臺灣博碩士論文加值系統

隨著預防老化等健康照護觀念的提升，皮膚的年齡變化問題成為現今社會關注的話題之一，又由於消費者對天然產品的濃厚興趣與需求性，使得近年來有關天然資源在皮膚保健之相關研究愈顯重要。因此，本研究之目的為利用酵素水解及微生物發酵方式，將帶鱗單角革單棘魨魚皮之蛋白質水解，期望能以低成本、簡便且具商業應用價值之較適方法與條件，產生具有皮膚保健功效之魚皮水解產物，並將其應用在預防及延緩皮膚老化保健產品開發。本研究之第一部份以清除 DPPH 自由基及促進皮膚纖維母細胞產生 typeⅠprocollagen 等能力作為指標，評估魚皮酵素水解與微生物發酵產物在皮膚保健應用之潛力，結果以 B. subtilis natto 之發酵產物具有最顯著清除 DPPH 自由基能力。第二部份之研究則利用 6.6 L 發酵槽探討 B. subtilis natto 分別在通氣量為 1.5 vvm、1.0 vvm、0.5 vvm 及 0.1 vvm 發酵帶鱗單角革單棘魨魚皮，尋找具最佳抗氧化能力以及清除因 H2O2 或太陽光模擬器誘導皮膚細胞產生 ROS 能力之發酵條件。結果顯示 DPPH、ABTS+、超氧陰離子及過氧化氫的清除能力，在發酵 72 小時之前均隨著發酵時間增加而提昇，而約在發酵 72 小時之後持平。在清除 DPPH 自由基之能力方面，以通氣量為 1.5 vvm 發酵 72 小時產物較佳；而清除ABTS＋ 自由基、過氧化氫、超氧陰離子之能力則以通氣量為 0.5 vvm 發酵 72 小時產物較佳。另在促進皮膚纖維母細胞增生方面，在濃度 1 mg/mL 時，以通氣量為 1 vvm 發酵 24 小時，與未發酵組相比，細胞增生率增加 16%；在清除細胞內活性氧物質 (Reactive oxygen species, ROS) 能力部分，通氣量 0.5 vvm 發酵 72 小時之產物分別在濃度為 2 mg/mL 及 1 mg/mL 可顯著性降低 H2O2 誘導皮膚纖維母細胞 (CCD-966SK) 及角質細胞 (HaCaT) 產生之 ROS；而以太陽光模擬光源誘導皮膚細胞產生 ROS 之試驗，通氣量 0.5 vvm 發酵 72 小時之產物分別在濃度為 2 mg/mL 及 3 mg/mL 時可顯著清除纖維母細胞及角質細胞內因太陽光模擬源照射所產生之 ROS。
綜言之，魚皮發酵產物以通氣量 0.5 vvm 發酵 72 小時之產物具清除 ABTS+、超氧陰離子、過氧化氫等自由基以及降低皮膚細胞因 H2O2 與太陽光模擬器產生 ROS 之能力，預期應可減少皮膚因光老化或自然老化所累積產生之 ROS，藉以降低膠原蛋白酶的活化，減少膠原蛋白分解，因而具有皮膚保健產品應用之潛力。

Currently, with the popularity of antiaging notion, the physiological changes of aged skin have become one of the main issues concerned by the public. Driven by rising consumer interest and demand for natural products, more and more natural raw materials have been investigated for the improvement of skin aging. The purpose of this research was to investigate the skin care potential of the products prepared by enzymatic hydrolysis or fermentation of the skin of Aluterus monoceros. This study was divided into two parts. The first part was the evaluation of DPPH free radical scavenging activity and the stimulating effect on collagen synthesis of fermented and enzyme hydrolyzed products of the skin of Aluterus monoceros. The results revealed B. subtilis natto-fermented fish skin products had higher DPPH free radical scavenging activity than other preparations.
The second part of this research was to find the optimal fermentation condition in 6.6 L fermentor for B. subtilis natto-fermented fish skin products on antioxidant activities and intracellular reactive oxygen species (ROS) scavenging effects induced by hydrogen peroxide and solar simulator in human skin fibroblasts and keratinocytes. It was found that fermented products, in comparison with unfermented samples showed a dramatic increase in DPPH, ABTS+, superoxide anion and hydrogen peroxide scavenging effects. The products fermented at 1.5 vvm for 72 h presented the best DPPH free radical scavenging effect, while the products fermented at 0.5 vvm for the same fermentation time appeared to have the best ABTS+, superoxide anion, and hydrogen peroxide scavenging effects. In addition, the growth rate of fibroblast treated with the products fermented at 1vvm for 24 h increased 16%, at the concentration of 1 mg/mL. Further, the products fermented at 0.5 vvm for 72 h significantly decreased the amount of hydrogen peroxide- and solar simulator- induced the accumulation of ROS in human skin fibroblasts and keratinocytes.
Taken together, it is cleared that the products fermented at 0.5 vvm for 72 h had free radical scavenging activities and intracellular ROS- scavenging effects on human skin fibroblasts and keratinocytes. Therefore, we expect that B. subtilis natto-fermented fish skin products might be a promising candidate for eliminating the age-dependent accumulation of ROS in human skin cells, and have a potential use for skincare application.

