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研究生:何公瑞
研究生(外文):Kung-Jui Ho
論文名稱:以固態發酵製備高麥角硫因之杏鮑菇穀類及其呈味性質與生理活性
論文名稱(外文):Preparation of High Ergothioneine of Pleurotus eryngii -Fermented Grains Using Solid State Fermentation and Their Taste Quality and Physiological Activities
指導教授:毛正倫毛正倫引用關係
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:162
中文關鍵詞:麥角硫因杏鮑菇固態培養呈味品質抗氧化性質抗發炎
外文關鍵詞:ergothioneinePleurotus eryngiisoild culturetast qualityantioxidant propertiesanti-inflammation
相關次數:
  • 被引用被引用:18
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麥角硫因(Ergothioneine)為一種稀有的胺基酸,也是天然的抗氧化劑,其在生物體內對氧化壓力傷害之預防扮演著重要角色,麥角硫因不能在動物體內合成,僅能由食物中攝取,近年來發現在菇類中含有高含量的麥角硫因,從28種食藥用菇類中篩選出麥角硫因含量較高的菌種,以杏鮑菇子實體最高(1521.6 mg/g dw),因此本論文以杏鮑菇為媒介,利用固態發酵方式,製備出高麥角硫因之杏鮑菇薏仁和杏鮑菇蕎麥,並針對薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體,進行一般成分、營養、物性、呈味、生理活性物質、抗氧化性質之分析及抗發炎反應之評估。
在不同發酵條件下生長所得之杏鮑菇薏仁和杏鮑菇蕎麥,探討獲得最大麥角硫因含量之最適培養組成。經探討後,以含水量40%之薏仁於30 ℃下培養17天後之麥角硫因量最高,可達574.0 mg/kg dw,再添加酵母萃取物為氮源可提高麥角硫因到824.8 mg/kg dw,生物質為139.0 mg/g dw;以含水量45%之蕎麥於30 ℃下培養17天後之麥角硫因含量最高,可達580.6 mg/kg dw,生物質為163.4 mg/g dw,添加酵母萃取物為氮源,麥角硫因含量可達651.6 mg/kg dw。在碳源方面,添加果醣能提高麥角硫因含量為721.4 mg/kg dw。
在一般成分分析方面,薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體主要由醣類為主(87.64%、85.55%、78.43%、78.77%、50.14%)。在生理活性方面,麥角固醇含量上以杏鮑菇菌絲體(2056.4 mg/kg dw)>杏鮑菇蕎麥(469.2 mg/kg dw)>杏鮑菇薏仁(443.4 mg/kg dw);麥角硫因含量也是以杏鮑菇菌絲體最高(1514.6 mg/kg dw)。機能性方面,蕎麥之芸香苷及槲皮素分別為198.56 mg/kg及10.77 mg/kg。
呈味方面,可溶性糖以杏鮑菇菌絲體總含量最高(411.70 mg/g dw)。而游離胺基酸部分,薏仁和蕎麥經過固態發酵後之總游離胺基酸分別從14.38 mg/g dw提升到46.24 mg/g dw以及24.06 mg/g dw提升到45.28 mg/g dw。核苷酸方面,以杏鮑菇菌絲體之核苷酸總量最高(1.70 mg/g dw)。
薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體熱水萃取在抗氧化性質方面,在濃度20 mg/ml時,以蕎麥和杏鮑菇蕎麥最高分別為92.55%和92.79 %;在還原力方面,濃度20 mg/ml時,蕎麥還原力最高(1.31);在清除DPPH自由基之能力上,在濃度20 mg/ml時,依序為杏鮑菇蕎麥(84.59%)>蕎麥(63.10%)>杏鮑菇薏仁(50.31%)>杏鮑菇菌絲體(48.56%)>薏仁(47.31%);而在螯合亞鐵離子之能力上,濃度20 mg/ml時,杏鮑菇蕎麥可達到90.52 %的螯合力。總酚含量以杏鮑菇菌絲體最高(17.27 mg/g),類黃酮則以蕎麥最高(6.13 mg/g)。
乙醇萃取物抗氧化性質方面,薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體乙醇萃取之抗氧化力在濃度20 mg/ml時分別為100%、39.14 %、92.03%、59.51%和33.93%;在還原力方面,濃度10 mg/ml時,以杏鮑菇菌絲體之還原力最高(0.55);在清除DPPH自由基,濃度20 mg/ml時,樣品排序為杏鮑菇薏仁(97.81%)、杏鮑菇菌絲體(96.89%)、薏仁(92.67%)、蕎麥(92.92%)、杏鮑菇蕎麥(91.25%)。在螯合亞鐵離子之能力上,在濃度20 mg/ml時,以薏仁螯合能力最好(100%),其次為蕎麥(87.05%)。總酚含量以杏鮑菇菌絲體最高(12.65 mg/g)。
