台灣土肉桂露(其為水蒸氣萃取土肉桂葉之水萃取物(water extracts; CWEts)，購自台灣南投中寮鄉)，而漢氏醋桿菌(Komagataeibacter hansenii) (來自台灣康普茶之茶菇菌所篩選出一種醋酸桿菌)發酵之基礎培養基(basic cultural medium; Bcm)包含有5 g/L酵母萃出物(yeast extract)、3 g/L蛋白腖(peptone)及10%紅糖(brown sucrose)。本研究探討發酵時間(0、3、7、10、14、 17、21、24天)與CWEts之不同添加濃度(0%、1%、5%, 10%、20%、40% (v/v)分別相對定義為C-0、C-1、C-5、C-10、C-20、C-40)對K. hansenii發酵Bcm上清液的 pH 值、糖度(°Brix)與抗氧化活性之影響。實驗結果顯示Bcm發酵上清液的 pH 值會隨發酵時間增加而逐漸下降，其C-0、C-1、C-5、C-10、C-20、C-40之起始pH值分別相對為5.42、5.54、5.33、5.53、5.54、 5.51而下降至分別相對為5.05、4.68、4.88、 4.86、4.81、4.89。在糖度方面，除了C-40的糖度未有變化並且維持在6.3OBrix之外，其它C-0、C-1、C-5、C-10、C-20的糖度由起始值分別相對為10.5、10.5、10.3、9.6、8.7分別略微下降至10.1、9.75、9.55、9.35、8.3OBrix。Bcm發酵上清液之抗氧化活性分別以DPPH(1,1-diphenyl-2-picrylhydrazyl)自由基清除率(DPPH scavenging %)、還原力(reducing power (RP)定義為vitamin C equivalent, Vit.CE)、總多酚含量(total phenolic content (TPC)定義為gallic acid equivalent; GAE)等活性為表示，其結果顯示，在最佳發酵14天與最適5%添加濃度(C-5)時，Bcm發酵上清液有最高DPPH自由基清除率與還原力及總多酚含量，其最高值分別相對為53.639 ± 3.219%與0.0441 ± 0.0014 mg Vit.CE/mL及0.0209 ± 0.0015 mg GAE/mL，同時發現20%與40%土肉桂露添加濃度似乎抑制K. hansenii生長。對Bcm發酵上清液之DPPH自由基清除率與還原力及總多酚含量進行線性迴歸關係探討其斜率與截距而言，其結果顯示DPPH-RP與DPPH-TPC的斜率值分別相對為0.0007與0.0004，而截距值分別相對為-0.0011與-0.0006，另外，對RP-TPC所得線性關係的斜率與截距值分別相對為0.5217與0.0002，這些線性迴歸關係結果顯示，例如要達到相同DPPH自由基清除率之抗氧化活性時，RP濃度必須幾乎2倍於TPC濃度，換言之，Bcm發酵上清液之RP活性幾乎是2倍高的TPC活性。

Water extracts of Cinnamomum leaves (CWEts), originally obtained from Nantou, Taiwan, were used to add into the basic cultural medium (5 g/L yeast extract, 3 g/L peptone, and 10 % (w/v) brown sugar, defined as Bcm) for Komagataeibacter hansenii fermentation. In this study, the effects of the fermentation time (0, 3, 7, 10, 14, 17, 21, 24 days) and the different concentrations (0%, 1%, 5%, 10%, 20%, and 40% (v/v) were defined as C-0, C-1, C-5, C-10, C-20, and C-40, respectively) of WEts on pH, sugar content (defined as OBrix), and antioxidant activities of the Bcm supernatants were investigated. The results showed that the pH values of Bcms (C-0, C-1, C-5, C-10, C-20, and C-40) gradually decreased with increasing fermentation time from the initial pH value of 5.42, 5.54, 5.33, 5.53, 5.54, and 5.51, respectively, down to 5.05, 4.68, 4.88, 4.86, 4.81, and 4.89, respectively. Aside from C-40 (the sugar content was unchanged with fermentation time and kept at 6.3 OBrix), those of Bcms (C-0, C-1, C-5, C-10, and C-20) slightly decreased from 10.5, 10.5, 10.3, 9.6, and 8.7 OBrix, respectively, to 10.1, 9.7, 9.5, 9.3, and 8.3 OBrix, respectively. The antioxidant activities were determined by the DPPH (1,1-diphenyl 2-picrylhydrazyl) radical scavenging activity (DPPH scavenging %), reducing power (RP defined as vitamin C equivalent, Vit.CE), and total phenolic content (TPC defined as gallic acid equivalent; GAE). Among the Bcm supernatant samples, the highest antioxidant activities were determined to those values of 53.64 ± 3.22% DPPH scavenging, 0.0441 ± 0.0014 mg Vit.CE/mL RP, and 0.0209 ± 0.0015 mg GAE/mL TPC for the optimal C-5 obtained at the optimal 14th-day fermentation time. Meanwhile, the higher concentrations (C-20 and C-40) seem to inhibit the growth of K. hansenii. The linear relationship slopes and intercepts among the values of DPPH scavenging%, RP, and TPC were investigated. The results showed the slope values were 0.0007 and 0.0004 for DPPH-RP and DPPH-TPC, respectively, while the intercept were -0.0011 and -0.0006 for DPPH-RP and DPPH-TPC, respectively. In addition, the slope and intercept values were 0.5217 and 0.0002 for RP-TPC, respectively. These linear relationship results indicated that the concentration of RP needs to be almost 2-fold of TPC for the same antioxidant activity, e.g. DPPH scavenging%, that is, the RP activity of the Bcm supernatant was almost doubly higher than the TPC one.

