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研究生:黃仕政
研究生(外文):Huang, Shih-Jeng
論文名稱:蛹蟲草之培養和其呈味性質及經γ-照射之巴西蘑菇子實體和樟芝菌絲體之抗氧化性質
論文名稱(外文):Cultivation of Cordyceps militaris and its taste quality and antioxidant properties of γ-irradiated Agaricus blazei fruit bodies and Antrodia camphorata mycelia
指導教授:毛正倫毛正倫引用關係
指導教授(外文):Mau, Jeng-Leun
學位類別:博士
校院名稱:國立中興大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:200
中文關鍵詞:蛹蟲草巴西蘑菇樟芝培養呈味品質γ-照射抗氧化性質
外文關鍵詞:Cordyceps militarisAgaricus blazeiAntrodia camphorataincubationtaste qualityγ-irradiationantioxidant property
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本研究為了解蛹蟲草培養的可行性和一般食用上的應用性,而探討其培養條件和呈味性質;另外,因γ-射線經常應用於食藥用菇相關產品的滅菌,為了解γ-照射過的食藥用菇品質,本研究也針對γ-照射過的巴西蘑菇子實體和樟芝菌絲體之抗氧化性質加以研究。
蛹蟲草 [Cordyceps militaris (L.) Link] 又稱北方蟲草,在台灣目前已經當做營養食品和機能性食品的配方使用。本研究則針對其菌種篩選、培養條件和生理活性成分加以探討。結果發現,經篩選發現蛹蟲草CM2因具有快速形成子實體的能力,而適合做為菌絲體液態培養和子實體固態培養時使用的菌株。根據培養皿培養的初步探討,CM2的較適培養基和條件為使用葡萄糖或蔗糖為碳源、酵母粉或酵母抽出物為氮源以及將其pH值調至5.5-6.0。而使用三角瓶震盪培養14天時,發現使用碳源和氮源分別為2%葡萄糖和1%胰蛋白腖時,並將酸鹼值調成pH 6.0且於20ºC的環境培養,可得到較高的產量。此外,子實體固態培養時,以蔗糖和酵母粉分別當作碳源和氮源,並在起始pH值為6.0和25ºC的培養溫度下,是較適且較合乎成本的培養條件。菌種保存方面,水是所使用的保護劑中最經濟且繼代培養步驟較簡單的。當培養完成時,所採收的子實體中腺苷、蟲草酸和多醣體的含量分別為0.88、117.66和23.21 mg/g,而在菌絲體中則為1.41、112.55和21.01 mg/g。整體而言,蛹蟲草CM2易於培養且具有生理活性物質,是值得開發的食藥用菇。
本研究進一步地針對蛹蟲草子實體和菌絲體之非揮發性呈味成分加以探討。結果發現,子實體和菌絲體兩者都含有多量的碳水化合物、粗纖維和粗蛋白。而子實體和菌絲體之總可溶性糖和糖醇含量分別為260.64和189.82 mg/g。其中甘露糖醇在子實體和菌絲體中的含量都是最多,分別為117.66和112.55 mg/g。子實體和菌絲體中總游離胺基酸的含量分別為48.15和67.63 mg/g。另外,類味精 (MSG-like) 成分的含量在子實體中為10.60 mg/g,多於菌絲體所含有的2.70 mg/g;但是,總5'-核苷酸和呈味5'-核苷酸的含量則是菌絲體中較多,分別為26.27和9.34 mg/g。以等價鮮味值來看,子實體和菌絲體的含量很接近,其相等的鮮味強度則分別為6.08和6.22 g的MSG。整體而言,蛹蟲草子實體和菌絲體擁有非常強的鮮味。
在γ-照射和抗氧化性質的關係方面,本研究將巴西蘑菇 (Agaricus blazei Murrill) 分別以劑量為2.5、5、10、15和20 kGy的 γ-射線照射後,針對其甲醇萃取物之抗氧化活性加以探討。當濃度為7.5和10 mg/mL時,巴西蘑菇經2.5-20 kGy之γ-照射後,其甲醇萃取物之抗氧化力顯著高於未照射組的甲醇萃取物。當濃度為0.5-7.5 mg/mL時,巴西蘑菇之2.5、10、15和20 kGy γ-照射組與未照射組,其甲醇萃取物間之還原力則相當。所有甲醇萃取物於濃度為0.5 mg/mL時都表現出極佳的清除1,1-二苯基-2-苦味肼基 (DPPH) 之能力 (95.2-100.7%)。關於清除羥自由基的能力方面,未照射組和照射組之甲醇萃取物間的能力無顯著差異。而巴西蘑菇甲醇萃取物中之總酚是最主要的天然抗氧化物,其含量範圍為18.77-21.48 mg/mL。此外,所有甲醇萃取物的EC50都低於10 mg/mL,而其中之還原力、清除DPPH能力和螯合亞鐵離子能力方面,其值都低於1 mg/mL。結果指出,未照射與照射的巴西蘑菇都具有良好的抗氧化性質,而且經20 kGy以下γ-照射的巴西蘑菇中總抗氧化物質的含量並無顯著之影響。
