最適化生產具美白活性之臺灣紫芝菌絲體

Abstract

靈芝已被證實具有抑制酪胺酸酶的活性。臺灣紫芝 (Ganoderma formosanum) 為臺灣特有種靈芝，已有文獻證實其菌絲體酒精萃取物之乙酸乙酯劃分層 (GFE-EA)，會透過抑制酪胺酸酶活性來顯著減少黑色素生成，達到與麴酸相似的美白效果。本實驗期望能通過生產條件的優化，提升菌絲體中酒精萃取物的美白活性。本實驗將利用液態深層發酵培養不同因子的菌絲體 (天數、不同碳源及濃度、起始 pH 值和溫度) 並以酒精萃取後，以酪胺酸酶比色法來進行粗萃物美白活性測定。根據單因子試驗結果，以 5 種不同濃度的葡萄糖、果糖、乳糖和蔗糖做為碳源進行培養，其中以 50 g/L 乳糖作為碳源得到的酪胺酸酶抑制率最高，而不同起始 pH 值及培養溫度的菌絲體產物皆具有酪胺酸酶抑制活性。最終選擇以 50 g/L 乳糖作為碳源、調整 pH 值為 7，在 25oC 下培養 9 天作為優化之生產條件進行培養，並將收成之菌絲體以酒精萃取得到粗萃物。在小鼠黑色素瘤細胞 (B16-F0) 實驗中，含有 0.1 mg/mL 粗萃物的培養液組別，細胞內黑色素含量會減少為控制組的 76%；在斑馬魚動物實驗中，斑馬魚胚在與含有 0.05 mg/mL 粗萃物的培養液反應 2 天後，斑馬魚胚內黑色素含量會減少為控制組的 62%。在斑馬魚表徵拍照中，可明顯觀察到體表黑色素生成的減少。經本實驗優化培養條件得到之臺灣紫芝菌絲體亦具有美白活性，可提供未來挑選最適化培養美白活性成分條件之基礎及相關研究之進行。

Ganoderma has been shown to have tyrosinase inhibitory activity. Ganoderma formosanum was an endemic species found in Taiwan. Recently, research showed that the ethyl acetate fraction of G. formosanum mycelium ethanolic extract (GFE-EA) can significantly reduce melanin production by inhibiting tyrosinase activity, achieving a skin-lightening effect similar to kojic acid. The aim of this experiment is to improve the skin-lightening activity of the alcohol extract in the mycelium through the optimization the production conditions. In this experiment, submerged fermentation will be used to cultivate mycelia of different factors (days, different carbon sources and concentrations, initial pH value and temperature), and then the mycelia were extracted with ethanol, and the skin-lightening activity was assessed by colorimetric screening assays. According to the single factor test results, five different concentrations of glucose, fructose, lactose and sucrose were used as a carbon source for the cultivation. Among them, 50 g/L lactose obtained the highest inhibition rate of tyrosinase. and the mycelium products with different initial pH and cultivation temperature show tyrosinase inhibition activity. Finally, 50 g/L lactose was selected as the carbon source, the pH value was adjusted to 7, and cultured at 25oC for 9 days as the optimized production conditions, then cultivating, extracting the alcohol extract of the mycelium of the harvest, and confirmed to be using melanoma cell and zebrafish animal platforms. In the mouse melanoma cell (B16F0) experiment, the intracellular melanin content reduced to 76% of the control group in 0.1 mg/mL crude extract group; while the melanin content in the zebrafish embryo could be reduced to 62% of the control group in 0.05 mg/mL crude extract group under 2-day treatment. For the body pigmentation of zebrafish, a reduction in melanin production on the body surface can be clearly observed under microscopy. The G. formosanum mycelium obtained by optimizing the cultivation conditions in this experiment can provide the basis for selecting the optimal conditions for cultivating the whitening active ingredients in the future.