台灣香檬Citrus depressa Hayata(又稱為Hirami lemon;其購自台灣花蓮有機農場)被認為有益人體健康之檸檬，而漢氏醋桿菌(Komagataeibacter hansenii)是來自台灣當地康普茶菇菌所篩選出一種醋酸桿菌，台灣香檬以20%(w/v)添加於10%(w/v)蔗糖(sucrose)而製成K. hansenii所需香檬蔗糖(Hirami lemon sucrose簡稱Hls)培養基質。另外，來自甘露醇基質(mannitol broth)前培養基質的K. hansenii被使用於探討有添加與無添加K. hansenii菌種發酵Hls基質(分別相對定義為HlswKh與HlswoKh)之不同發酵天數(0, 3, 7, 10, 14, 17, 21, 24, 28 days)對Hls上清液之pH值、糖度(°Brix)及抗氧化活性與維生素C含量之影響。對HlswKh上清液pH值與糖度而言，其值會隨發酵時間增長而略微緩慢下降，由第0天的pH 3.28與糖度9.40 oBrix降至第28天的pH 2.50與糖度8.80 oBrix，但對HlswoKh而言，其pH與糖度值在28天的儲存期間安定地分別在pH 3.02 與大約糖度 10.00~10.40oBrix之間。對Hls上清液之抗氧化活性定義為還原力 (reducing power; RP定義為維生素C相當量 (vitamin C equivalent; Vit.CE))，一般而言，HlswKh上清液RP高於HlswoKh上清液RP，這歸因於起始菌(starter) K. hansenii與起始培養基質所致。對HlswKh上清液而言，若其RP值(0.374 mg Vit.CE/mL)減去HlswoKh上清液的RP值(0.282 mg Vit.CE/mL)時，其最高差值(0.092 mg Vit.CE/mL)的最佳發酵天數被視為在第14天。接著對HlswKh上清液的維生素C含量(vitamin C content (VCC)定義為mg Vit.C/mL) 而言，其值在28天發酵期間也高於HlswoKh上清液者，這也歸因於K. hansenii起始菌所致。相類似對HlswKh上清液而言，若其VCC值(0.044 mg Vit.C/mL)減去HlswoKh上清液的VCC值(0.035 mg Vit.C/mL)時，其最高差值(0.009 mg Vit.CE/mL)的最佳發酵天數被視為在第14天。接著分別對HlswKh與HlswoKh上清液的RP與VCC值進行線性迴歸關係的斜率與截距探討， HlswKh上清液所得線性關係的斜率與截距值(分別接近於14.48與0.020 mg Vit.C/mL)皆高於HlswoKh者(分別接近於14.27與0.016 mg Vit.C/mL)，這可能也歸因於K. hansenii起始菌前培養狀態所致。本研究結果顯示，K. hansenii起始培養於甘露醇基質可改進提高發酵香檬上清液的抗氧化活性能力及維生素C含量，而且這些K. hansenii發酵HlswKh上清液的發現將貢獻於具有抗氧化活性的新健康型康普茶之研究發展。