Abstract ........................................................................................................................................ i
摘要 ........................................................................................................................................... iii
Acknowledgments ...................................................................................................................... v
Table of Contents ...................................................................................................................... vi
List of Tables ............................................................................................................................. ix
List of Figures ............................................................................................................................. x
Abbreviations ........................................................................................................................... xii
Chapter 1. Introduction ............................................................................................................... 1
1.1. Background ...................................................................................................................... 1
1.2. Research object ................................................................................................................ 2
Chapter 2. Literature Review...................................................................................................... 3
2.1. Beverage fermentation ..................................................................................................... 3
2.2. Kombucha fermentation .................................................................................................. 3
2.2.1. Traditional kombucha fermentation ......................................................................... 3
2.2.2. Kombucha fermentation using alternative ingredients ............................................. 4
2.3. Microbial composition of kombucha ............................................................................... 5
2.4. Bacterial and yeast symbiotic culture (SCOBY) ............................................................. 8
2.5. Microbial metabolism during kombucha fermentation ................................................. 10
2.6. Chemical composition in Kombucha ............................................................................ 12
2.7. Benefits of Kombucha Fermentation ............................................................................. 14
2.8. Cinnamomum ................................................................................................................. 15
2.8.1. Overview ................................................................................................................ 15
2.8.2. Cinnamomum osmophloeum (C. Osmophloeum) ................................................... 17
Chapter 3. Materials and methods ............................................................................................ 19
3.1. Materials ........................................................................................................................ 19
3.1.1. Substrate and microorganism ................................................................................. 19
3.1.2. Equipment and tools .............................................................................................. 19
3.1.3. Chemicals ............................................................................................................... 20
3.2. Experimental design ...................................................................................................... 21
3.2.1. Culture of K. hansenii ............................................................................................. 21
3.2.2. Fermentation ........................................................................................................... 21
3.3. Changes in pH value and sugar content during fermentation ........................................ 23
3.4. Antioxidant analysis ...................................................................................................... 23
3.4.1. DPPH radical scavenging activity .......................................................................... 23
3.4.2. Total phenolic content ............................................................................................ 24
3.4.3. Reducing power ...................................................................................................... 25
3.5. Statistical analysis ......................................................................................................... 26
Chapter 4. Results and discussion ............................................................................................ 27
4.1. The appearance of bacteria cellulose during fermentation ............................................ 27
4.2. The changes of pH values and sugar contents of Bcms with CWEts fermented by K. hansenii ................................................................................................................................. 30
4.2.1. The pH changes of Bcms with CWEts fermented by K. hansenii .......................... 30
4.2.2. The sugar content changes of Bcms with CWEts fermented by K. hansenii ......... 31
4.3. The antioxidant activities changes of Bcms with CWEts fermented by K. hansenii .... 32
4.3.1. DPPH radical scavenging activity assay ................................................................ 33
4.3.2. Total phenolic content assay .................................................................................. 34
4.3.3. Reducing power assay ............................................................................................ 36
4.4. The relationship between antioxidant activities ............................................................ 37
4.4.2. The relationship between DPPH and TPC ............................................................. 37
4.4.3. The relationship between DPPH and RP ................................................................ 38
4.4.4. The relationship between TPC and RP ................................................................... 39
4.4.5. The meaning of relationships ................................................................................. 39
4.5. Effect of dilution concentrations on antioxidant activity .............................................. 40
Chapter 5. Conclusion and Recommendation .......................................................................... 44
5.1. Conclusions ................................................................................................................... 44
5.2. Significance ................................................................................................................... 45
5.3. Limitation ...................................................................................................................... 45
5.4. Recommendations ......................................................................................................... 45
References ................................................................................................................................ 46
APPENDIX I ............................................................................................................................ 53
APPENDIX II ........................................................................................................................... 54
APPENDIX III ......................................................................................................................... 55
Extended Abstract..................................................................................................................... 65

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