臺灣博碩士論文加值系統

樟芝(Taiwanofungus camphoratus)是台灣特有的一種藥用菇類，已知主要的有效成份為多醣體以及三萜類。近期我們發現小分子量(小於30,000 Da)的樟芝菌絲體深層培養的胞外多醣體具有抑制基質金屬蛋白酶（Matrix Metalloproteinases, MMPs）的活性。因此，本研究利用醣類水解酵素降解樟芝菌絲體深層培養中所含有的大分子量胞外多醣體，使之成為較低分子量的多醣體，且在總多醣中增加具有抑制MMPs幼蘆漱p分子量多醣體。藉由本研究我們找出最佳的醣類水解酵素降解大分子量多醣體的反應條件，產生具有抑制MMPs幼蘆漱p分子量多醣體，可作為抗老化化粧品的有效性成分。
本研究將樟芝菌絲體深層培養中所含有的大分子量胞外多醣體，於40℃下以40 units/ml endo-β-1,3-glucanase降解6小時，可得到小分子量胞外多醣體，且這些胞外多醣體之分子量約小於31,542 Da。這些小分子量多醣體再應用於抑制3T3纖維母細胞所分泌之MMPs，並藉由gelatin-based zymography進行MMP-2及MMP-9之活性分析。我們發現小分子量多醣體在濃度於500 ?慊/ml之劑量下，處理3T3纖維母細胞培養液，可以抑制細胞培養液中所含pro-MMP-2、MMP-2及MMP-9之活性。
　　本研究繼續擴大至5公升醱酵槽培養，乾菌體量及胞外多醣量分別可增加至27.8 g/L及6.9 g/L，將這不同深層培養的胞外多醣體，也利用醣類水解酵素降解為小分子量多醣體，並且評估抑制MMP-2及MMP-9活性之能力。經實驗結果顯示，所產生的小分子量多醣體亦具有顯著的抑制pro-MMP-2、MMP-2及MMP-9活性之能力。
　　綜合以上研究結果，我們認為利用酵素降解胞外多醣體所產生的牛樟芝低分子量多醣體具有抑制MMPs抗老化能力，可視為一種應用於化粧保養品的有效成份。

　Taiwanofungus camphoratus is an medicinal mushroom in Taiwan. It is well known that the major effective components in this medical fungus are polysaccharides and triterpenoids. Recently we found that low molecular weight (less than 30,000) of exopolysaccharides from the mycelium culture of T. camphoratus could suppress the activities of matrix metalloproteinases (MMPs). Therefore, this study was designed to utilize endogenous enzymes to degrade high molecular weight of expolysaccharides from the submerged culture of T. camphoratus into low molecular weight of polysaccharides. The optimal condition of polysaccharide-degrading enzymes for the production of low molecular weight of polysaccharides with the anti-aging properties will be explored.
　　By using endo-β-1,3-glucanases (40 units/ml) at 40℃ for 6 h, high molecular weight of exopolysaccharides was transformed into low molecular weight, and less than 31,542 Da of polysaccharides was obtained. These low molecular weight of polysaccharides were applied to investigate the capacities for inhibiting MMPs secreted from 3T3 fibroblasts by gelatin-based zymography. The results showed that at the concentration of 500 ?慊/ml, low molecular weight of polysaccharides did inhibit the activities of pro-MMP-2, MMP-2, and MMP-9.
　　The experiment for the production of exopolysaccharides from T. camphoratus was further scale-up to 5-L fermentor. The biomass and exopolysaccharides content could be increased to 27.8 g/L and 6.9 g/L, respectively. The different submerged culture of exopolysaccharides were also applied to utilize endogenous enzymes to decompose into low molecular weight of polysaccharides, and the inhibitory ability on the activities of MMP-2 and MMP-9 was evaluated. The results presented here show that low molecular weight of polysaccharides exhibit the markedly repressive ability on the activities of pro-MMP-2, MMP-2, and MMP-9.
　　Consequently, we suggest that the low molecular weight of polysaccharides derived from exopolysaccharides of T. camphoratus mycelial culture degraded with glucanases exhibit the antiaging capacities and can be an effective ingredient used in skincare products.

目錄...................................................I
圖次.................................................III
表次..................................................VI
縮寫表...............................................VII
中文摘要............................................VIII
英文摘要...............................................X

第一章 緒論............................................1
第二章 文獻回顧........................................3
2.1 樟芝介紹...........................................3
2.2 樟芝生理活性之成分.................................7
2.3 多醣體在化粧品上的應用............................10
2.4 β-1,3-glucanase的特性............................11
2.5 皮膚老化及抗老化..................................12
2.6 基質金屬蛋白酶....................................18
2.7 應用多醣體抑制基質金屬蛋白酶之研究................21
第三章 材料與方法.....................................23
3.1 藥品..........................................23
3.2 儀器..........................................26
3.3 菌株之培養....................................27
3.4 菌株培養方式..................................28
3.5 樟芝萃取與分離................................29
3.6 酵素降解樟芝胞外多醣體之反應測試..............32
3.7 體外細胞毒性測試..............................34
3.8 基質金屬蛋白酶(MMPs)之活性抑制分析............36
3.9 基質金屬蛋白酶之酵素活性電泳分析..............37
第四章 結果與討論...................................42
4.1 0~50%酒精濃度沉澱樟芝胞外多醣體之單醣分析.........42
4.2 利用酵素降解樟芝大分子量之胞外多醣體..............45
4.3 樟芝胞外多醣體對基質金屬蛋白酶之抑制分析..........56
4.4 樟芝5公升醱酵培養之抑制基質金屬蛋白酶之研究.......65
4.5 小分子量樟芝胞外多醣體延緩膠原蛋白降解之分析......72
第五章 結論..........................................74
第六章 參考文獻......................................75

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