本研究使用培養七天的康普茶菌群發酵 Gracilaria sp. 之 β-agarase 或 βagarase 加 cellulase 水解液，並觀察其發酵前後抗氧化能力及揮發性氣味化合物之組成。一般成分分析顯示 Gracilaria sp. 粉末中總碳水化合物含量為 (63.11 ± 0.54)%。在地康普茶菌群於含有 10% (w/v) 蔗糖的 2% (w/v) 紅茶中在 25℃ 下培養七天，ITS I 及 16S 全長定序分別顯示培養七天的康普茶中主要真菌和細菌分別為 Zygosaccharomyces sp. 和 Komagataeibacter rhaeticus，接種 10% (v/v)康普茶菌群至 5% (w/v) 海藻水解液中，於 25℃ 150 rpm 發酵 7 天。微生物分析顯示兩組海藻康普茶中的總酵母菌數與總細菌菌數於第一天快速上升至 7 及
6 Log CFU/mL 以上，並於發酵第七天分別達到 8 及 7 Log CFU/mL。理化特性分析顯示康普茶菌群於發酵期間會利用基質中部分的還原糖及有機酸。將發酵 7 天之海藻康普茶的 1% 乙酸乙酯萃取物進行抗氧化能力測試，結果顯示使用 βagarase 水解之 Gracilaria sp. 溶液經康普茶菌群發酵七天後，其銅離子還原抗氧化能力 (Cupric ion (II) reducing antioxidant capacity, CUPRAC)、DPPH 自由基清除能力及總酚含量分別提升 30%、400% 及 115%，並且保留 99.3% 氧自由基清除力 (Oxygen radical absorbance capacity, ORAC) 及 96.8% 亞鐵離子螯合力；使用 β-agarase 加 cellulase 水解之 Gracilaria sp. 溶液經康普茶菌群發酵七天之1% 乙酸乙酯萃取物，其 ORAC、CUPRAC、DPPH 自由基清除能力、亞鐵離子螯合力及總酚含量分別提升 100.3%、38%、39%、43.6% 及 18%。ORAC、CUPRAC、DPPH 自由基清除能力、亞鐵離子螯合力及總酚含量最大值分別為 66.12 ± 5.33 TE μmol/g dw、76.40 ± 11.33 TE mg/g dw、90.04 ± 3.51 TE mg/g dw、1.48 ± 0.02 EDTAE mg/g dw 及 14.59 ± 1.38 TE mg/g dw，出現在發酵第 0 或第 7 天的 β-agarase 組別。後續研究預計使用頂空固相微萃取結合氣相層析質譜儀分析海藻康普茶發酵前後之揮發性氣味化合物組成變化。

In this study, Gracilaria sp. was hydrolysed with β-agarase or β-agarase plus cellulase. The hydrolysates were then fermented by using a 7-day culture of kombucha consortium. Changes of their antioxidant capacity and composition of volatile compounds were observed after 7-day of fermentation. Proximate analysis showed that the total carbohydrate content of Gracilaria sp. powder was 63.11% ± 0.54%. Local kombucha culture was grown in 2% (w/v) of brewed black tea contain 10% of sucrose for 7 days at 25℃.The Internal transcribed spacer I and 16S full-length sequencing showed that the dominant fungi and bacteria genera in the kombucha were Zygosaccharomyces sp. and Komagataeibacter rhaeticus, respectively. 10% (v/v) of
consortium were applied to 5% of Gracilaria sp. hydrolysate. The fermentation was carried out at 25°C 150 rpm for 7 days . Microbiological analysis showed that the total yeast count and total bacterial count in the two groups of seaweed-based kombucha increased rapidly to over 7 and 6 Log CFU/mL on the first day, and reached 8 and 7 Log CFU/mL, respectively, on the seventh day of fermentation. Physical and chemical
analysis showed that the kombucha tea flora used some of the reducing sugars and organic acids in the matrix during fermentation. The antioxidant capacity of the 1% ethyl acetate extract from seaweed-based kombucha hydrolysed by β-agarase was investigated and the results showed that cupric ion (II) reducing antioxidant capacity (CUPRAC), DPPH radical scavenging capacity and total phenolic content (TPC) were increased by 30%, 400% and 115% respectively, and 99.3% Oxygen radical absorbance capacity (ORAC) and 96.8% ferrous ion chelating power (FIC) were retained after 7 days of fermentation. The ORAC, CUPRAC, DPPH radical scavenging capacity, FIC and TPC were increased by 100.3%, 38%, 39%, 43.6% and 18% respectively for the 1% ethyl acetate extract of seaweed-based kombucha with its matrix hydrolysed by βagarase plus cellulase before inoculation. The maximum values of ORAC, CUPRAC, DPPH, free radical scavenging capacity, ferric ion chelating power and total phenol
content were 66.12 ± 5.33 TE μmol/g dw, 76.40 ± 11.33 g/g dw, 90.04 ± 3.51 TE mg/g dw, 1.48 ± 0.02 EDTAE mg/g dw and 14.59 ± 1.38 TE mg/g dw, respectively, and all of which appeared on β-agarase-treated group by day 0 or 7 of fermentation. It is expected that the change in composition of volatile compounds before and after fermentation of the seaweed-based kombucha will be analysed by headspace solidphase microextraction coupled with gas chromatography mass spectrometry (HSSPME/GC/MS).