中文摘要 I
Abstract III
圖目錄 VIII
表目錄 X
第一章 前言 1
第二章 文獻回顧 3
2.1 自由基與活性氧 3
2.1.1 自由基的種類 3
2.2 人體皮膚之結構組成與功能 5
2.2.1 表皮層 6
2.2.1.1 皮膚角質細胞 7
2.2.2真皮層 7
2.2.2.1 皮膚纖維母細胞 8
2.2.3 皮下組織層 8
2.3 皮膚老化 8
2.3.1 皮膚之自然老化與光老化 8
2.3.1.1 皮膚老化的自由基學說 11
2.3.1.2 紫外線與皮膚老化 11
2.3.2 皮膚老化之調控 13
2.3.2.1 抗氧化物質與皮膚老化 14
2.3.2.2 胜肽與皮膚老化 16
2.3 帶鱗單角革單棘魨魚皮 20
2.4 發酵菌種 21
2.4.1 Aspergillus oryzae 21
2.4.2 Bacillus subtilis natto 22
第三章 實驗構想及設計 23
3.1 構想 23
3.2 探討帶鱗單角革單棘魨魚皮酵素水解與微生物發酵產物在皮膚保健之應用潛力 24
3.3利用攪拌式發酵槽探討帶鱗單角革單棘魨魚皮之納豆菌發酵物的最佳抗氧化與清除因H2O2 或 UV 誘導皮膚細胞產生ROS之發酵條件 25
第四章 材料與方法 26
4.1. 實驗材料 26
4.1.1 實驗原料 26
4.1.2 發酵菌種 26
4.1.3 水解酵素 26
4.2 實驗細胞株 26
4.3 實驗藥品與試劑 26
4.4 實驗器材與儀器 28
4.5 實驗方法 29
4.5.1 菌種保存與活化 29
4.5.2 發酵及水解魚皮 30
4.5.3 攪拌式發酵槽發酵魚皮 31
4.5.4 菌數測定 31
4.5.5 胜肽含量測定 31
4.5.6 抗氧化活性測定 32
4.5.6.1 清除DPPH自由基能力 32
4.5.6.2 總抗氧化能力 32
4.5.6.3 清除氫氧自由基能力 33
4.5.6.4 清除超氧陰離子能力 33
4.5.6.5 清除過氧化氫能力 34
4.5.7 細胞實驗 35
4.5.7.1 發酵產物之製備 35
4.5.7.2 細胞株之冷凍、解凍及培養 35
4.5.7.2.1 皮膚纖維母細胞 (CCD-966SK) 35
4.5.7.2.2 皮膚角質細胞 (HaCaT) 36
4.5.8 細胞存活率分析 (MTT assay) 37
4.5.9 對纖維母細胞與角質細胞增生之影響 37
4.5.10 對纖維母細胞及角質細胞毒性分析 37
4.5.11 促進纖維母細胞合成 typeⅠprocollagen能力 38
4.5.12 清除細胞內活性氧物質之能力 39
4.5.12.1 利用H2O2誘導 39
4.5.12.2 利用太陽光模擬光源誘導 41
4.6 統計分析 42
第五章 結果與討論 43
5.1 探討帶鱗單角革單棘魨魚皮酵素水解與微生物發酵產物在皮膚保健之應用潛力 43
5.1.1 胜肽含量 43
5.1.2 促進纖維母細胞合成 typeⅠprocollagen能力 45
5.1.3 清除DPPH自由基能力 46
5.2 利用攪拌式發酵槽探討帶鱗單角革單棘魨魚皮之納豆菌發酵物的最佳抗氧化與清除因H2O2 或 UV 誘導皮膚細胞產生ROS之發酵條件 49
5.2.1 不同通氣量對 B. subtilis natto 發酵期間各項指標之影響 49
5.2.1.1 菌數 49
5.2.1.2 胜肽含量 50
5.2.1.3 發酵液 pH 值 50
5.2.2 抗氧化活性分析 51
5.2.2.1 清除DPPH自由基能力 51
5.2.2.2 總抗氧化能力 52
5.2.2.3 清除氫氧自由基能力 53
5.2.2.4 清除超氧陰離子能力 53
5.2.2.5 清除過氧化氫能力 54
5.2.3 對纖維母細胞與角質細胞增生之影響 54
5.2.4清除細胞內活性氧物質之能力 55
5.2.4.1 利用H2O2誘導 56
5.2.4.1.1 H2O2 誘導纖維母細胞與角質細胞產生 ROS 及其存活率 56
5.2.4.1.2 發酵物清除H2O2 誘導細胞內產生 ROS 之能力 57
5.2.4.2 利用太陽光模擬光源誘導 58
5.2.4.2.1 太陽光模擬光源誘導纖維母細胞及角質細胞產生 ROS 及其存活率 58
5.2.4.2.2 發酵物清除太陽光模擬光源誘導細胞內產生 ROS 之能力 58
第六章 結論 63
第七章 參考文獻 95

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