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研究生:李呈浩
研究生(外文):Cheng-Hao Li
論文名稱:牛樟芝發酵萃取物在美白功效的評估
論文名稱(外文):Assessment of Whitening Efficacy of Antrodia cinnamomea Fermentation Extract
指導教授:吳建一
指導教授(外文):Jane-Yii Wu
口試委員:吳建一陳晉照顏裕鴻
口試委員(外文):Jane-Yii WuChin-Chao ChenYu-Hong Yan
口試日期:2014-07-20
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物產業科技學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:227
中文關鍵詞:牛樟芝抗氧化美白酪胺酸酶
外文關鍵詞:A. cinnamomeaantioxidant activitywhiten effecttyrosinase
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野生和人工培養的菇類子實體中含有藥物化合物成分,常被使用作為傳統藥物與化妝品。蕈類中常含有多種具有發展潛力的藥用產物,因此可用於功能化妝品。牛樟芝(Antrodia cinnamomea)灣發現的特有物種,是一種藥用菇類,廣泛應用於傳統中藥上。因為牛樟子實體含有高濃度三萜類,具有抗癌以及免疫作用,因此大都數研究都著重在如何快速獲得牛樟芝子實體之培養方法、提升多醣體以及三萜類之產量並廣泛應用在保健食品與醫學領域。至目前,牛樟芝液態或固態培養後之萃取物仍未被應用於美白化妝品原料上。而酪胺酸酶是黑色素合成的關鍵酵素,藉由酪胺酸酶之抑制作用可以達到美白功效。因此,本研究目的擬期望從牛樟芝液態與固態發酵之萃取物中可以得到抑制酪胺酸酶抑制劑之有效成分,並將其應用在美白化妝品上,因此未來應用於化妝品工業上將具有很大的發展潛力。
結果顯示,在所有測試的牛樟芝菌株中,僅有Antrodia cinnamomea AC6菌株具有較強的抑制酪胺酸酶活性,而牛樟芝固態發酵比液態發酵萃取物具有較強的抗氧化與抑制酪胺酸酶活性。所有的固態發酵萃取物抑制菇類酪胺酸酶作用皆屬於競爭型抑制機制,根據Lineweaver-Burk plots可獲得最大的反應速率(Vmax)為0.256 OD475/min。另外,以牛樟發酵萃取物配製成的美白精華液、乳霜及面膜,經使用75天後,可有效改善皮膚黑色素含量分別為8.1、7.4和4.2%,這也意味著牛樟芝萃取物具有皮膚美白之作用。

Antrodia cinnamomea, an endemic species in Taiwan, is known as a medicinal mushroom and currently used as a traditional Chinese medicine. Most of studies have focused on how to quickly get the fruiting bodies, to enhance the production of polysaccharides and triterpenoids, and applications in health food and medical field. Up to now, those extracts from submerged cultivation and solid-state fermentation of A. cinnamomea are not used in whitening cosmetics materials. The purpose of this study is to try to produce tyrosinase inhibitor obtained from the extracts from submerged cultivation and solid-state fermentation of A. cinnamomea in this study, and can be effective used in whitening cosmetics.
Among the tested A. cinnamomea strains, A. cinnamomea AC6 showed the strongest inhibition of tyrosinase activity. Extractions from A. cinnamomea AC6 mycelia produced by solid and liquid stage fermentation were further used in the tests of tyrosinase inhibition activities and antioxidant effects. However, A. cinnamomea AC6 solid-state fermentation extracts exhibited significant inhibition of tyrosinase activity, compared to those prepared from submerged cultivation.
Among the tested grains species, the extracts from solid-state fermentation displayed competitive inhibition of the mushroom tyrosinase activity, for which Lineweaver-Burk plots revealed a maximum reaction rate (Vmax) of 0.256 OD475/min. After testing with skin analyzer, the results suggestsds that extract from A. cinnamomea AC6 decrease melanin index using the cosmetic products containing with A. cinnamomea extracts for 75 days. It means that A. cinnamomea extracts has skin whitening effects.