此外,樟芝 [Antrodia camphorata (Chang & Chou) Wu, Ryvvarden & Chang] 菌絲體亦分別以劑量為2.5、5、10、15 和20 kGy的γ-射線照射後,同時也針對其甲醇萃取物之抗氧化性質加以探討。當濃度為2.5 mg/mL 時,菌絲體經10-20 kGy γ-照射後,其甲醇萃取物之抗氧化力顯著高於未照射者。其中除了經20 kGy γ-照射的菌絲體之外,未照射者及經0.5和7.5 kGy照射者之甲醇萃取物間之還原力相當。而當濃度為2.5 mg/mL時,所有甲醇萃取物表現出極佳之清除DPPH自由基能力為92.3-103%。此外,菌絲體經2.5-20 kGy γ-照射者,其甲醇萃取物於濃度為10 mg/mL時之清除羥自由基能力都比未照射者顯著為高;而經5-20 kGy 照射之菌絲體,其螯合亞鐵離子之能力也比未照射者顯著為高。再者,除了未照射組之清除羥自由基之能力外,所有組別之抗氧化性質之EC50值都小於 15 mg/mL。另一方面,所測得的天然抗氧化物質中,總酚類是最主要的成分,其含量範圍為12.97-15.47 mg/mL。研究結果顯示,γ-照射的處理不僅保有菌絲原本之抗氧化性質,而且能提升其部份抗氧化性質至較高的程度。
In order to know the feasibility of cultivation of Cordyceps militaris on and its application in edibility, the object of this research was to study its culture conditions and taste quality. In addition, because γ-ray is often applied to the sterilization of the products of edible and medicinal mushrooms, in the cause of understanding the quality of γ-irradiated mushrooms, this study also focused on the antioxidant properties of γ-irradiated Agaricus blazei fruit bodies and Antrodia camphorata mycelia.
Cordyceps militaris (L.) Link (Clavicipitaceae), northern Cordyceps, is currently available in Taiwan for use in the formulation of nutraceuticals and functional foods. The research reported herein was designed to study the screening of C. militaris, conditions of cultivation and physiologically active components. Cordyceps militaris CM2 is a optimal strain for submerged culture and solid culture, because it can fruit quickly on solid medium. Incubation on Petri dish with potato dextrose agar, the more ideal medium and conditions for CM2 were glucose or sucrose as the carbon source and yeast powder or yeast extract as the nitrogen source at pH 5.5-6.0. Using shaken flask incubation for 14 days, CM2 mycelia grew well on the liquid medium with 2% glucose as the carbon source and 1% trypton as the nitrogen source at pH 6.0 and 20ºC. In addition, for the production of fruit bodies, using sucrose as the carbon source and yeast powder as the nitrogen source at pH 6.0 and 25ºC is a suitable and economical incubation condition. For maintaining CM2 culture, water is the most economical preservation reagent and the easiest method for continuous subculture. After incubation, harvesting and assaying, the contents of adenosine, polysaccharide and cordycepic acid of fruit bodies were 0.88, 117.66 and 23.21 mg/g and those of mycelia were 1.41, 112.55 and 21.01 mg/g. Overall, C. militaris CM2 is worth developing, because it is easy to incubate and possesses physiologically active components.