在抗發炎部分,薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體之熱水萃取物,在測試劑量範圍內(10~1000 μg/ml),對小鼠巨噬細胞(RAW 264.7)皆不具有生長抑制和毒性。在內毒素(LPS)誘導巨噬細胞之NO生成量方面,在濃度為500 μg/ml時,依序為杏鮑菇薏仁(39.58 μM)>杏鮑菇蕎麥(29.77 μM)>蕎麥(29.16 μM)>薏仁(29.89 μM)>杏鮑菇菌絲體(29.48 μM)。而對於LPS誘導巨噬細胞產生的TNF-α的生成量,並無抑制的作用。
在乙醇萃取物方面,在濃度為1000μg/ml時,其中以杏鮑菇薏仁(73.71 %)、杏鮑菇蕎麥(80.12%)和杏鮑菇菌絲體(81.28 %)對巨噬細胞有抑制的作用。在LPS誘導巨噬細胞產生之NO生成量方面,在濃度為500 μg/ml時,以薏仁(33.56 μM)、杏鮑菇薏仁(35.43 μM)和杏鮑菇菌絲體(21.96 μM)對NO有抑制的作用。在TNF-α方面,薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體對TNF-α在最高濃度500 μg/ml時皆有抑制作用,分別為4.64 ng/ml、4.49 ng/ml、4.82 ng/ml、3.91 ng/ml和0.41 ng/ml。
綜合以上結果,薏仁與蕎麥經固態發酵製備成高麥角硫因之杏鮑菇薏仁和杏鮑菇蕎麥,具有提高營養、呈味特性、生理活性物質含量、抗氧化及抗發炎性質。
Ergothioneine is a kind of precious amino acid, and it is also a natural antioxidant. It plays an important role to prevent the oxidative damage in organism. Ergothioneine cannot be biosynthesized in humans, only can be absorbed from the diet. In recently the research discovered the high content of ergothioneine in the mushroom. Content of ergothioneine of Pleurotus eryngii is the highest from 28 kinds of mushrooms, therefore the research untilized P. eryngii to make the products of P. eryngii-fermented adlay(PFA) and P. eryngii-fermented buckwheat(PFB) which content high ergothioneine. Also, we analyzed their proximate composition, nutritional components, taste and physiologically active components, antioxidant properties and evaluation of anti-imflammatory effect.
Ergothioneine of PFA and PFB at various culture condition, investigated optimal incubation to intent to get hight content of ergothioneine. Results showed that optimal conditions for high content of ergothioneine were moisture content at 40%, temperature at 30℃and polished adlay (PA) as medium, cubation 17 days (574.0 mg/kg dw), biomass was 139.0 mg/g dw. The addition of yeast extract for nitrogen source can increase content of ergothioneine (824.8 mg/ml dw);Fermentation of Pleurotus eryngii was polished buckwheat (PB) as medium, moisture content at 45%, temperature at 30℃and cubation 17 days could get high content of ergothioneine (580.6 mg/kg dw), and biomass was 163.4 mg/g dw, yeast extract as nitrogen source (651.6 mg/kg dw), Fructose as carbon source (721.4 mg/kg dw).
