臺灣博碩士論文加值系統

老化於現今社會中越來越受到重視，而皮膚是老化最明顯的器官，其徵兆包括皺紋的產生、皮膚暗淡灰黃、萎縮、鬆弛、乾燥和色素沉澱等等。老化的成因主要可分為兩種，先天基因隨著時間的變化而產生的老化稱作自然老化(內因性老化)；長期暴露在紫外線下造成的老化稱做光老化(外因性老化)，其中光老化是加快老化的主要原因。過去的研究顯示，清酒(sake)製程中之產物能降低紫外光的傷害，減少基質金屬蛋白&;#37238;-1(MMP-1)的表現量以及增加Type-Ⅰprocollagen的合成，能夠恢復皮膚彈性以及減少皺紋的產生。此外，也有文獻指出胜&;#32957;和醣類同樣具有抗皮膚老化的功效。本實驗目的乃利用米及大豆粕當作發酵基質，擬建立一個發酵方法，生產出具有抗皮膚老化功效的產品。實驗分成兩個部分，第一部分選擇適當的基質組成、比例和培養時間，利用米麴菌(Aspergillus oryzae BCRC30428)進行米麴(RA)及大豆麴(SA)的製備；第二部分則利用兩種不同的酵母菌(Saccharomyces cerevisiae K901及YCL1087)，接種於米麴及大豆麴中，完成發酵物的製備，並將兩部份樣品進行水萃取(W)及酒精萃取(E)，探討發酵萃取物對人類皮膚纖維母細胞之抗皮膚老化功效。結果顯示，各組發酵產物之萃取液大部分於濃度50 μg/mL以下，細胞存活率達85％以上。在清除ROS能力的試驗上，發現經由Saccharomyces cerevisiae YCL1087發酵的產物之酒萃組別，具有良好ROS清除效果，達20％以上。而促進第一型原膠原蛋白增生的結果顯示，經由Saccharomyces cerevisiae YCL1087發酵之樣品，在低濃度下，米麴菌未發酵組(RACY)、米麴菌發酵36小時(RA36Y)米酒萃組以及米麴菌未發酵而酵母菌發酵72小時(SACY72)之大豆酒萃組，與控制組相比仍可維持第一型原膠原蛋白之含量。因此在不具有抑制第一型原膠原蛋白合成的發酵物，且又能夠清除細胞內的ROS含量，有利於預防因紫外線造成的皮膚細胞所造成的氧化壓力之情形，仍具有抗皮膚老化產品之潛力。

Skin is the most visible organ in the aging process. The characteristics of skin aging include wrinkling, sallowness, laxity and dryness. Aging may result from both the passage of time (intrinsic aging) and from ultraviolet light exposure (extrinsic aging). Past research showed that sake (rice wine) has positive effect on skin, including prevention of UV damage, reduction of matrix metalloproteinase-1 (MMP-1) expression and increase of typeⅠprocollagen synthesis. However, rice wine production requires tedious procedures and longtime processing. In this study, we attempt to simulate the fermentation conditions of rice wine production but with much shorter time, and hope that the fermentation product also possess anti-aging effect on skin. Since both peptide and carbohydrate are all considered to possess anti-aging effect for skin, other than rice, soybean meal was also used as substrates for fermentation. The fermentation experiment was divided into two parts. First, the optimal composition and fermentation time of koji prepared by Aspergillus oryzae were determined. Then, two kind of Saccharomyces cerevisiae were added to complete the fermentation process. The human skin fibroblast (CCD-966SK) was used as in vitro model to evaluate the bioactivity of the fermentation product. Results showed that the dose below 50μg/mL would not exert cytotoxic effect on the human skin fibroblast. The samples derived from ethanol extract of fermented product which fermented by Aspergillus oryzae and Saccharomyces cerevisiae YCL1087 could significantly reduce about 20％ of the accumulation of UV-induced ROS production in skin fibroblast. But the samples with higher concentration could inhibit typeⅠprocollagen synthesis. If treated with low concentration of the product fermented with Aspergillus oryzae for a shorter time, compared with control group, there was no significant effect on typeⅠprocollagen synthesis. Therefore, this fermented extract could scavenge ROS production significantly and reduce UV-induced oxidative stresses in skin cells. But this product is not a comprehensive skin care product, because of they cannot stimulate typeⅠprocollagen synthesis.

目錄
中文摘要 &;#618;
英文摘要 Ⅱ
口試委員審定書 Ⅳ
目錄 Ⅴ
表次 Ⅷ
圖次 Ⅸ
第一章、 緒論 1
第二章、 文獻回顧 2
一、人體皮膚 2
(一) 皮膚功能與結構 3
1. 表皮層 3
2. 真皮層 5
3. 皮下組織 6
(二) 皮膚老化特徵 6
1. 皮膚老化的外觀變化 6
2. 皮膚老化的生理變化 8
(三) 皮膚中的膠原蛋白 8
1. 膠原蛋白之結構 8
2. 膠原蛋白之生合成 9
(四) 皮膚老化之形成原因 11
1. 自然老化與光老化 11
2. 皮膚老化之自由基學說 13
3. 皮膚老化與紫外線的關係 13
4. 皮膚老化的預防與治療 19
二、發酵菌種與皮膚保健 21
(一) 米麴菌 21
(二) 酵母菌 21
(三) 清酒 22