Furthermore, the non-volatile components in the form of C. militaris fruit bodies and mycelia were studied. Both fruit bodies and mycelia were high in contents of carbohydrate, crude fiber and crude protein. Content of total sugars and polyols were 260.64 and 189.82 mg/g for fruit bodies and mycelia, respectively. Mannitol content was the highest in both fruit bodies and mycelia (117.66 and 112.55 mg/g, respectively). Contents of total free amino acids in fruit bodies and mycelia were 48.15 and 67.63 mg/g, respectively. The content of monosodium glutamate (MSG)-like components in fruit bodies (10.60 mg/g) was higher than that in mycelia (2.70 mg/g). The contents of total and flavor 5'-nucleotides were high in mycelia (26.27 and 9.34 mg/g, respectively). Equivalent umami concentrations of fruit bodies and mycelia were similar, and equivalent to the umami intensity given by 6.08 and 6.22 g of MSG, respectively. Overall, both fruit bodies and mycelia of northern Cordyceps possessed highly intense umami taste.
In addition, Agaricus blazei Murrill (Agariaceae) was irradiated with gamma rays at doses of 2.5, 5, 10, 15 and 20 kGy and the antioxidant properties of its methanolic extracts were studied. At 7.5 and 10.0 mg/mL, antioxidant activities of methanolic extracts from 2.5-20 kGy γ-irradiated A. blazei were significantly higher than those of methanolic extract from the non-irradiated control. At 0.5-7.5 mg/mL, reducing powers of methanolic extracts from A. blazei with 2.5, 10, 15 and 20 kGy of irradiation and without irradiation were comparable. All methanolic extracts showed excellent scavenging abilities of 95.2-100.7% against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at 0.5 mg/mL. With regard to the scavenging ability against hydroxyl radicals, unirradiated and γ-irradiated A. blazei were comparable. Total phenols were the major naturally occurring antioxidant components found in the range of 18.77-21.48 mg/g. All EC50 values were below 10 mg/mL, except values in reducing power, scavenging ability against DPPH radicals and chelating ability against ferrous ions were below 1 mg/mL. That indicates the unirradiated and irradiated A. blazei were good in antioxidant properties. Summarily, up to 20 kGy of irradiation did not remarkably affect the amounts of total antioxidant components in A. blazei.
Besides, the mycelia of Antrodia camphorata (Chang & Chou) Wu, Ryvarden & Chang were irradiated with gamma rays at doses of 2.5, 5, 10, 15 and 20 kGy and the antioxidant properties of its methanolic extracts were also studied. At 2.5 mg/mL, antioxidant activities of methanolic extracts from 10-20 kGy irradiated mycelia were significantly higher than the non-irradiated control. Reducing powers of methanolic extracts from unirradiated and 0.5 to 7.5 kGy irradiated mycelia were comparable except 20 kGy irradiated mycelia. At 2.5 mg/mL, all methanolic extracts showed excellent scavenging abilities of 92.3-103% on DPPH radicals. Scavenging abilities of methanolic extracts from 2.5-20 kGy irradiated mycelia on hydroxyl radical were better than that of the unirradiated control at 10 mg/mL. With irradiation at 5-20 kGy, mycelia possessed higher chelating ability on ferrous ions than the unirradiated control. The EC50 values were below 15 mg/mL, except values in scavenging ability of the unirradiated control on hydroxyl radicals. Total phenols were the major naturally occurring antioxidant components found in the range of 12.97-15.47 mg/g. Summarily, γ-irradiation not only maintained the antioxidant properties of mycelia but also enhanced some of its antioxidant properties to higher extent.
