基團自由基之能力 95
圖10、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物清除羥自由基能力 98
圖11、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物對亞鐵離子之
螯合能力 101
圖12、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物之抗氧化力 109
圖13、香杉芝菌絲、香杉芝燕麥和燕麥乙醇取物之還原力 112
圖14、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物清除1,1-二苯
基-2-苦味肼基團自由基之能力 114
圖15、香杉芝菌絲、香杉芝燕麥和燕麥乙醇萃取物對亞鐵離子之
螯合能力 116
圖16、香杉芝菌絲、香杉芝燕麥和燕麥水萃取物對巨噬細胞存活
率之影響 126
圖17、香杉芝菌絲、香杉芝燕麥和燕麥熱水萃取物對LPS誘導巨
噬細胞生成NO之影響 129

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