米蛋白之磷酸化與應用於米蛋白磷酸胜肽之製備

Phosphorylation of Rice Protein and its Application on Preparation of Rice Phosphopeptides

10.6342/NTU201704379

陳立於

米蛋白 ； 米蛋白水解物 ； 磷酸化 ； 三偏磷酸鈉 ； 雙硫鍵 ； 蛋白水溶性 ； 溶鈣能力 ； Rice protein ； Rice protein hydrolysates ； Phosphorylation ； Sodium trimetaphosphate ； Disulfide bond ； Protein solubility ； Calcium-solubilizing capacity

國立臺灣大學園藝暨景觀學系學位論文

2017年

博士

許輔

繁體中文

本研究嘗試以磷酸化法將水難溶之米蛋白予以修飾，並探討磷酸化對於米蛋白的低水溶性和其溶鈣能力方面的改變與賦予。米蛋白之磷酸化是將米蛋白分散於pH 11.5、溫度35°C之鹼性溶液中，並和法規認可之食品添加物：三偏磷酸鈉一併反應進行磷酯化之作用，試驗結果顯示20.6%之米蛋白絲胺酸可於STMP之反應下被進行磷酸化。然而，不同於過去其他蛋白質在經由磷酸化修飾後可在蛋白水溶性方面有顯著之提升效果，磷酸化修飾對於米蛋白其低水溶性 (2.5%, pH 7) 之性質並未給予明顯之改善。 分析研究米蛋白分子結構內部之疏水性與雙硫鍵兩大作用力，對於磷酸化修飾提升米蛋白水溶性之效果影響，試驗結果顯示以尿素減緩米蛋白結構內部之疏水性作用力，對於後續磷酸化米蛋白之磷酸化程度 (22.0 %) 與水溶性 (3.6%, pH 7) 方面並無明顯之提升效果。相較之下，預先將米蛋白結構內部之雙硫鍵進行部分之還原斷裂，則可顯著增加後續磷酸化米蛋白之磷酸化程度 (31.3%) 與水溶性 (8.3%, pH 7) ，上述結果顯示分子結構內大量之雙硫鍵應為導致米蛋白在經過磷酸化修飾後水溶性無法提升之原因。 另一方面，相較於對蛋白水溶性無明顯之改善效果，磷酸化修飾可顯著提升米蛋白於磷酸鹽溶液中之溶鈣能力 (由90.4 mg/g提升至147.2 mg/g) 。本研究以同樣具有優異溶鈣能力的酪蛋白磷酸胜肽 (casein phosphopeptide, CPP) 作為仿效對象，初步比較以先磷酸化後水解 (Type 1) 、先水解後磷酸化米蛋白 (Type 2) 兩種製備途徑製備含有米蛋白磷酸胜肽 (rice phosphopeptide, RPP) 之米蛋白水解物。試驗結果顯示Type 1磷酸化米胜肽於低水解度之情況下 (以鹼性蛋白酶處理時水解度為6.7%) 可因磷酸化修飾呈現較好之溶鈣能力，過度將Type 1磷酸化米胜肽進行水解，則可能使其對於鈣離子之螯合結構受到破壞，降低Type 1磷酸化米胜肽之溶鈣能力。對於Type 2磷酸化米胜肽之製備方面，預先以蛋白酶水解米蛋白之後有助於提升後續米蛋白樣品進行磷酸化修飾時之磷酸化程度，例如以水解度3.4%之米胜肽進行磷酸化修飾時，其磷酸化程度 (50.4%) 可明顯高於以未水解之米蛋白進行磷酸化時之磷酸化程度 (20.6) 。而相較於Type 1磷酸化米胜肽僅能於低水解程度之情況下相比於未磷酸化米胜肽呈現較好之溶鈣能力，Type 2磷酸化米胜肽於水解程度較高之條件時，其相比於未磷酸化米胜肽仍可因較高之磷酸化程度而展現較佳之溶鈣能力。然而，Type 2磷酸化米胜肽在製備上被發現有樣品回收不易之缺點，將米蛋白先行水解成米胜肽在將其進行磷酸化修飾，米胜肽容易因分子量較小而於磷酸化反應後之透析脫鹽步驟中與STMP等無機鹽類一併流失，造成後續分析Type 2磷酸化米胜肽其磷酸化程度與溶鈣能力有低估之跡象，此結果顯示Type 2磷酸化米胜肽之製備方式可能不利於應用至工業上之生產，相較之下，先磷酸化後水解米蛋白之方式 (Type 1) 則可由於樣品流失量小，在工業製程上有操作簡易且可從中進一步精緻純化出具良好溶鈣能力之米蛋白磷酸胜肽之優點。 本研究中應證米蛋白可經由磷酸化修飾賦予更多磷酸基團，且對於磷酸化米蛋白亦表現出更好的鈣離子螯合能力而言，發現其具有作為原料製得類似酪蛋白磷酸胜肽之應用潛力。基於目前產業上已有相對較成熟之米蛋白生產製程，實際從製程中導入磷酸化步驟在操作上並無太大之困難，因此利用磷酸化修飾在未來將可望製得有別於一般缺乏可利用功能特性之米蛋白配料，進一步拓展米蛋白在食品產業上之應用價值。

