Effects of cultivation modes and conditions on microbial activities, antioxidant potential and tea polyphenol levels of kombucha

Kombucha have in vivo antioxidant activity. Recently, many researches reported that metabolites from degradation of tea polyphenols by colonic microflora were the major active substances in vivo. This study focused on the variations of microbial activities, antioxidant activities and tea polyphenol levels etc. during kombucha fermentation. There are six topics included as followed. Results showed that (1) Antioxidant activities of kombucha were dependent on starter sources and fermentation time. The average antioxidant potentials of eight kombucha samples after fermentation of 15 days were raised to about 70%, 40%, 49% determined respectively by the assays of DPPH, ABTS radical scavenging, and inhibition of linoleic acid peroxidation, while the ferrous ion binding ability was inversely diminished by 81%. The total phenol content increased up to 98%. (2) Efficiency of kombucha fermentation was enhanced under shaken environment. Compared to 4 g/l tea media, 10 and 20 g/l black or green tea media enhanced sugar consumptions, productions of ethanol and organic acids, and polyphenol degradations by kombucha starter, but accelerated the decline of viable acetic acid bacteria other than yeast counts. Antioxidant activities of black tea media at 4, 10 and 20 g/l against ABTS or DPPH radicals decreased during fermentation; the final retained ratios in 20 g/l media were 64% and 63%, respectively. Antioxidant activities of 4, and 10 g/l green tea media remained constant throughout fermentation; however, the final retained ratios in 20 g/l medium decreased to 51% and 42%, respectively. (3) The antioxidant ability and total color (index of thearubigin level) of kombucha both decreased with fermentation time while cultivation temperatures (27-36℃) showed less effect on these variations; however, higher values were seen when cultivated at 27°C within 6 d. Initial pH values from 4.5 to 6.5 of black tea decoctions, had no influence on variations of antioxidant activities of kombucha during fermentation. However, kombucha had lower antioxidant potential when fermented in media of pH 7.5. Initial pH values also showed no effect on changes in total color during fermentation. Total color of kombucha decreased more obviously when fermented in black tea media at increased concentrations. However, the retained antioxidant ability of kombucha ranged from 92% to 104% when cultivated in media below 30 g/l. No differences were seen in variations of antioxidant activities and total color when kombucha was prepared in media containing various carbon sources. Kombucha cultivated in decoctions containing 4% and 7% sucrose (w/v) retained higher antioxidant activities than that in medium, supplemented with 1%. However, most decrease in total color of kombucha was observed when fermented in sugared media (4% sucrose) within the first 6 days. Decreases of antioxidant activity and total color of kombucha during fermentation were enhanced when nitrogen sources were present in media. Kombucha, prepared from media containing 0.29% and 1.0% peptone (w/v) had higher antioxidant activities than that from medium containing 0.50%. More decreases in total color of kombucha were