目 錄
第一章 文獻整理 1
壹、食藥用菇之機能性 2
一、一次機能 2
二、二次機能 3
三、三次機能 4
四、四次機能 4
貳、自由基與氧化 5
一、自由基之種類與生成 5
二、脂質過氧化物的產生 5
(一) 起始期 6
(二) 連鎖反應期 6
(三) 終止反應期 7
三、氧化與食品和疾病之關係 7
四、氧化的抑制 8
參、食藥用菇之呈味性質 9
一、游離胺基酸 9
二、核苷酸 10
三、可溶性糖及糖醇 14
肆、食藥用菇之培養 14
一、菇類子實體之栽培 18
(一) 木生菌 18
(二) 土生菌 18
(三) 蟲生菌 18
二、菇類菌絲體之培養 19
(一) 液態發酵技術 19
(二) 固態發酵技術 20
伍、加馬射線於食品上之應用 20
一、食品輻射照射技術的源由 20
二、食品輻射照射技術 21
三、食品照射之效應 22
(一) 生物學效應 22
(二) 生理學效應 22
(三) 化學效應 22
四、影響照射效果之因素 22
五、照射技術在食品上之應用 23
(一) 提高葡萄渣中花青素的回收率 23
(二) 降解碳水化合物,增加生物資源的回收 23
(三) 促進米酒的熟成 27
(四) 促進菇類培養基質的利用 27
(五) 促進類黃酮的生合成 27
(六) 減少食物過敏症 27
(七) 減少食物中抗營養因子的含量 27
(八) 化學單體的接枝 28
(九) 脫色 28
陸、蛹蟲草之簡介 28
一、蛹蟲草之現況 28
二、分布情形 28
三、安全性和毒性研究 30
四、化學組成 30
(一) 硒 30
(二) 脂肪酸 30
(三) 胺基酸 30
(四) 蟲草素 30
(五) 甘露糖醇 30
(六) 超氧歧化酶 31
(七) 麥角固醇 31
(八) 腺苷 31
(九) 多醣體 31
五、生物活性研究 31
(一) 抑制腫瘤作用 31
(二) 抗氧化作用 32
(三) 抗疲勞作用 32
(四) 保肝作用 32
(五) 抑菌作用 32
(六) 抗發炎作用 32
(七) 降血糖作用 32
六、一般食用方法 33
七、發展蛹蟲草之優點 33
柒、巴西蘑菇之簡介 34
一、巴西蘑菇的近況 34
二、巴西蘑菇的組成 34
三、巴西蘑菇的生理活性 36
(一) 抗腫瘤作用 36
(二) 抗癌作用 37
(三) 降血糖作用 37
(四) 抗血栓作用 37
(五) 降血壓和降膽固醇作用 38
(六) 免疫調節作用 38
(七) 抗病毒作用 38
(八) 抗致突變作用 38
(九) 保肝作用 39
四、巴西蘑菇的儲藏性 39
捌、樟芝之簡介 40
一、樟芝之分類及分佈 40
二、樟芝的近況 42
三、樟芝的化學組成 42
四、生物活性研究 45
(一) 抗氧化作用 45
(二) 保肝作用 46
(三) 抗腫瘤作用 47
(四) 抗發炎作用 48
(五) 抗血小板凝集作用 48
(六) 降血糖作用 48
玖、研究目的 49
拾、實驗架構 50
第二章 蛹蟲草子實體之栽培及其菌絲體之液態培養 51
摘要 52
Abstract 53
壹、前言 54
貳、材料與方法 55
一、蛹蟲草 55
二、藥品與培養基 55
三、較適培養條件探討 56
(一) 固態培養基中碳、氮源和 pH 值對菌絲體生長之影響 56
(二) 液態培養基中碳、氮源、pH 值和溫度對菌絲體生長之影響 56
(三) 子實體固態培養 57
四、菌種保存 57
五、生理活性物質之分析 58
(一) 多醣體 58
(二) 蟲草素和腺苷 58
(三) 甘露糖醇 59
六、生菌數測定 59
七、統計分析 60
參、結果與討論 61
一、菌種篩選 62
二、較適培養條件之培養皿試驗 62
三、菌絲體之液態培養 68
四、子實體之固態培養 70
五、菌種保存 72
六、生理活性物質和生菌數 74
肆、結論 77
第三章 蛹蟲草子實體及菌絲體之非揮發性呈味性質 78
摘要 79
Abstract 80
壹、前言 81
貳、材料與方法 82
