改善米蛋白水解物的水溶性與苦味減量之研究

Abstract

米蛋白具高營養價值、低致敏性、產量大且價格低廉等優勢，為極具潛力的植物性蛋白質，然而，水溶性低造成應用上的限制，尤其飲料產業之發展限制。已有許多酵素修飾法提升米蛋白功能性之研究，但對其水解後苦味程度之變化則較少著墨，故本研究將以米蛋白水解物進行實驗與討論。研究架構分為三部分，第一部分為替換酵素修飾法中的蛋白酶，並篩選適合用於產生低苦味且高水溶性的米蛋白水解物者，第二部分為分析水解液之物化特性與苦味程度之間的關係，第三部分為利用其他物化方法降低苦味。 第一部分篩選適合生產低苦味米蛋白水解物的市售蛋白酶，其中以 Neutrase®、FF104、MF101、PHT、51FP 等五種蛋白酶水解的效果最好，可得苦味程度低 (分數低於 5 分，最高為 10分) 且水溶性高於 20% (w/v) 的水解物。將本研究中主要樣品進行苦味強度排序，結果如下：以 Alcalase 水解者 (RPH_A) > 以 Neutrase 水解者 (RPH_N) = 市售米蛋白水解物 (CPRH) > 以 Alcalase + Protease P 水解者 (RPH_A+PP) = 以 Neutrase + Protease P 水解者 (RPH_N+PP) = 未水解的米蛋白 (RP)，發現將水解液接續使用外肽酶 (Protease P) 水解可使苦味降低至與未水解的米蛋白相同，等同於無苦味。 第二部分為分析水解物物化特性與苦味程度間的關係，先將水解物分散於水中並離心後，可得上清液與沉澱物，研究結果指出苦味胜肽主要存在於上清液；另外，造成苦味的原因與水解液上清液的胺基酸組成無關。在疏水性方面，以液相層析/質譜儀分析可得，低疏水性胜肽含量與水解液苦味強度呈負相關，RPH_A (8.8%) < RPH_N (10.8%) < RPH_A+PP (17.1%)；高疏水性胜肽含量與水解液苦味強度成正相關：RPH_A (29.1%) > RPH_N (20.6%) > RPH_A+PP (10.6%)。從水解液分子量分布中，發現 RPH_N 與 RPH_A+PP 的苦味都低於 RPH_A，但兩者的分子量分布趨勢卻截然相反，因此從本研究中無法看出分子量分布與苦味程度之間的關係。 由於前述的樣品 RPH_A 及 RPH_N，與未水解的米蛋白 (RP) 相比都還有苦味，因此第三部分是以此兩者作為樣品，進行其他物理化學方式去除水解物苦味之研究。研究結果發現，先以酸法去醯胺化再進行水解無法有效降低 RPH_A 的苦味；以活性碳吸附或是以 β-環糊精處理都能顯著降低 RPH_A 與 RPH_N 之苦味程度，兩者皆是利用移除或屏蔽疏水性胜肽，以達到去除苦味的效果，此部分結果與第二部分之研究結果相符。 本研究之貢獻為提出多種降低米蛋白苦味之方法，做為食品產業中多元的配方替代與混合使用之參考，使米蛋白產品能在提升功能性之際，同時具有消費者可接受的風味特性，進而提升其產業上的附加價值與應用潛力。

Rice proteins are considered as valuable plant proteins due to their unique nutritional and hypoallergenic properties, high yield, and low cost. The key limitation of rice proteins is their poor solubility. Many studies have attempted to use enzymatic modification to improve the functional properties of rice proteins, but only few has been done on the lowering of bitterness of rice protein hydrolysates. Therefore, different treatments for rice protein hydrolysates of higher solubility and lower degree of bitterness of rice proteins were evaluated. There were three parts in this study. First, protease were selected and protein solubility was used as an indicator to determine the appropriate enzyme formula to reduce the bitterness of rice protein hydrolysates. Second, the relationship between rice protein and its bitterness were analyzed. Third, other physico chemical approaches to remove bitterness from hydrolyzates. In the first part, single commercial protease was used to hydrolyze rice proteins. The treatment that produced hydrolysates with their bitterness intensity less than 5 points (the highest score: 10 point) and solubility higher than 20% were selected. The results indicated that commercial proteases such as Neutrase®, FF104, MF101, PHT, and 51FP were suitable for hydrolyzing rice protein. Bitterness intensity of major samples were as follows: RPH_A > RPH_N = CRPH > RPH_A + PP = RPH_N + PP = RP. Hydrolysates with exopeptidase after a single step of commercial protease could reduce the bitterness to zero, the same intensity as rice protein without hydrolysis. In the second part, the factor resulting in bitter hydrolysates were analyzed. It was found that bitter peptides existed in the supernatants. The bitterness was not related to amino acid composition. After liquid chromatography/mass spectrometry analysis, the results showed that the amount of hydrophilic peptides was inversely correlated to the bitterness intensity, for example: RPH_A (8.8%) < RPH_N (10.8%) < RPH_A+PP (17.1%). The content of hydrophobic peptides were positively correlated to the bitterness intensity: RPH_A (29.1%) > RPH_N (20.6%) > RPH_A+PP (10.6%). In terms of molecular weight, the molecular weight of RPH_N was greater than that of RPH_A, following by RPH_A+PP. Both RPH_N and RPH_A+PP were less bitter than RPH_A, indicating that molecular weight was irrelevant to bitterness inrensity. The third part of this study focused on the other physico chemical approaches to remove bitterness from hydrolyzates, including deamidation, activate carbon adsorption, and incorporation of β- cyclodextrin. Since the treatment with acid deamination before hydrolysis could not reduce the bitterness of RPH_A, the use of activate carbon or β- cyclodextrin significantly reduced the bitterness of RPH_A and RPH_N. This study took water solubility and bitterness as the indexes for the estimation of functional property. In the evaluation of suitable commercial proteases for rice protein hydrolysates, treatment with exopeptidase after a single commercial protease could reduce the bitterness to zero level. In addition, treatment with activated carbon adsorption or β-cyclodextrin also reduced the bitterness of rice protein hydrolysates. In this study, we have verified that the hydrophobicity of peptides in the hydrolysates was one of the main factors that influenced the intensity of bitterness. The physico chemical method used in the third part was probably an appropriate application to remove or mask the hydrophobic structure of peptides. The contribution of this study was to provide various approaches to reduce bitterness of rice protein hydrolysates, and present more flexible choice of alternatives and formulas for industry. Furthermore, produce high quality rice protein hydrolysates as formulas with good fuctional properties and consumer-acceptable flavor may increase its added value and potential.