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|標題:||Production of ß-glucosidase from Aureobasidium pullulans NCH-218 and its application for aroma enhancing capabilities in cold-brewed green tea|
以Aureobasidium pullulans NCH-218液態培養製備ß-葡萄糖苷酶及其增香特性於冷泡綠茶之應用
Aureobasidium pullulans NCH-218
Aureobasidiium pullulans NCH-218
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|摘要:||茶飲中含有大約六百種揮發性香味化合物，主要為類胡蘿蔔素、脂類、糖苷(glycosides)以及梅納反應(Maillard reaction)產物。其中，糖苷可被茶葉中的酵素如β-D-glycosidase水解，而釋放出揮發性香味化合物。但glycosidase常在茶葉製作過程中，因高溫加工程序而失活，導致許多香氣化合物仍以結合態保留在茶葉內。近年來，利用額外添加β-glycosidase以增進果汁或葡萄酒香氣之技術引起廣泛注意。本研究目的為探討以Aureobasidium pullulans NCH-218液態培養生產之β-glucosidase應用於冷泡綠茶增香之可行性。
首先使用內含5%麥麩、2.5%乳糖、1%酵母萃取物之培養基，未調整起始pH值下 (pH 6.25-6.27)，接種1% A. pullulans NCH-218活化菌液，於30°C、150 rpm下培養4天生產β-glucosidase。粗酵素液經離心與過濾後，再經超過濾濃縮，得到之酵素活性為136.8 U/mL。次以不同種類飲料做初步試驗，以挑選出最適合應用β-glucosidase的飲料。結果顯示經酵素處理過的飲料，與未經處理者相比都有較高的葡萄糖含量，尤其是綠茶組，其增加率可達到14%，此外，綠茶在香氣上也具有最佳的喜好程度，因此後續實驗選擇以冷泡綠茶為進行β-glucosidase應用的飲料。
根據GC-MS質譜圖顯示冷泡綠茶以β-glucosidase酵素處理後所釋放出來的芳香化合物中以水樣酸甲基酯(methyl salicylate)增加量最高，其次是芳樟醇(linalool)。使用10% (v/v)之 β-glucosidase 350 U/ mL，於20% (w/v) 之冷泡綠茶在50°C反應3小時，可獲得最高的水樣酸甲基酯(16.23 ppm)以及芳樟醇(1.14 ppm)。另外，在抗氧化活性與成分試驗中，結果顯示酵素處理對冷泡綠茶之抗氧化活性並無造成任何不利影響，而處理前後之兒茶素及咖啡因含量變化並不明顯。總而言之，經過β-glucosidase酵素處理的冷泡綠茶，可以達到增進香氣的目的，但不影響原本的抗氧化能力與兒茶素及咖啡因等成分。|
There are more than 600 volatile tea aroma compounds available, which are generated from four main precursors such as carotenoids, lipids, glycosides, and maillard reaction. Glycoside precursors can be hydrolyzed by endogenous enzyme such as β-D-glycosidase to release the free aroma compounds. However, tea plant glycosidases are often destroyed by thermal deactivation during tea manufacturing process, thus there are many bound aroma compounds left in tea product. Additional β-glucosidase has been widely used in aroma enhancement due to its roles in releasing the aroma compounds. Hence, this study focus on the aroma enhancing capabilities by β-glucosidase from Aureobasidium pullulan NCH-218 in green tea. The study starts with production of the enzyme under optimal condition based on the previous study: 5% wheat bran (w/v), 2.5% lactose (w/v), initial pH at 6.25-6.27, after that 1% (v/v) A. pullulans NCH-218 was inoculated and cultured at 30°C and 150 rpm for 4 days. Moreover, the produced crude enzyme was centrifuged and filtered then proceed further by using ultrafiltration which could obtain 136.8 U/ml of enzyme activity. At first, preliminary study was conducted to decide which beverage is the most suitable for the enzyme application. The result demonstrated that enzymatic treatment was resulted in higher amount of glucose content in all of the beverages, especially in green tea with the highest increasing rate for about 14% and also highest preference score (aroma profile) for the sensory evaluation test compare to other beverages. Furthermore, GC-MS chromatogram of enzyme-treated green tea indicated the highest increase of methyl salicylate (glycoside-based) and another increase was also detected in the amount of linalool. The highest amount methyl salicylate and linalool was obtained after mixing 10% of 350 U/ mL of β-glucosidase to 20% (w/v) cold-brewed green tea and was reacted for 3 hours at 50°C. The enzymatic treatment was resulted in the highest concentration of 16.23 ppm methyl salicylate and 1.14 ppm linalool, respectively. Some antioxidant activity assays as well as catechin and caffeine content determination was also conducted in order to examine the enzymatic treatment effect towards the composition and antioxidant properties of the green tea. Overall, there wasn't any remarkable changes in the antioxidant properties and the composition of catechin and caffeine after the enzymatic treatment with β-glucosidase. Therefore, this enzymatic treatment by β-glucosidase from A. pullulans NCH-218 was proved to not only offer a promising aroma enhancement effect but also did not give any negative impact on the antioxidant properties as well as the composition of the green tea.
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