With regard to proximate composition, carbohydrate (87.64%, 85.55%, 78.43%, 78.77%, 50.14%) were major components found in PA, PFA, PB, PFB and mycelia. With regard to physiologically active components, the contents of ergosterol and ergothioneine of mycelia (2056.4 mg/kg dw and 1514.6 mg/kg dw) were higher than others. With regard to bioactive components, rutin and quercetin of polished buckwheat was 198.56 mg/kg and 10.77 mg/kg.
With regard to taste characteristics, total soluble sugar contents of P. eryngii mycelia was the highest at 411.70 mg/g dw. PA and PB by mycelia after its solid- state fermenrtation, free amino acid contents increase from 14.38 mg/g dw to 46.24 mg/g dw and 24.06 mg/g dw to 45.28 mg/g dw. Total nucleotide contents of mycelia was highest at 1.70 mg/g dw.
The antioxidant of hot water extracts were highest at 92.55% and 92.79% at 20mg/ml for PB and PFB. The reducing powders of PB showed an great(1.31) at 20 mg/ml. The scavenging abilities on 1,1-diphenyl-2- picrylhydrazyl radicals were in the descending order PFB (84.59%)>PB (63.10%)>PFA (50.31%)>mycelia (48.56%)>PA (47.31%). With regard to chelating abilities on ferrous ions, the chelating abilities of PFB can reach 90.52% at 20 mg/ml.
With regard to ethanolic extracts, the antioxidant activities of PA, PFA, PB, PFB and mycelia at 20mg/ml were 100%, 39.14%, 92.03%, 59.51% and 33.93%. The reducing power of mycelia (0.55) was the highest at 10 mg/ml. At 20mg/ml, the scavenging abilities on 1,1-diphenyl-2- picrylhydrazyl radicals were in the descending order: PFA (97.81%), mycelia (96.89%), PA (92.67%), PB (92.92%), PFB (91.25%). With regard to chelating abilities on ferrous ions, the chelating ability of PA was the highest (100%) at 20 mg/ml, PB was the second (87.05%).
In the anti-inflammation effect test, the inhibition on the growth of RAW 264.7 cells was studied in a MTT test using hot water extracts from PA, PFA, PB, PFB and mycelia. Increased of five samples concentrations did not inhibit the growth of RAW 264.7 cells. Effect of hot water extracts from PA, PFA, PB, PFB and mycelia on LPS-induced NO production in RAW 264.7 cell in the descending: PFA(39.58 μM)>PFB(29.77 μM)>PB(29.16 μM)>PA(29.89 μM)>mycelia(28.48 μM). And it is no effect to inhibit LPS-induced TNF-α production in RAW 264.7 cell.
With regard to ethanolic extracts, the effect of PFA(73.71%), PFB(80.12%) and mycelia(81.28%) on the growth of RAW 264.7 cell showed inhibition at 1000 μg/ml. Effect of hot water extracts from PA(33.56 μM), PFA(35.43 μM) and mycelia(21.96 μM) on LPS-induced NO production in RAW 264.7 cell showed inhibition at 500 μg/ml. The effect of PA(4.64 ng/ml), PFA(4.49 ng/ml), PB(4.82 ng/ml), PFB(3.91 ng/ml) and mycelia(0.41 ng/ml) on the LPS-induced TNF-α production in RAW 264.7 cell showed inhibition at 500 μg/ml.
In conclution, PFA and PFB prepared from the solid state fermentation of polished adlay and buckwheat have better nutritional, taste quality and physiologically active components, antioxidant properties and anti-inflammation activity.