Taiwan organic Citrus depressa Hayata is known as Hirami lemon with the beneficial effects on human health, from Hualian, Taiwan, was used in 20% (w/v) and added into 10% (w/v) sucrose as Hirami lemon sucrose (defined as Hls) cultural medium for Komagataeibacter hansenii (known as one acetic acid bacteria and isolated from Taiwan local kombuch tea fungus) which was precultured in the mannitol broth. In this study, the effect of fermentation time (0, 3, 7, 10, 14, 17, 21, 24, 28 days) on the pH, sugar content (oBrix), antioxidant activity, and vitamin C content of Hls supernatant with and without K. hansenii fermentation (defined as HlswKh and HlswoKh, respectively) was investigated. The pH and sugar content of Hls with K. hansenii fermentation (defined as HlswKh) were little slowly decreased with increasing fermentation time, that is, the values at the 0th day were pH 3.28 and 9.40 oBrix, respectively, and those at the 28th day were pH 2.50 and 8.80 oBrix, respectively, while those of Hls without K. hansenii fermentation (defined as HlswoKh) were pH 3.02 stably and around 10.00~10.40oBrix, respectively, during the 28-day storage. The antioxidant capabilities were analyzed by reducing power (RP defined as vitamin C equivalent, Vit.CE) assay for its activity. The RP of HlswKh was generally higher than that of HlswoKh, which was due to K. hansenii starter and the initial cultural medium. After the RP value (0.374 mg Vit.CE/mL) of HlswKh minus that (0.282 mg Vit.CE/mL) of HlswoKh, the optimal fermentation day was observed at the 14th day with the highest different value (0.092 mg Vit.CE/mL). Moreover, the values of vitamin C content (VCC defined as mg Vit.C/mL) for HlswKh were also higher than those HlswoKh during 28-day fermentation, which also was due to K. hansenii starter. Similarly, after the VCC value (0.044 mg Vit.C/mL) of HlswKh minus that (0.035 mg Vit.C/mL) of HlswoKh, the optimal fermentation day was also observed at the 14th day with the highest different value (0.009 mg Vit.C/mL). In addition, the linear relationship slopes and intercepts between the RP and VCC values of HlswKh and HlswoKh was investigated, the results showed the slope (closed to 14.48) and intercept (0.020 mg Vit.C/mL) values of HlswKh were higher than those (closed to 14.27 and 0.016 mg Vit.C/mL) of HlswoKh, while those of the pure vitamin C is 1.0 and 0.000 mg Vit.C/mL, respectively. This may be also due to the precultural condition of K. hansenii starter. The results of this study indicated the antioxidant capabilities and VCC of the supernatants of Hirami lemon can be improved by the fermentation with K. hansenii starting in the mannitol broth. These findings will contribute to the research and development of the HlswKh supernatants fermented by K. hansenii to the new healthy kombucha tea with antioxidant activity

Abstract i
摘要 iii
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures xi
Abbreviations xv
Chapter 1. Introduction 1
1.1. Background 1
1.2. Research Object 2
Chapter 2. Literature Review 3
2.1. Hirami lemon 3
2.1.1. Geography and history 3
2.1.2. Description and utility 3
2.1.3. Chemical composition 4
2.2. Microorganism symbiotic 8
2.2.1. Komagataeibacter hansenii 8
2.2.2. Bacterial cellulose 10
2.3. Sugar source 14
2.4. The composition changes during fermentation. 15
2.4.1. Formation of ethanol 16
2.4.2. Organic acids 17
2.4.3. Antioxidant compounds 18
2.4.4. The other compositions 20
2.5. Contraindications of fermentation 21
Chapter 3. Materials and methods 22
3.1. Materials 22
3.1.1. Substrate and microorganism 22
3.1.2. Equipment 22
3.1.3. Chemicals 23
3.2. The cultural media of K. hansenii 24
3.3. Experimental design 25
3.4. Measure pH value and sugar content 27
3.5. Reducing power assay 27
3.6. Determine Vitamin C content by iodometric titration 29
3.7. Statistical analysis 31
Chapter 4. Results and discussion 32
4.1. The appearance of bacteria cellulose during fermentation 32
4.2. The pH and sugar content changes of mannitol broth fermented by K. hansenii for precultivation 32
4.3. The pH changes of kombucha lemon fermented by K. hansenii 34
4.4. The sugar content changes of kombucha lemon fermented by K. hansenii 35
4.5. The antioxidant activity changes of kombucha lemon fermented by K. hansenii 36
4.6. The changes of vitamin C content during kombucha lemon fermentation 40
4.7. Linear correlation between vitamin C content and antioxidant activity 44
Chapter 5. Conclusion 46
5.1. Conclusion 46
5.2. Significance 47
5.3. Limitation 47
5.4. Recommendations for future research 47
Reference 48
APPENDIX I 57
APPENDIX II 58
APPENDIX III 59
Extended Abstract 63

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