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

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.Acknowledgements i 摘要 iii Abstract v Table of Contents vii List of Figures xii List of Tables xiv Chapter 1 Introduction 1 Chapter 2 Literature Review 3 2.1 Introduction to Aureobasidium pullulans 3 2.1.1 Potential application of Aureobasidium pullulans 3 2.1.1.1 Pullulan 3 2.1.1.2 Single cell protein 5 2.1.1.3 Extracellular enzyme 6 2.2 Introduction to cellulase 6 2.2.1 General description of β-glucosidases 10 2.2.1.1 Distribution and function of β-glucosidase 10 2.2.1.2 Classification of β-glucosidases 10 2.2.1.3 β-glucosidases mode of action 11 2.2.1.4 Industrial application of β-glucosidases 14 2.3 Wheat bran as fermentation substrate 15 2.4 Introduction to tea beverages 16 2.4.1 Classification of tea 16 2.4.2 Cold-brewed tea 19 2.4.3 Health benefit of tea 19 2.4.4 Formation of tea aroma 22 2.4.4.1 Glycosidically-bound volatile compounds in tea 22 Chapter 3 Materials and Methods 26 3.1 Materials 26 3.1.1 Raw materials 26 3.1.2 Microorganism 26 3.1.2.1 Aureobasidium pullulans NCH-218 26 3.1.2.2 Culture media 26 3.2 List of chemicals 27 3.3 Instruments 28 3.4 Experimental design 29 3.5 Experimental methods 30 3.5.1 Stock culture and inoculum preparation 30 3.5.1.1 Stock culture 30 3.5.1.2 Inoculum preparation 30 3.5.2 Proximate analysis of wheat bran 30 3.5.2.1 Moisture content 30 3.5.2.2 Crude fat 31 3.5.2.3 Crude protein 31 3.5.2.4 Crude ash 33 3.5.2.5 Crude fiber 33 3.5.3 β-glucosidase production 34 3.5.4 Determination of β-glucosidase activity and substrate specificity 35 3.5.4.1 Substrate specificity 36 3.5.5 Preparation of the beverages 38 3.5.5.1 Tea infusion 38 3.5.5.2 Wheat bran extract 38 3.5.5.3 Concentrated and fresh fruit juices 38 3.5.6 Application of β-glucosidase in various beverages 38 3.5.6.1 First trial 38 3.5.6.2 Second trial 39 3.5.7 Analysis of aroma enhancement 39 3.5.7.1 Determination of reducing sugar content 39 3.5.7.2 Determination of glucose content by HPLC 40 3.5.7.3 Sensory evaluation 40 3.5.7.4 Solid-phase microextraction (SPME) 41 3.5.7.5 Gas Chromatography-Mass Spectrometry (GC-MS) 43 3.5.7.6 Gas Chromatography-Flame Ionization Detector (GC-FID) 43 3.5.8 The optimum condition for β-glucosidase application 45 3.5.8.1 Reaction time 45 3.5.8.2 β-glucosidase addition 45 3.5.8.3 Reaction temperature 45 3.5.8.4 Green tea concentration 46 3.5.9 Antioxidant activity assays 46 3.5.9.1 Total phenolic content 46 3.5.9.2 Total flavonoid content 46 3.5.9.3 DPPH free radical scavenging ability 47 3.5.9.4 Reducing power assay 48 3.5.9.5 Metal chelating activity 48 3.5.9.6 Superoxide radical scavenging activity 49 3.5.10 Determination of catechin and caffeine contents 50 Chapter 4 Results and Discussion 52 4.1 Proximate analysis of wheat bran 52 4.2 β-glucosidase production by A. pullulans NCH-218 52 4.2.1 The colony morphology and microscopic appearance of A. pullulans NCH-218 52 4.2.2 The growth curve of A. pullulans NCH-218 58 4.2.3 β-glucosidase substrate specificity 59 4.2.4 Partial purification of β-glucosidase 63 4.3 Determination of the selected sample 63 4.3.1 Preliminary sensory evaluation 63 4.3.2 Glucose content determination 69 4.4 Aroma enhancement effect on tea beverage 74 4.5 Optimum treatment condition 77 4.5.1 Reaction time 77 4.5.2 β-glucosidase addition 79 4.5.3 Reaction temperature 81 4.5.4 Green tea concentration 83 4.6 Antioxidant activity assays 85 4.7 Determination of individual catechins and caffeine 88 Chapter 5 Conclusions 92 5.1 Proximate analysis of wheat bran 92 5.2 Partial purification of β-glucosidase 92 5.3 Determination of the selected sample 92 5.4 Aroma enhancement effect on tea beverage 92 5.5 Optimum treatment condition 93 5.6 Antioxidant activity assays 93 5.7 Determination of individuals catechin and caffeine 93 Chapter 6 Future prospects 94 Chapter 7 References 9