- 學位論文
以Paddy Oats 種子皮(Gnetum gnemon) 作為蛋白酶的新來源及其應用
Paddy Oats (Gnetum gnemon) as A New Source of Protease and Its Application
摘要
對具有特定性質的蛋白酶的需求增加已經導致生物技術專家探索新的蛋白酶來源。由於蛋白酶在生物體中是生理上必需的，因此它們無處不在，存在於植物，動物和微生物等多種來源中。植物來源的蛋白酶因為其特性和功能而受到藥物和食品生物技術的特別關注。數百年前，從植物皮層萃取之蛋白酶已廣泛應用於肉品嫩化。這項研究的目的是研究Gnetum gnemon種子皮作為蛋白酶來源的可能性，並進行特性分析，研究其作為嫩肉劑，椰子油提取酶以及洗滌劑添加劑之應用性。 完全成熟的果皮具有最高蛋白水解活性343 U / mg的提萃物，評估溫度和pH對提萃物的蛋白酶活性的影響。三個成熟階段的最佳pH和溫度曲線表明了相似的趨勢。Gnetum gnemon籽皮萃取物最適作用溫度範圍廣，最大活性在45-55℃，粗萃物的最適pH為6.0-8.5。為了鑑定蛋白酶的類別，已經研究了不同蛋白酶抑製劑的作用。硫醇試劑，二硫代-雙-硝基苯甲酸（DTNB）抑制蛋白酶活性，而EDTA顯示刺激作用。該抑製曲線表明粗萃物含有半胱胺酸蛋白酶。結果顯示，成熟和完全成熟的Gnetum gnemon種子皮，可作為蛋白酶的來源。 60％硫酸銨沉澱可增加三個不同成熟階段（未成熟，成熟和完全成熟果皮）的Gnetum gnemon種子皮中萃取的蛋白酶活性。完全成熟的果皮之蛋白酶萃取物具有最高活性343 U/mg，與未成熟果皮和成熟果皮的活性具有顯著差異。完全成熟的果皮萃取物具有最高蛋白水解活性892 U/mg。SDS-PAGE下蛋白酶提取物的蛋白質模式產生25和28kDa的兩個主要蛋白質條帶。果皮成熟的三個階段之間的分子量沒有差異。結果表明，使用60％硫酸銨進行純化可有效提高Gnetum gnemon籽皮蛋白酶的純度。 針對Gnetum gnemon種子皮蛋白酶萃取物對肉嫩度的影響，並與商業木瓜蛋白酶，商品菠蘿蛋白酶和對照物進行了比較。Gnetum gnemon蛋白酶萃取物顯示出高肉嫩化活性，因為它導致肉品樣本的剪切力值比對照低46％，與商業木瓜蛋白酶和商業菠蘿蛋白酶相似（p ≤0.05）。使用10點特徵量表評量肉品柔軟度。Gnetum gnemon蛋白酶萃取物處理的肉樣品（7.4）比對照組（5.8）更柔軟，與商業木瓜蛋白酶和商業菠蘿蛋白酶沒有差別（p≤0.05）得分分別為7.3和7.7。因此，Gnetum gnemon蛋白酶萃取物可作為肉嫩化劑，以改善肉品嫩度。 Gnetum gnemon蛋白酶萃取物在萃取椰子油中表現出良好的性能。將來自Gnetum gnemon的蛋白酶的功效與各種處理的功效進行比較。Gnetum gnemon蛋白酶顯示出萃取初榨椰子油的高性能，因為它具有高產率（25.7％）和優質油，游離脂肪酸為0.092％，密度為0.924 g/mL。用蛋白酶處理萃取的初榨椰子油，其感官分析香氣得分為7.6，味道得分為7.3，顏色得分為9.2。整體而言，完全成熟的 Gnetum gnemon種子皮，可作為萃取初榨椰子油的蛋白酶來源。 從Gnetum gnemon種皮中萃取的蛋白酶具有作為洗滌劑添加劑的潛力。為模擬洗滌條件，將洗滌劑在蒸餾水中稀釋至終濃度為2mg/mL。加熱溶液，然後將蛋白酶萃取物加入洗滌劑溶液中。結果表明，在65分鐘內，洗滌劑-蛋白酶溶液可以很好地去除織物上的血跡，比其他處理快得多。為了評估作為洗滌劑添加劑的蛋白酶相容性，將蛋白酶與當地市場上可獲得的不同洗滌劑品牌混合。然而，這種蛋白酶與某些洗滌劑的相容性相對較低。
並列摘要
Market demand for proteases that continue to grow rapidly has stimulated researchers to find new sources of proteases with specific properties as needed. Since proteases are enzymes that are always present in creatures, they can be explored from various sources both from microorganisms, animals and plants. Each enzyme source has advantages and has its own specifications so that it has the opportunity to get different types of proteases from different sources. Protease from plant origin is widely explored by biotechnologists in the pharmaceutical, food sector because of its superior properties and functions. Plant protein, papain for example, is one of the ensimes that has been known for hundreds of years to improve the tenderness of meat. The aim of this research was to investigate the possibility of paddy oats fruit peel as a source of protease. The study was conducted to characterize protease extracted from paddy oats fruit peel, and also studied its application as a meat tenderizer, coconut oil extraction enzyme, as well as a detergent additive. The highest proteolytic activity was yielded from ripe peel extract which was 343 U/mg. Different temperatures and pH substrate was evaluated to determine optimum temperature and pH of protease. The optimum temperature was in over a broad temperature range from 45 to 55oC, and optimum pH of protease from paddy oats fruit peel was 6.0-8.5. Protease from paddy oats fruit peel included in cysteine class protease. Since the protease was inhibited by dithio-bis-nitrobenzoic acid (DTNB) and slightly stimulated by EDTA. The result of study proved paddy oats