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|標題:||Study on physiological activities of tannase-modified green tea and cultivation conditions for tannase production by Aspergillus awamori|
|作者:||Lu, Min- Jer|
The consumption of tea drinks in the market of Taiwan is growing each year, and is now the largest consumption, compared with all other drink products in the local market. Yet, precipitation in drinks of green tea starts taking place during manufacturing process, while the intake of green tea by large amount inhibits the activity of digestive enzyme, reducing the amount of nutrient absorption as the result. These are the problems aimed for in this study, where green tea leaves treated with commercially available tannase were hydrolyzed and extracted with hot water at 85℃ for 20 minutes for storage related test and physiological functioning study, while allowing the tannase to be produced by Aspergillus awamori. Our test results showed that by means of the enzymic action of the tannase, EGCG of the catechins was hydrolyzed into EGC, that by adding various concentration of Bovine Serum albumin (BSA), the tannase-treated tea was found to have a relatively lower protein binding rate. This hydrolyzed tannase-treated tea was further studied after storage over 4 weeks for the contents of catechins, formation of tea cream, and extent of turbidity, showing no significant difference in the contents of catechins (p >0.05), while the control group of untreated green tea showed significant decrease of catechin content (p<0.05) and, in the meantime, lower quality in terms of physical appearance and clarity due to the gradual darkening of the color of the tea infusion during storage and the formation of tea cream (p<0.05). In terms of the sensory evaluation, the analysis results showed that the fragrance, flavor and the overall acceptance of the tannase-treated green tea were all better than that of the control group. The antioxidant capacity was evaluated by using the assays of DPPH for determining the amount of superoxide anion, TEAC, hydrogen peroxide, and chelating of metal ions, and the results showed that tannase-treated green tea at concentration of 200 ppm had higher antioxidant capacity than the untreated green tea, having scavenging effects on superoxide anion, hydrogen peroxide and DPPH as high as 97%, 83% and 62%, respectively. Tannase-treated green tea of concentration at 200 ppm also showed a significantly higher level of TEAC antioxidant activity, with 2.4% versus 1.0% of the untreated green tea. Furthermore, higher chelating activities for metal ions Fe+2 and Cu+2, at 42.6% and 33.5%, respectively, were observed in tannase-treated green tea at 150 ppm, whereas only 14.6% and 7.6% of chelating activities were observed in the untreated green tea. The RAW 264.7 cells incubated in tannase-treated green tea at the presence of copper ions after 144 h was found to have higher rate of viability than those without any treatment. The tannase-treated green tea was found to inhibit nitrosation, or the formation of N-nitrosodimethylamine (NDMA), much better than green tea treated with ascorbic acid or without enzymic treatment, as indicated by the testing with four major catechins, where epigallocatechin (EGC) was found to block the N-nitrosation more efficiently, and, compared with epigallocatechin, epigallocatechin gallate (EGCG) was found to be more unstable at pH 2.0 and pH 8.0. Furthermore, digestive enzyme activity inhibitors, such as α-amylaser, pepsin, trypsin and lipase, were found to be less in experment groups than in control groups(p<0.05). The producing of tannase enzyme by Aspergillus awamori pointed to the finding about the characteristics of enzyme that pH 5.0 is the optimum for enzamic reaction, 30℃is the thermal optimum for purified tannase enzyme, and that enzamic activity declines sharply when temperature goes beyond the 30℃ to 60℃ stability range. Besides, the Aspergillus awamori produced tannase enzyme disclosed by this experiment can be applied effectively in tea-making process to solve turbidity problem, while enhancing the physiological function of green tea.
Key words: catechins, tannase enzyme, gallic acid, nitrosamine, Apergillus awamori|
中文摘要 茶飲料在國內飲料市場的消耗量逐年上升，目前已居國內各飲料市場消耗量的首位。但綠茶飲料在製造過程中造成的沈澱，及大量攝取綠茶飲料會抑制消化酵素的活性，降低營養素的吸收。以上所造成的問題將是本實驗研究重點，有鑑於此。故本實驗方法利用商業化單寧酶處理綠茶後以85℃沸水萃取20分鐘後，做儲存試驗和生理功能探討。並同時利用Aspergillus awamori生產單寧酶。由結果顯示經酵素作用後能使綠茶兒茶素中的EGCG水解成EGC。並添加不同濃度的牛血清蛋白發現酵素處理的綠茶對蛋白質的結合度較低，同時經過4週的儲存試驗對兒茶素含量的分析，和茶湯茶乳及濁度的分析，顯示水解後的茶湯，兒茶素含量並無明顯差異(p>0.05)，但控制組之兒茶素卻明顯降低(p<0.05)，同時在外觀品質和澄清度，控制組茶湯在儲放過程中外觀逐漸變深，而在茶乳的形成控制組明顯增加(p<0.05)。官能評品和總體接受性的比較以實驗組較好(p<0.05)。在抗氧化測定，比較總抗氧化能力、超氧離子、過氧化氫和DPPH。由實驗結果顯示單寧酶處理綠茶在 200 ppm時測定超氧離子、過氧化氫和DPPH依序為97%, 83%, 62％,較控制組為高。實驗組濃度在 200ppm時，總抗氧化能力的比較由結果顯示實驗組為2.4％較控制組1.0％為高。清除鐵離子和銅離子實驗組濃度在 150ppm顯示實驗組清除效率較控制組佳。以RAW 264.7細胞株在銅離子存在下培養經144小時後，發現實驗組細胞存活率較高。對抑制亞硝胺的形成，由結果顯示比維生素C和未經酵素處理的綠茶更有效率。當在pH=2和8時比較四種兒茶素，發現EGC較EGCG更能抑制NDMA形成，因EGCG在pH=2和8是不穩定。進一步比較消化酵素發現α-amylaser、pepsin、trypsin、lipase發現實驗組對酵素活性抑制較低（p<0.05）。以Aspergillus awamori生產單寧酶發現，酵素特性如下：最適反應pH值為5.0，最適反應溫度為30℃，超過60℃活性明顯下降。因此由本實驗證明經單寧酶處理綠茶將能改善製茶時的混濁問題，同時更能強化綠茶飲料的生理活性。 關鍵詞：兒茶素、單寧酶、酚酸、亞硝胺、Aspergillus awamori
|Appears in Collections:||食品暨應用生物科技學系|
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