一、子實體與菌絲體 82
(一) 菌種來源與活化 82
(二) 子實體之培養 82
(三) 菌絲體之培養 82
(四) 子實體與菌絲體樣品粉末 83
二、藥品及培養基 83
三、一般組成分析 84
(一) 水分之測定 84
(二) 脂質之測定 84
(三) 蛋白質之測定 84
(四) 灰分之測定 85
(五) 粗纖維之測定 85
(六) 還原糖之測定 85
四、可溶性糖與糖醇之測定 86
(一) 可溶性糖類與糖醇之萃取 86
(二) 可溶性糖類與糖醇之分析 86
五、游離胺基酸之測定 87
(一) 游離胺基酸之萃取 87
(二) 游離胺基酸之分析 87
六、5'-核苷酸之測定 87
(一) 核苷酸之萃取 87
(二) 核苷酸之分析 88
七、等價鮮味值之換算 88
八、統計分析 89
參、結果與討論 90
一、一般組成 91
二、可溶性糖與糖醇 93
三、游離胺基酸 95
四、5'-核苷酸 98
五、等價鮮味值 98
肆、結論 100
第四章 巴西蘑菇子實體經γ-射線照射後之甲醇萃取物的抗氧化性質 103
摘要 104
Abstract 105
壹、前言 106
貳、材料與方法 108
一、巴西蘑菇樣品製備與萃取 108
二、藥品 108
三、抗氧化力 109
四、還原力分析 110
五、清除 1,1-二苯基-2-苦味肼基能力分析 110
六、清除羥自由基能力分析 111
七、螯合亞鐵離子能力分析 111
八、抗氧化物質分析 112
(一) 抗壞血酸含量測定 112
(二) β-胡蘿蔔素測定 112
(三) 生育酚測定 113
(四) 總酚類化合物測定 113
九、統計分析 114
參、結果與討論 115
一、萃取率 115
二、抗氧化力 117
三、還原力 120
四、清除 1,1-二苯基-2-苦味肼基能力 123
五、清除羥自由基能力 126
六、螯合亞鐵離子能力 128
七、抗氧化性質之EC50 128
八、抗氧化物質 132
肆、結論 135
第五章 樟芝菌絲體經γ-射線照射後之甲醇萃取物的抗氧化性質 136
摘要 137
Abstract 138
壹、前言 139
貳、材料與方法 141
一、樟芝菌絲體 141
二、藥品 141
三、抗氧化力分析 142
四、還原力分析 143
五、清除 1,1-二苯基-2-苦味肼基能力分析 143
六、清除羥自由基能力分析 144
七、螯合亞鐵離子能力分析 144
八、抗氧化物質分析 145
(一) 抗壞血酸含量測定分析 145
(二) β-胡蘿蔔素測定 145
(三) 生育酚測定 146
(四) 總酚類化合物測定 146
九、統計分析 147
參、結果與討論 148
一、萃取率 148
二、抗氧化力 150
三、還原力 154
四、清除 1,1-二苯基-2-苦味肼基能力 157
五、清除羥自由基能力 160
六、螯合亞鐵離子能力 162
七、抗氧化性質之EC50 165
八、抗氧化物質 167
肆、結論 170
參考文獻 171

表 次
附表 1、L-胺基酸之呈味特性 11
附表 2、胺基酸之相對甜度 12
附表 3、鮮味胺基酸及鮮味核苷酸之個別相對鮮味強度 13
附表 4、菇類子實體的等價鮮味值 15
附表 5、菇類菌絲體之等價鮮味值 17
附表 6、食品輻射照射處理標準 24
表 2-1、蛹蟲草培養於不同三角瓶液態培養條件下之菌絲體產量 69
表 2-2、蛹蟲草培養於不同的固態培養條件下之子實體產量 71
表 2-3、蛹蟲草菌絲體經覆蓋不同保護劑後於-20ºC的環境下存放一段時間之存活力 73
表 2-4、蛹蟲草子實體和菌絲體之生理活性物質和總生菌數含量 75
表 3-1、蛹蟲草子實體和菌絲體之ㄧ般組成 92
表 3-2、蛹蟲草子實體和菌絲體中可溶性糖和糖醇之含量 94
表 3-3、蛹蟲草子實體和菌絲體中游離胺基酸之含量 96
表 3-4、蛹蟲草子實體和菌絲體中游離胺基酸之呈味特性 97
表 3-5、蛹蟲草子實體和菌絲體中5'-核甘酸之含量 99