fruit peel is a potential source of protease. Ammonium sulphate precipitation 60% increased protease activity extracted from paddy oats fruit peel from three different maturity stages (immature, mature and ripe peel). The highest activity of protease extract was 343 U/mg belong to ripe peel and differed significantly from those of mature peel and immature peel. Purification process using 60 percent ammonium sulphate was increasing protease activity (2.47; 2.53; 2.60 fold purity) for immature, mature and ripe peel respectively. The ripe peel yielded the extract with the highest proteolytic activity of 892 U/mg. Protein pattern of the protease extracts under SDS-PAGE resulted in two major protein bands of 25 and 28 kDa. There was no difference in molecular weights among the three stages of peel maturity. The results revealed that purification using ammonium sulphate 60% effectively increases the purity of paddy oats fruit peel protease. To examine the efficacy of paddy oats protease extract as a meat tenderization, protease was compared to those commercial bromelain, papain and control. Sample treated meat with paddy oats protease resulted 46% lower in shear force value of meat sample than that of control. It was not significant different (p˂0.05) from those of commercial bromelain and papain. Sample treated meat with protease also gained high score of 7.4 (using a 10-point hedonic scale), while meat treated bromelain and papain of 7.7 and 7.3 respectively. There were not significant different among them, and they considered much more tender than that of control. There is no doubt that protease from paddy oat fruit peel have the great efficacy to be a meat tenderizer to improve the quality of meat tenderness. Paddy oats protease was used as a bioactive to extract coconut oil from coconut milk. It produced high yield of coconut oil (25.7%), and all of Psysico-chemical parameters observed met with both of Malaysian Standars as well as Asian and Pacific Coconut Community Standard for free fatty acids of 0.092%, and density of 0.924 g/mL. Sensory parameter of coconut oil extracted using paddy oats protease consistently ranked high. The aroma, taste and color of coconut oil evaluated using a 10-point hedonic scale, and they were scored 7.6, 7.3 and 9.2 respectively. In overall this study proved that protease from paddy oats fruit peel is a protease that has excellent performance to extract coconut oil, since it produced the high yield and high quality of coconut oil. Protease extracted from paddy oats fruit peel has potential as a detergent additive. To simulate washing conditions, the detergent was diluted in distilled water up to a final concentration of 2 mg/mL. The solution was heated and then paddy oats protease extract was added to the detergent solution. The result showed blood stain on the fabric could be removed perfectly by detergent-protease solution in 65 minutes, much faster than those of other treatments. To evaluate the protease compatibility as a detergent additive, protease was mixed with a different detergent brand that available in Taiwan local market. However, the compatibility this paddy oats protease against some detergent was relatively low.