第三章、 實驗目的與架構 24
一、研究目的 25
二、實驗架構 25
(一) 米麴菌Aspergillis oryzae發酵及萃取 26
(二) 酵母菌Saccharomyces cerevisiae K901及Saccharomyces cerevisiae發酵 及萃取 27
(三) 抗老化活性測定 29
第四章、 材料與方法 29
一、實驗材料 29
(一) 實驗原料 29
(二) 實驗菌種 29
(三) 實驗細胞株 29
(四) 實驗藥品與試劑 29
(五) 實驗器材與儀器 30
二、實驗方法 31
(一) 樣品前處理 31
(二) 微生物發酵 31
(三) 發酵萃取液製備 33
(四) 細胞實驗 33
(五) 成分分析 40
(六) 統計分析 42
第五章、 結果與討論 43
一、麴菌發酵過程變化 43
(一) pH 43
(二) 水分含量 48
二、酵母菌生長曲線 50
三、細胞實驗 51
(一) 皮膚纖維母細胞(CCD-966SK)毒性試驗 51
(二) 清除由UVA誘導皮膚纖維母細胞產生之ROS能力分析 62
(三) 第一型原膠原蛋白(typeⅠprocollagen)含量分析 74
四、成分分析 77
(一) 總糖含量分析 77
(二) 胜&;#32957;含量分析 77
(三) 總酚含量分析 78
五、結論 85
第六章、 參考資料 86

表次
表2-1、光老化死自然老化症狀 12
表5-1、不同浸米時間對蒸米水分含量之影響 48
表5-2、不同大豆粕與水比例對蒸煮大豆水分含量之影響 49

圖次
圖2-1、皮膚的結構 2
圖2-2、表皮的結構 5
圖2-3、原膠原蛋白的結構 9
圖2-4、膠原蛋白生合成 11
圖2-5、紫外光誘導MMPs表現之訊息傳導路徑 18
圖4-1、MTT結構及反應原理 34
圖4-2、由DCFDA轉變螢光物質DCF示意圖 36
圖4-3、UV照射器 37
圖4-4、三明治酵素連結免疫吸附法原理 38
圖5-1、米麴菌發酵米及大豆過程中pH值變化量 45
圖5-2、米麴菌發酵米及大豆之萃取液pH值變化量 45
圖5-3、酵母菌發酵米及大豆之萃取液pH值變化量 45
圖5-4、米麴菌發酵米及大豆36小時之萃取液pH值變化量 46
圖5-5、米麴菌發酵米及大豆48小時之萃取液pH值的變化量 46
圖5-6、米麴菌發酵米及大豆72小時之萃取液pH值的變化量 47
圖5-7、米麴菌發酵米及大豆之基質水分含量變化 49
圖5-8、Saccharomyces cerevisiae K901及Saccharomyces cerevisiae YCL1087之生長曲線 50
圖5-9、米麴菌發酵物之萃取液對人類纖維母細胞之毒性測試 53
圖5-10、酵母菌K901發酵物之萃取液對人類纖維母細胞之毒性測試 54
圖5-11、酵母菌YCL1087發酵物之萃取液對人類纖維母細胞之毒性測試 55
圖5-12、米麴菌發酵36小時及酵母菌K901發酵之萃取液對人類纖維母細胞之毒性測試 56
圖5-13、米麴菌發酵36小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞之毒性測試 57
圖5-14、米麴菌發酵48小時及酵母菌K901發酵之萃取液對人類纖維母細胞之毒性測試 58
圖5-15、米麴菌發酵48小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞之毒性測試 59
圖5-16、米麴菌發酵72小時及酵母K901菌發酵之萃取液對人類纖維母細胞之毒性測試 60
圖5-17、米麴菌發酵72小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞之毒性測試 61
圖5-18、以UVA照射人類皮膚纖維母細胞內活性氧物質及細胞存活率之影響 64
圖5-19、米麴菌發酵米之萃取液對人類纖維母細胞照射UVA之ROS生成量 65
圖5-20、酵母菌K901發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 66
圖5-21、酵母菌YCL1087發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 67
圖5-22、米麴菌發酵36小時及酵母菌K901發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 68
圖5-23、米麴菌發酵36小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 69
圖5-24、米麴菌發酵48小時及酵母菌K901發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 70
圖5-25、米麴菌發酵48小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 71
圖5-26、米麴菌發酵72小時及酵母菌K901發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 72
圖5-27、米麴菌發酵72小時及酵母菌YCL1087發酵之萃取液對人類纖維母細胞照射UVA之ROS生成量 73
圖5-28、發酵物酒萃液之對人類皮膚纖維母細胞生成typeⅠ procollagen之能力 76
圖5-29、發酵物經由酵母菌YCL1087發酵酒萃取之總糖及胜&;#32957;含量 81
圖5-30、發酵物經由酵母菌K901發酵酒萃取及水萃取之總糖及胜&;#32957;含量 82
圖5-31、發酵物經由酵母菌YCL1087發酵酒萃取之總酚含量 83
圖5-32、發酵物經由酵母菌K901發酵酒萃取及水萃取之總酚含量 84

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