seen when cultivated in decoctions, containing 0.50% or 1.0% peptone. (4) From the results of RSM analysis, it was found that antioxidant activity of kombucha increase to 1.78 times when cultivated in 30 g/l black tea medium, containing 2% sucrose at 27°C for 48 h. Decreased ratios of total color were up to 74% when kombucha were fermented in medium, containing 10% sucrose and 0.93% peptone at 30°C for 120 h or in medium, containing 10% sucrose and 0.93% peptone at 33°C for 72 h. (5) More decreases in total color of kombucha were observed when 10 or 20 g/l black tea media (residual ratio of total color: 72-73%), aeration rate of 0.5 l/min (52%), agitation rate of 0 rpm (52%), 0.21% peptone (47%), seeding ratio of 2.5% (28%), medium feeding at day 1 (38%), feeding rate of 2250 ml/d (37%), seeding ratio of 1.25% in feed batch fermentation (38%), and inoculation only with yeasts were used for kombucha fermentation with the bioreactor. Decreases in tea polyphenol levels of kombucha were also enhanced when 20 g/l black tea media, aeration rate of 0.5 l/min, agitation rate of 130 rpm, 0.36% peptone, seeding ratio of 10%, medium feeding at day 1, feeding rate of 750 ml/d, seeding ratio of 10% in feed batch fermentation, inoculation with kombucha starter, and alternative green tea medium at 10 g/l were used for fermentation with the bioreactor. (6) Residual ratio of total color of black tea medium was 93% after being reacted to crude enzyme solution from black tea kombucha at 30°C for 48 h; ECG level in this reactant increased while EC and gallic acid levels remained unchanged. EC, ECG, ECG, EGCG and gallic acid levels in green tea medium were not changed after being treated with above crude enzyme solution. Residual ratio of total color of black tea medium was 95% after being reacted to crude enzyme solution from green tea kombucha at 30°C for 48 h; EC, ECG, gallic acid levels of this reactant showed no variations. Decreased EC level and raised gallic acid level of green tea medium were observed while ECG, ECG, and EGCG contents remained constant after being treated with above crude enzyme solution. Tea polyphenol levels of black tea medium were not changed after being treated with crude enzyme solution from fermented broth by yeasts; consistent results were obtained when green tea medium was reacted to above enzyme solution with the exception of observed decrease of EGCG content. Furthermore, all types of crude enzyme solution show no tannase activities. This study might elucidate the relationships among starter activities, and tea polyphenol levels and antioxidant potential of kombucha. Cultivation conditions which increased antioxidant activity or enhanced degradations of tea polyphenols of kombucha might be the reference information for further production. As tea polyphenol hydrolysis activities of crude enzyme solution were not apparent and thearubigin could not be absorbed directly by starter, it was possible that enzymes on cell membrane of starters were major contributors to tea polyphenol degradation.已知紅茶菇具有體內抗氧化活性。近來研究也指出，大腸微生物降解茶多酚所生成代謝物才是提供其體內活性的主要物質。本研究即針對紅茶菇於發酵中的微生物活性、抗氧化力、茶多酚量等變化進行探討，共分為六部分。 結果顯示：（一）紅茶菇抗氧化力取決於菌種來源與發酵時間的長短。八種不同來源的紅茶菇經靜置發酵15天後，其捕捉DPPH與ABTS自由基及抑制亞麻油酸氧化的能力平均各增加70％、40％及49％，總多酚量增加98％，螯合亞鐵離子的能力則降低81％。（二）震盪培養有助提升紅茶菇菌的發酵速率。