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標題: Aspergillus oryzae NCH 42 固態發酵生產單寧酶 之探討與應用
Application and production tannase from Aspergillus oryzae NCH 42 by solid state fermentation
作者: Hsieh, Li-Chun
關鍵字: 固態發酵
Solid-state fermentation
Aspergillus oryzae
Water caltrop hull
Gallic acid
Aspergillus oryzae
出版社: 食品暨應用生物科技學系
摘要: 單寧酶 (tannase EC 是一種可水解單寧酯鍵與縮酚酸鍵;生成沒食子酸與葡萄糖之水解酵素,普遍存在於植物性食品及飼料中。一般可將利用單寧酶分解食物中之單寧,阻礙其與蛋白質及消化酵素結合,進而增進食物之消化吸收率,而沒食子酸亦具有良好之抗氧化力與抑菌能力。 本研究所使用之菌株Aspergillus oryzae NCH 42具分泌較高活性單寧酶能力,可降解水解型單寧產生沒食子酸。故本試驗利用農業廢棄物香蕉皮、龍眼殼、龍眼子與菱角殼等為基質,以固態發酵方式,探討A. oryzae NCH 42分離株生產單寧酶之較適培養條件;並測定菱角殼萃取物之單寧酶水解產物中成分變化、抗氧化力與抑菌能力。結果顯示,以菱角殼為基質可獲得活性較高之單寧酶,其較適培養條件為粒徑20 mesh之2 g基質,與水分比例為1:8,另補充0.2% NH4NO3、0.1% KH2PO4、0.05% MgSO4.7H2O、0.05% KCl之營養素,並於pH 3.5,30°C培養5天後,以20 ml之0.3% Tween 80,於120 rpm萃取1小時,可獲最佳活性為325 U/g, db之單寧酶。 A. oryzae NCH 42單寧酶經超過濾與硫酸銨 (飽和度80-90%) 之沉澱劃分後,分析酵素分子量約為54.58 kDa;酵素最適pH為5.0與7.0,且於pH 5-7之間仍具穩定之高活性;最適溫度為30°C,熱失活活化能 (Ea) 約為13.16 kcal/mole。金屬離子Mg2+、Mn2+及Na+對酵素活性具提升之效果;而Ba2+、Ca2+、Cu2+則具有明顯抑制單寧酶活性之現象。Tween 80對A. oryzae NCH 42單寧酶活性具提升之效果;基質特異性以單寧酸之酵素表現活性較高,由酵素動力學分析結果得知,單寧酶以單寧酸為基質之Km值為1.49 mM,Vmax值為14.90 µmol/ml/min。 菱角殼萃取物經單寧酶作用後可降解生成沒食子酸,且單寧酶粗酵素液之水解能力較商業酵素高,經硫酸銨劃分後單寧酶之水解能力更為提升;且經酵素作用後沒食子酸含量增加,其抗氧化力亦顯著提升。經四種酵素作用後之抑菌能力相較於未經酵素作用之能力,對革蘭氏陽性菌之抑菌效果較佳,其中以Listeria monocytogenes BCRC 14848敏感性最高。
Tannase (tannin acyl hydorlase, E.C., which presents commonly in plant origin foods and feeds, catalyzes the hydrolysis of ester and depside bonds in hydrolysable tannins such as tannic acid, and releases glucose and gallic acid. Tannins can be degraded by tannase to prevent them from binding with proteins or digestive enzymes, thus leading to improve the digestibility of foods; moreover, the gallic acid is also a strong antioxidant and a potent antibacterial agent. In this study, an extracellularly tannase-producing fungal isolate, Aspergillus oryzae NCH 42 was used. Agricultural residues such as banana peel, longan peel, longan seed and water caltrop hull were tested for the production of tannase under solid-state fermentation (SSF). Results showed that, water caltrop hull was the best substrate among four substrate tested for enzyme production under SSF. The optimum medium composition and cultivation condition for tannase production, changes in components in the enzymatic hydrolysates of water caltrop hull, and the antioxidant and antibacterial activities of hydrolysates were studied.The optimum particle size of water caltrop hull (2 g) in 20 mesh, tap water containing 0.2% NH4NO3, 0.1% KH2PO4, 0.05% MgSO4.7H2O, 0.05% KCl, at pH 3.5 was found to be the best moistening agent, and the ratio of substrate to the moistening agent was 1:8 (w/v). After 5 day of incubation at 30°C, 20 ml of 0.3% (w/v) Tween 80 was added and enzyme was extracted by shaking at 30°C, 120 rpm for 1 h. The activity of tannase obtained was 325 U/g, db. Tannase from A. oryzae NCH 42 was partially purified by a two-step purification strategy comprising of ultra-filtration and a subsequent precipitation with solid ammonium sulphate (80-90% saturation). Molecular weight of the partially purified tannase was estimated to be 54.58 kDa by SDS PAGE. The optimal pH and temperature of the enzyme were 5 and 50°C, respectively. However, the enzyme remains stable for pH from 5.0 to 7.0, and temperature between 30 and 60°C, respectively. Activation energy (Ea) of the enzyme by thermal inactivation was 13.16 kcal/mole. The effect of metal ions on tannase activity was studied, presence of Mg2+, Mn2+and Na+activated tannase, while Ba2+, Ca2+and Cu2+ inhibited the activity. Tween 80 enhanced tannase activity. Based on the kinetic study, tannic acid is the best substrate for the enzyme with Km of 1.49 mM and Vmax of 14.90 µmol/ml/min. Gallic acid was end-product from the enzymatic hydrolysis of the water caltrop hull as the substrate, and the hrdrolysis efficiency was superior to commercial ones. The hydrolysates showed higher scavenging activity and antioxidant capacity compared to the control (water caltrop hull). In addition, the hydrolysates also showed stronger antimicrobial activity against gram positive bacterial strains, especially Listeria monocytogenes BCRC 14848 than that untreated.
Appears in Collections:食品暨應用生物科技學系



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