The present study tried to phosphorylate rice protein (RP), a known insoluble food ingredient, and to improve its solubility and calcium-solubilizing capacity (CSC). RP was allowed to react with an approved non-toxic food additive; sodium trimetaphosphate (STMP) in an alkaline aqueous solution (pH 11.5, 35°C). The results indicated that 20.6% of the RP seryl residues were phosphorylated. Interestingly, RP did not show a considerable increase in solubility (2.5%, pH 7) after phosphorylation as compared to those studies that have confirmed the protein solubility improvement via chemical phosphorylation. The involvement of hydrophobic interactions and disulfide bonds in phosphorylated RP solubility was further evaluated. The phosphorylation of RP in the presence of urea as a chaotropic agent for weakening the hydrophobic effect resulted in 22.0% phosphoseryl residues but still did not increase RP solubility. The reduction of RP disulfide bonds prior to phosphorylation resulted in 31.3% phosphoseryl residues and increased RP solubility to 8.3% at pH 7, indicating that disulfide bonds within RP could be responsible for the failure to increase its solubility after phosphorylation. On the other hand, it was found that the phosphorylation could enhance the CSC of RP from 90.4 mg/g to 147.2 mg/g. To follow the example of casein phosphopeptide (CPP), a hydrolysate that features an extraordinary CSC, the present study has demonstrated two modification procedures for the preparation of rice phosphopeptides (RPP) contained phosphorylated rice protein hydrolysates (PRPH), which included chemical phosphorylation followed by the proteolysis of RP (Type 1 PRPH) or inversely the procedure of proteolysis before the phosphorylation of RP (Type 2 PRPH). In terms of Type 1 PRPH, the results showed that the superior CSC of Type 1 PRPH could be exhibited to be below the range of the degree of hydrolysis (DH) after proteolysis (e.g., DH below 6.7% for alcalase treatment). The phosphorylated chelating structure of Type 1 PRPH might be interrupted by excessive hydrolysis and caused a decrease in its CSC. In the case of Type 2 PRPH, pre-hydrolysis of RP with alcalase prior to phosphorylation consequently introduced more phosphoryl residues (e.g., 50.4% at a DH of 3.4%) compared to that of Type 1 PRPH (20.6%). Type 2 PRPH could exhibit a superior CSC at a higher DH, whereas the Type 1 PRPH only showed a better CSC than that of native RP hydrolysates at a DH below 6.7%. However, the pre-hydrolysis of RP was found to add difficulty to sample recovery in Type 2 PRPH preparation process. The low MW peptides of Type 2 PRPH could be easily lost during the desalting procedure after phosphorylation. Therefore, considering the economics and ease of operation, the pre-phosphorylation-post-hydrolysis process (Type 1) might be the better procedure for preparing CSC-fortified rice protein (RP) peptides. In conclusion, the present study has verified that the chemical phosphorylation could introduce more negatively charged phosphate groups and thus enhance the CSC of RP. These findings have shown the potential use of RP for preparing the CPP-like bioactive peptides. The CSC-fortified RP might be utilized as promising calcium-absorption enhancers and bioactive agents in future functional food ingredients.

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