稻米儲藏安定性與米糠脂肪?之研究

Studies on storage stability of rice and rice bran lipase

莊敦智(Chuang-Tun Chih)

指導教授 ： 陳駿季 ； 李雅琳

朝陽科技大學/理工學院/應用化學系生化科技碩士班/碩士(2013年)

摘要

米糠 (rice bran) 為糙米碾磨後的副產物，富含油脂及多種生理活
性物質 (如生育酚、生育三烯酚、米糠醇、植物固醇等等)，然而米
糠亦含有酯?(carboxylesterase, EC 3.1.1.1) 與脂肪?(lipase, EC
3.1.1.3)，會水解三酸甘油脂形成游離脂肪酸及甘油，是導致油脂酸敗
的主因。本研究先進行18 種稻米儲藏實驗，觀察酯?活性與稻米游
離脂肪酸含量的相關性，接著再以水稻台?9 號米糠為材料，分離純
化其脂肪?並研究其特性。米糠蛋白質萃取液以硫酸銨沉澱劃分蛋白
質，並以酯?基質p-nitrophenyl butyrate 與脂肪?基質p-nitrophenyl
palmitate 進行活性分析。酯?活性最高出現於30-50% 硫酸銨飽和度
沉澱區間，比活性為2.3×103 U/mg。脂肪?活性最高則出現於0-30%
飽和度，比活性為8.6 U/mg。然而以蛋白質電泳SDS-PAGE 分析，
酯?與脂肪?活性染色的最高活性區，均出現在0-30%硫酸銨飽和度，
取此區間之蛋白質進行酵素純化，發現純化過程導致脂肪?失去活性，
然而酯?則不受到影響，可維持高活性的狀態。逐步分析研究發現，
酯?是親水性高且相當的安定的酵素蛋白質；脂肪?性質則為高疏水
性蛋白質，分子間疏水性作用強、容易聚集沉澱，不易與水互溶，不
需要二價金屬離子維持或提高活性。脂肪?酵素安定性低，容易在實
驗過程失活，導致純化困難，故取出電泳分離、具有脂肪?活性的蛋
白質條帶，利用全系統蛋白質檢定，結果鑑定得到152 條蛋白質，其
中含有14 條熱休克蛋白和2 條油膜蛋白，驗證了0-30%硫酸銨飽和
區的蛋白質，含有高量的輸水性蛋白質，故推測脂肪?與這些蛋白質
相似，其表面擁有較大面積的疏水性區塊。

關鍵字

台?9 號水稻 ； 脂肪? ； 酯? ； 米糠

並列摘要

Rice bran, rice milling by-product, is rich in lipids and various
bioactive components (such as tocopherols, tocotrienols, γ-oryzanol,
phytosterols, etc). However, carboxylesterase (EC 3.1.1.1) and lipase (EC
3.1.1.3) in rice bran may hydrolyze triacylglycerols releasing glycerol and
free fatty acids which result in rancidity of the bran. This study conducted
rice storage experiments with 18 cultivars to study the relationship
between bran esterase activities and free fatty acid contents. Then rice
cultivar Taikeng 9 (Tk9) was used as material to explore the lipases
activities and their characteristics. Ammonium sulfate precipitation
method was employed to fractionate rice bran proteins. p-Nitrophenyl
butyrate was used as esterase substrate and the highest activity was
observed at the fraction of 30-50% ammonium sulfate with a specific
activity of 2.3×103 U/mg. p-Nitrophenyl palmitate used as lipase
substrate, the highest activity was observed at the fraction of 0-30%
ammonium sulfate with a specific activity of 8.6 U/mg. However,
SDS-PAGE separation showed the highest activities of both enzymes
were at the fraction of 0-30% ammonium sulfate. This fraction of proteins
was further purified and found that the lipase activity lost fast but the
esterase’s remained stable and high. It was found that esterase proteins
were hydrophilic and very stable; the lipase proteins were hydrophobic
and the protein-protein interaction made them prone to aggregation and
precipitation, but need not metal ions to maintain or increase their activity.
Because of lability the lipase proteins were not amenable for traditional
purification procedures. Therefore the lipase proteins with activity
staining on the gel of SDS-PAGE were isolated for LC-MS/MS analysis
to identify their proteins sequences. There were 152 proteins identified.
Of them fourteen were heat-shock proteins and two oleosin protein. It
suggested that the lipase proteins have a similar feature with those
precipitated at the fraction of 0-30% ammonium sulfate, that they bear
significant hydrophobic areas on the protein surface.

並列關鍵字

Taikeng 9 rice cultivar ； lipase ； esterase ； rice bran

參考文獻

colorimetric method for the determination of non-esterified fatty

林文龍(1983)環境與稻米品質。臺中區農業改良場研究彙報。7:

許志聖、楊嘉凌(2009)台中區農業改良場特刊94 號。水稻篇：

Ueki, J., Morioka, S., Komari, T., Kumashiro, T., Purification and

Prabhu, A. V., Tambe, S. P., Gandhi, N. N., Sawant, S. B., Joshi, J. B.,