謝誌 iv
摘要 v
Abstract vi
圖目錄 ix
表目錄 x
附錄目錄 xi
縮寫表 xii
壹、前言 1
貳、文獻整理 2
2.1. 香氣化合物 2
2.1.1 香氣化合物簡介 2
2.1.2 香氣化合物分類 2
2.2 藻類 7
2.2.1 藻類簡介 7
2.2.2 藻類香氣化合物 7
2.2.3 發酵海帶選擇 10
2.2.4 龍鬚菜 (Gracilaria sp.) 10
2.2.5 自由基與藻類抗氧化活性 11
2.2.6 抗氧化能力測定 14
2.2.7 抗氧化方式選擇 16
2.3 康普茶簡介 16
2.3.1 康普茶中微生物 17
2.4 揮發性成分萃取方式 18
2.4.1 溶劑萃取 (SE) 19
2.4.2 蒸餾 (HD) 19
2.4.3 聚焦微波輔助水蒸餾 (FMA-HD) 19
2.4.4 超臨界流體萃取 (SFE) 20
2.4.5 頂空萃取 (HS) 20
2.5 頂空固相微萃取於揮發性化合物鑑定 22
2.5.1 GC-MS 23
參、實驗設計 24
肆、材料與方法 25
4.1 實驗材料 25
4.1.1 原料-康普茶與龍鬚菜 25
4.1.2 培養基 25
4.1.3 化學藥品 26
viii
4.1.4 酵素 27
4.1.5 實驗耗材 27
4.1.6 實驗儀器 27
4.2 實驗方法 28
4.2.1 海藻龍鬚菜前處理 28
4.2.2 海藻粉末一般組成分分析 29
4.2.3 康普茶製備與保存 30
4.2.4 康普茶菌叢鑑定 30
4.2.6 海藻溶液水解 32
4.2.7 康普茶菌群發酵海藻龍鬚菜水解液 32
4.2.10 海藻發酵液萃取物抗氧化能力測定 33
4.3 統計分析 35
伍、結果與討論 36
5.1 海藻龍鬚菜一般成分分析 36
5.2 康普茶菌叢鑑定結果 36
5.3 洋菜酶粗酵素液生產 37
5.3.1 粗酵素液蛋白質濃度定量與酵素活性測定 38
5.4 海藻康普茶發酵期間微生物計數 38
5.4.1 發酵期間康普茶菌群於 β-agarase 水解液之生菌 39
5.4.2 發酵期間康普茶菌群於 β-agarase 及 cellulase 水解液之生菌數 39
5.5 海藻康普茶發酵期間理化特性分析 40
5.5.1 pH 值 40
5.5.2 還原糖含量 40
5.5.3 可滴定酸度 40
5.5.4 理化特性討論 41
5.6 海藻康普茶萃取液抗氧化能力測試 42
5.6.1 ORAC 42
5.6.2 CUPRAC 43
5.6.3 DPPH 自由基清除力 43
5.6.4 亞鐵離子螯合力 43
5.6.5 總酚含量測定 (Folin-Ciocalteu assay, F-C assay) 43
5.6.6 抗氧化能力結果討論 44
陸、結論 45
柒、參考文獻 46
捌、圖表 62
玖、附錄 80

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