目錄

表次 ix
圖次 xii
前言 1
文獻整理 3
一、麥角硫因簡介 3
二、杏鮑菇簡介 9
三、食藥用菇之機能性 10
四、固態發酵 12
五、食藥用菇之呈味特性 16
六、抗氧化 22
七、發炎反應與相關因子 27
材料與方法 37
一、實驗材料 37
二、實驗方法 37
(一)菌種活化及接種原製備 37
1. 試藥 37
2. 步驟 38
(二)固態發酵 38
1. 杏鮑菇薏仁生長條件之探討 38
2. 杏鮑菇蕎麥生長條件之探討 39
3. 生理活性物質分析 39
4. 樣品粉末之製備 40
(三)一般成分分析 41
1. 試藥 41
2. 水分測定 41
3. 灰分測定 42
4. 粗脂質測定 42
5. 粗纖維測定 42
6. 粗蛋白測定 42
7. 還原糖測定 43
(四)品質評估 43
1. 試藥 43
2. 物理性質分析 43
3. 生理活性物質分析 44
(五)機能性分析 44
1. 試藥 44
2. 芸香苷 44
3. 槲皮素 44
(六)呈味物質分析 45
1. 試藥 45
2. 可溶性糖 45
3. 游離胺基酸 46
4. 核苷酸 47
5. 等價鮮味值 47
(七)抗氧化性質分析 48
1. 試藥 48
2. 樣品製備 48
3. 抗氧化力 49
4. 還原力 49
5. 清除1,1-二苯基-2-苦味肼基團(DPPH)能力 49
6. 螯合亞鐵離子之能力 50
7. 抗氧化成分分析 50
(八)發炎反應之評估 50
1. 化學藥品與試劑 50
2. 細胞株 51
3. 熱水萃取物之製備 51
4. 乙醇萃取物之製備 51
5. 對巨噬細胞存活率之測定 52
6. 一氧化氮(NO)含量之測定 53
7. 腫瘤壞死因子-α(TNF-α)含量之測定 53
(九)統計分析 54
結果與討論 55
一、高麥角硫因之菌種篩選 55
二、固態發酵培養條件探討 55
三、一般成分分析 77
(一)水分 77
(二)碳水化合物 77
(三)粗灰分 77
(四)粗脂肪 79
(五)粗纖維 79
(六)粗蛋白 79
四、品質評估 80
(一)物理性質分析 80
(二)生理活性物質分析 84
五、機能性成分分析 86
(一)芸香苷 86
(二)槲皮素 86
六、呈味特性成分分析 88
(一)可溶性糖 88
(二)游離胺基酸 90
(三)核苷酸 94
七、抗氧化性質分析 96
(一)熱水及乙醇萃取物之萃取率 96
(二)熱水萃取物之抗氧化性質 96
1. 熱水萃取物之抗氧化力 96
2. 熱水萃取物之還原力 99
3. 熱水萃取物之清除1,1-二苯基-2-苦味肼基團之能力 102
4. 熱水萃取物之螯合亞鐵離子之能力 107
5. 熱水萃取物抗氧化性質之EC50 110
6. 熱水萃取物之抗氧化成分分析 110
(三)乙醇萃取物之抗氧化性質 116
1. 乙醇萃取物之抗氧化力 116
2. 乙醇萃取物之還原力 116
3. 乙醇萃取物之清除1,1-二苯基-2-苦味肼基團之能力 116
4. 乙醇萃取物之螯合亞鐵離子之能力 123
5. 乙醇萃取物抗氧化性質之EC50 123
6. 乙醇萃取物之抗氧化成分分析 128
八、抗發炎反應之評估 128
(一)對巨噬細胞(RAW 264.7)存活之影響 128
1. 熱水萃取物對巨噬細胞存活之影響 131
2. 乙醇萃取物對巨噬細胞存活之影響 131
(二)對LPS誘導巨噬細胞(RAW 264.7)生成一氧化氮
之影響 136
1. 熱水萃取物對LPS誘導巨噬細胞生成NO之影響 136
2. 乙醇萃取物對LPS誘導巨噬細胞生成NO之影響 136
(三)對LPS誘導巨噬細胞(RAW 264.7)生成腫瘤壞死
因子-α之影響 141
1. 熱水萃取物對LPS誘導巨噬細胞生成TNF-α之影響 141
2. 乙醇萃取物對LPS誘導巨噬細胞生成TNF-α之影響 142
結論 147
參考文獻 149

表次

附表1、L-胺基酸之呈味特性 18
附表2、胺基酸之相對甜度 19
附表3、鮮味胺基酸及鮮味核苷酸之個別相對鮮味強度 21
附表4、菇類八碳化合物之值及氣味特性 23
附表5、各種抗氧化物質的成分和其來源與功用 28
表1、食藥用菇子實體麥角硫因之含量 56
表2、食藥用菇菌絲體麥角硫因之含量 57
表3、杏鮑菇薏仁固態發酵期間,麥角硫因和生物質之變化 58
表4、杏鮑菇蕎麥固態發酵期間,麥角硫因和生物質之變化 63
表5、杏鮑菇薏仁與杏鮑菇蕎麥之麥角硫因含量和生物質
之相關係數 67
表6、不同培養條件所得杏鮑菇薏仁之麥角硫因含量和生物質 69
表7、不同培養條件所得杏鮑菇蕎麥之麥角硫因含量和生物質 72
表8、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲體
之一般組成 78
表9、不同溫度下薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和
杏鮑菇菌絲體之溶解度 81
表10、不同溫度下薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和
杏鮑菇菌絲體之膨潤力 82
表11、不同溫度下薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和
杏鮑菇菌絲體之吸水指標 83