10及20g/l 的紅茶或綠茶湯液較其4 g/l濃度明顯促進茶菇菌的糖類利用、乙醇和醋酸的生成及茶多酚的降解，但也導致醋酸菌數的快速下降。各濃度之紅茶液於發酵中其捕捉ABTS及DPPH自由基的能力皆逐降；20 g/l紅茶發酵液其最終抗氧化力分別僅剩64%及63%。4及10 g/l 綠茶液於發酵中其抗氧化力無明顯變化；20 g/l綠茶發酵液之最終抗氧化力則分別降至51%及42%。（三）由培養因子的影響中可知，紅茶菇的抗氧化力與總色度（茶紅素量指標）皆於靜置培養中逐降，培養溫度（27-36℃）的影響不大，但於27℃下培養6天內測定值皆較高。起始pH值在4.5-6.5間，不影響紅茶菇發酵時抗氧化力的變化，但於pH 7.5時，紅茶菇的抗氧化力顯著降低；起始pH值對總色度變化並無影響。茶液濃度增加可促進總色度降低；茶液濃度為30 g/l以下時，經發酵後仍保有92-104％的抗氧化力。碳源種類不影響紅茶菇抗氧化力及總色度變化；碳源濃度（蔗糖）於4％及7％時，可較1％保留較高抗氧化力；但於4％時（發酵6天內），總色度下降量最大。氮源添加會促進抗氧化力與總色度的減少；氮源濃度(peptone)為0.29％及1.00％時，較0.50％可保留較高抗氧化力，但總色度的降幅以0.50％及0.10％組較高。 （四）經反應曲面法探討後得知，發酵時間 48 h、溫度 27°C、茶液濃度 30 g/l與蔗糖濃度 2% 的條件下，發酵液抗氧化力增加的比率達78.7％。發酵時間 120 h、溫度 30°C、蔗糖濃度 10%及peptone濃度 0.93% 與發酵時間 72 h、溫度 33°C、蔗糖濃度 10%及peptone濃度 0.93%條件下，發酵液總色度的降低比率均可達74％。（五）於發酵槽操作中，茶濃度為10與20 g/l（總色度殘留率：72-73％），通氣量為0.5 l/min（52％），攪拌速率為0 rpm（52％），peptone濃度為0.21％（47％），接種量為2.5％（28％），於發酵第1天進行饋料（38％），饋料速率為2250 ml/day（37％），饋料發酵的接種量為1.25％（38％）及以酵母純菌接種（34％）時，發酵液總色度有較大的降低量。茶濃度為20 g/l，通氣量為0.5 l/min，攪拌速率為130 rpm，peptone濃度為0.36％，接種量為10％，於發酵第1天進行饋料，饋料速率為750 ml/day，饋料發酵的接種量為10％，以酵母及醋酸菌混菌接種，替代培養基為10 g/l綠茶液時，發酵液的茶多酚降低量較大。（六）紅茶液經紅茶菇之粗酵素液作用後（30℃，48 hr），其總色度殘率為93％，ECG量增加但EC與gallic acid量則無變化；綠茶液經同等條件作用後，其EC、ECG、EGC、EGCG與gallic acid量皆無變化。紅茶液經綠茶菇之粗酵素液作用後（30℃，48 hr），其總色度殘率為95％，但EC、ECG與gallic acid量均無變化；綠茶液經同等條件作用後，其EC與gallic acid量分別出現減少與增加的現象，但ECG、EGC、EGCG量則無變化。紅茶液經酵母發酵液之粗酵素液作用後（30℃，48 hr），其總色度與茶多酚量均無變化；綠茶液經同等條件作用後，茶多酚量中僅EGCG下降。此外，這三種粗酵素液均無單寧酶活性。 本研究可解明茶菇菌活性與發酵液中茶多酚量及抗氧化力的相關性。提升發酵液抗氧化力或促進其茶多酚降解的條件也可供作日後生產的參考。由於粗酵素液水解茶多酚的能力不顯著，且茶菇菌應無法直接吸收茶紅素，因此不排除茶菇菌降解茶多酚的主要酵素可能是位於其細胞膜上的酵素。中文摘要………………………………………...……….…………………I 英文摘要………………………………………...………………………..III 研究目的………………………………………...…………………………1 實驗架構………………………………………...…………………………3 第一章、文獻整理……………………………..……….………………….4 一、紅茶菇特性………………………………..….……..…………………4 二、類黃酮特性………………………………..….………………………10 三、類黃酮於體內的吸收與代謝……………..….………………………19 四、微生物在多酚代謝上的角色……………..….………………………26 五、反應曲面法………………………………..…….……………………27 六、微生物培養………………………………..….………………………30 第二章、紅茶菇菌種來源及發酵時間對其發酵液抗氧化力的影響…...33 一、前言………………………………………….………………..………33 二、材料與方法……………………………………….…………..………33 三、結果與討論…………………………………….……………..………35 四、結論…………………………………………….……………..………38 五、實驗結果圖表………………………………….……………..………38 第三章、茶液種類及濃度對紅茶菇於震盪培養下其微生物生長、茶多酚含量及抗氧化力之影響……………….……..…………...44 一、前言……………………………………….….…….…………………44 二、材料與方法………………………………..……….…………………44 三、結果與討論………………………………….……..…………………48 四、結論……………………………………………...……………………55 五、實驗結果圖表………………………………………...………………56 第四章、紅茶菇較適發酵條件之探討…………………...………………62 一、前言…………………………………………………...………………62 二、材料與方法…………………………………………...………………62 三、結果與討論…………………………………………...………………64 四、結論…………………………………………………..………………79 五、實驗結果圖表………………………………...………………………79 第五章、以反應曲面法探討增加紅茶菇抗氧化力或促進其茶紅素降解的較適條件……………………....………...…..…………….116 一、前言……………………………………………...…………………..116 二、材料與方法…………………………………….………...………….116 三、結果與討論………………………………………………..………..117 四、結論………………………………………………………..………..121 五、實驗結果圖表…………………………………….……………....…121 第六章、紅茶菇於發酵槽中較適發酵條件之探討…..………...…....…140 一、前言……………………………………………….………...…….…140 二、材料與方法………………………………………..………...…....…140 三、結果與討論……………………………………….………..…….…143 四、結論……………………………………………….………..…….…165 五、實驗結果圖表……………………………………..………..………166 第七章、紅茶菇、綠茶菇及酵母菌之紅茶發酵液其粗酵素液水解紅茶或綠茶多酚能力之探討…………………….….………..…..217 一、前言………………………………………….………………………217 二、材料與方法………………………………….………………………217 三、結果與討論………………………………….………………………218 四、結論………………………………………….………………………221 五、實驗結果圖表……………………………….………………………222 第八章、參考文獻……………………………….………………………232 附錄…………………………………………...…………………………24