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vi
目錄 vii
圖目錄 xii
表目錄 xviii

1.緒論 1
1.1 前言 1
1.2 研究目的 2
2.文獻回顧 6
2.1 牛樟芝簡介 6
2.2 牛樟芝的培養方法 8
2.2.1 液態發酵 9
2.2.2 固態發酵 11
2.2.3 皿培式培養 12
2.2.4 椴木培養 13
2.3 牛樟芝的生物活性成分及藥理功效 16
2.3.1 多醣體 17
2.3.2 三萜類化合物 19
2.3.2.1 羊毛甾烷三萜類 20
2.3.2.2 麥角甾烷三萜類 21
2.3.3 苯類化合物 22
2.3.4 Antroquinonol 23
2.4 黑色素 24
2.4.1 黑色素簡介 24
2.4.2 酪胺酸酶 26
2.4.3 酪胺酸酶抑制劑 27
2.5 市售的真菌化妝產品 33
3.材料方法 37
3.1 實驗藥品 37
3.2 菌種來源與保存 38
3.3 菌種鑑定 39
3.4 菌種活化 40
3.5 牛樟芝培養條件探討 40
3.5.1 液態培養 40
3.5.2 固態培養 41
3.6 分析方法 42
3.6.1 抗氧化活性測定 42
3.6.1.1 總酚含量測定 42
3.6.1.2 DPPH自由基清除效果測定 43
3.6.1.3 還原力之測定 44
3.6.2 體外抑制酪胺酸酶活性測定 45
3.6.2.1 單酚氧化酶抑制(Monophenolase inhibition) 45
3.6.2.2 雙酚氧化酶抑制(Diphenolase inhibition) 45
3.7 不同萃取方法之建立 47
3.8 抑制酪胺酸酶動力學解析 47
3.9 牛樟穀物萃取液皮膚測試 47
3.10 萃取物穩定性試驗 48
3.11 人體皮膚有效性試驗 49
3.12 統計分析 49
3.12.1 重複試驗數據統計分析 49
3.12.2 抗氧化能力之EC50值 49
3.12.3 抑制酪胺酸酶之IC50值 50
4.結果討論 51
4.1 牛樟芝菌株篩選及鑑定 51
4.1.1 牛樟芝菌株篩選 51
4.1.2 牛樟芝菌種鑑定 65
4.2 萃取方法探討 66
4.2.1 牛樟芝穀物萃取物之選擇 66
4.2.2 最佳萃取方法的建立 67
4.3 牛樟芝最佳液態培養條件建立及其萃取物之美白效果 75
4.3.1 不同碳源種類對A. cinnamomea AC6液態培養之美白功效探討 75
4.3.2 不同氮源種類對A. cinnamomea AC6液態培養之美白功效探討 82
4.3.3 不同麥芽萃取物濃度對A. cinnamomea AC6液態培養之美白功效探討 88
4.3.4 不同Yeast extract濃度對A. cinnamomea AC6液態培養之美白功效探討 94
4.3.5 不同初始pH值對A. cinnamomea AC6液態培養之美白功效探討 100
4.3.6 不同培養溫度對A. cinnamomea AC6液態培養之美白功效探討 106
4.3.7 添加不同精油種類對A. cinnamomea AC6液態培養之美白功效探討 111
4.3.8 添加不同牛樟精油濃度對A. cinnamomea AC6液態培養之美白功效探討 117
4.4 牛樟芝固態發酵最佳條件選擇與美白效果之探討 122
4.4.1 牛樟芝固態發酵基質種類選擇 122
4.4.1.1 基質種類對牛樟芝萃取物之抗氧化能力 122
4.4.1.2基質種類對牛樟芝萃取物之抑制酪胺酸酶活性 128
4.4.2 牛樟芝最佳固態發酵培養條件探討 134
4.4.2.1 不同穀物培養深度對牛樟芝菌株生長之影響 134
4.4.2.2 不同濃度MEY培養基對牛樟芝固態發酵之影響 139
4.4.2.3 不同穀物基質含水率對牛樟芝固態發酵之影響 144
4.4.2.4 不同穀物基質浸泡處理時間對牛樟芝固態發酵之影響 149
4.5 牛樟芝萃取物抑制酪胺酸酶反應動力學解析 153
4.5.1 不同牛樟芝萃取物對酪胺酸酶生成黑色素之變化 153
4.5.2 不同牛樟芝萃取物對酪胺酸酶抑制活性 154
4.5.3 不同牛樟芝萃取物對酪胺酸酶之Michaelis-Menten動力學作圖 155
4.5.4 不同牛樟芝萃取物對酪胺酸酶之Lineweaver-Burk作圖 156
4.5.5 不同牛樟芝萃取物對酪胺酸酶之動力學抑制參數影響 157
4.6 牛樟芝萃取物動物及人體皮膚試驗 192
4.7 牛樟萃取物液態及粉末形態之抗氧化能力與酪胺酸酶抑制活性比較 196
4.8 牛樟芝萃取物貯存性測試 199
4.9 牛樟芝美白乳霜產品穩定性測試 202
4.10 牛樟美白化妝品人體皮膚有效性試驗 204
5.結論 208
參考文獻 211

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