表12、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之麥角固醇與麥角硫因含量 85
表13、蕎麥和杏鮑菇蕎麥之芸香苷和槲皮素含量 87
表14、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之可溶性糖類組成 89
表15、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之游離胺基酸組成 91
表16、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之游離胺基酸之呈味特性 93
表17、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之核苷酸組成 95
表18、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體之等價鮮味值 97
表19、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水和乙醇萃取物之萃取率 98
表20、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物之抗氧化力 100
表21、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物之還原力 103
表22、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物清除1,1-二苯基-2-苦味肼基團自由基之能力 105
表23、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物對亞鐵離子之螯合能力 108
表24、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物抗氧化性質EC50 111
表25、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體粉末抗氧化性質EC50(熱水) 112
表26、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物中總酚與類黃酮之含量 114
表27、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體粉末中總酚與類黃酮之含量(熱水) 115
表28、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物之抗氧化力 117
表29、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物之還原力 119
表30、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物清除1,1-二苯基-2-苦味肼基團自由基之能力 121
表31、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物對亞鐵離子之螯合能力 124
表32、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物抗氧化性質EC50 126
表33、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體粉末抗氧化性質EC50(乙醇) 127
表34、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物中總酚與類黃酮之含量 129
表35、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體粉末中總酚與類黃酮之含量(乙醇) 130
表36、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物對巨噬細胞(RAW 264.7 cell)生長之影響 132
表37、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物對巨噬細胞(RAW 264.7 cell)生長之影響 134
表38、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取對LPS誘導巨噬細胞(RAW 264.7 cell)生成
一氧化氮之產量 137
表39、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物對LPS誘導巨噬細胞(RAW 264.7 cell)生成
一氧化氮之產量 139
表40、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體熱水萃取物對LPS誘導巨噬細胞(RAW 264.7 cell)生成
腫瘤壞死因子-α之含量 143
表41、薏仁、杏鮑菇薏仁、蕎麥、杏鮑菇蕎麥和杏鮑菇菌絲
體乙醇萃取物對LPS誘導巨噬細胞(RAW 264.7 cell)生成
腫瘤壞死因子-α之含量 145
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