表 3-6、蛹蟲草子實體和菌絲體之等價鮮味值 101
表 4-1、巴西蘑菇子實體經不同劑量γ-照射後之甲醇萃取率 116
表 4-2、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之抗氧化力 118
表 4-3、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之還原力 121
表 4-4、巴西磨菇子實體經不同劑量之γ-照射後,其甲醇萃取物之清除 1,1-二苯基-2-苦味肼基能力 124
表 4-5、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之清除羥自由基能力 127
表 4-6、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之螯合亞鐵離子能力 129
表 4-7、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物中抗氧化性質之EC50 131
表 4-8、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物中抗壞血酸、β-胡蘿蔔素、生育酚和總酚類之含量 133
表 5-1、樟芝菌絲體經不同劑量之γ-照射後其甲醇萃取率 149
表 5-2、樟芝菌絲體經不同劑量γ-照射後,其甲醇萃取物之抗氧化力 151
表 5-3、樟芝菌絲體經不同劑量γ-照射後,其甲醇萃取物之還原力 155
表 5-4、樟芝菌絲體經不同劑量照射後,其甲醇萃取物之清除1,1-二苯基-2-苦味肼基能力 158
表 5-5、樟芝菌絲體經不同劑量之γ-照射後,其清除羥自由基之能力 161
表 5-6、樟芝菌絲體經不同劑量之γ-照射後,其甲醇萃取物之螯合亞鐵離子能力 163
表 5-7、樟芝菌絲體經不同劑量之γ-照射後,其甲醇萃取物中抗氧化性質之 EC50 值 166
表 5-8、樟芝菌絲體經不同劑量照射後,其甲醇萃取物中β-胡蘿蔔素、生育酚和總酚類之含量 168

圖 次
附圖1、輻射照射食品標章 26
附圖2、蛹蟲草子實體 29
附圖3、巴西蘑菇子實體 35
附圖4、樟芝子實體 41
圖 2-1、不同來源的蛹蟲草於 PDA 培養基培養期間之菌絲體絕對生長半徑 63
圖 2-2、蛹蟲草 (CM2) 於含不同碳源培養基之培養皿中培養時的菌絲體絕對生長半徑 (20ºC) 64
圖 2-3、蛹蟲草 (CM2) 於含不同氮源培養基之培養皿中培養時的菌絲體絕對生長半徑 (20ºC) 66
圖 2-4、蛹蟲草 (CM2) 於調至不同酸鹼值培養基之培養皿中培養時的菌絲體絕對生長半徑 (20ºC) 67
圖 4-1、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之抗氧化力 119
圖 4-2、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之還原力 122
圖 4-3、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之清除 1,1-二苯基-2-苦味肼基能力 125
圖 4-4、巴西蘑菇子實體經不同劑量之γ-照射後,其甲醇萃取物之螯合亞鐵離子能力 130
圖 5-1、樟芝菌絲體經不同劑量γ-照射後,其甲醇萃取物之抗氧化力 152
圖 5-2. 樟芝菌絲體經不同劑量γ-照射後,其甲醇萃取物之還原力 156
圖 5-3、樟芝菌絲體經不同劑量照射後,其甲醇萃取物之清除1,1-二苯基-2-苦味肼基能力 159
圖 5-4、樟芝菌絲體經不同劑量之γ-照射後,其甲醇萃取物之螯合亞鐵離子能力 164
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