以液相層析串聯高解析質譜同步分析豆漿中營養成分及梅納反應產物

Abstract

豆漿具有完整的必需胺基酸且富含大豆異黃酮為眾人重視，但目前並無良好的保存期限評估；過短的保存期限造成食物資源的浪費，過長的保存期限或不當的貯存環境則使功效成分降解、影響營養品質。貯存過程中，豆漿中胺基酸的胺基可能與醣的羰基進行梅納反應，代表了胺基酸的降解及潛在的有害物質生成，其中以帶有ε-胺基的離胺酸最易發生反應產生糠胺酸(furosine)、carboxymethyl-lysine (CML)及carboxyethyl-lysine (CEL)。為了全面研究豆漿中品質指標，本研究開發無衍生化，直接以 HPLC-ESI-Q-Orbitrap 同步分析20種胺基酸與3種梅納反應產物之分析平台。利用4-胺基丁酸做為內標及胺基酸之半定量，偵測極限及定量極限分別為0.4及5.0 µM；梅納反應產物利用糠胺酸進行定量，偵測極限及定量極限分別為1.1及5.0 µM。基質匹配檢量線之相關係數皆大於 0.99，且準確度及精密度皆通過食品化學檢驗之確效規範。另以HPLC-DAD分析大豆異黃酮，與胺基酸及梅納反應產物共同作為豆漿於貯存期間品質評估之指標。一般及加速貯存試驗將豆漿分別貯存於5、25、35、45℃ 環境，分析不同時間點下，豆漿中胺基酸、梅納反應產物及大豆異黃酮之含量，藉此了解豆漿在貯存期間的營養品質變化。結果顯示豆漿中離胺酸之含量隨貯存期間先上升而後下降，接著為糠胺酸含量上升而後下降，並伴隨 CML 的出現，代表豆漿於貯存期間會發生梅納反應、導致營養品質之下降。單變量反應動力學結果顯示三項品質指標於豆漿貯存前期多呈現一級反應，胺基酸之 Q10 為 0.3-2.3，活化能為 9.7-62.9 kJ/mol；梅納反應產物之 Q10 為 1.7-2.1，活化能為 41.8-55.5 kJ/mol；大豆異黃酮之 Q10 為 1.2-2.8，活化能為 13.8-78.3 kJ/mol。本研究建立無衍生化，直接以 HPLC-ESI-Q-Orbitrap 同步分析胺基酸及梅納反應產物，用於未來評估豆漿保存期限之研究。並期望未來有更多研究豆漿中不同品質指標於貯存期間之反應動力學，給予豆漿保存期限更全面之評估。

Soymilk is a highly nutritional food because of it contains all essential amino acids and riches in isoflavones. However, there is no adequate way to evaluate the shelf life of soymilk. A short shelf life causes the waste of food, while an inappropriate shelf life leading to the degradation of the active components; thereby influences the nutritional quality of food. Maillard reaction, reactive amino group of the amino acid reacts with the carbonyl group of the sugar, leads to the degradation of amino acids and may generates hazardous substances in food during the storage. Among the amino acids, the ε-amine in lysine is prone to undergo Maillard reaction, which generates furosine, N(ε)-carboxymethyl-lysine (CML), and N(ε)-carboxyethyl-lysine (CEL). Nevertheless, there are less analytical platform focus on the simultaneously analyze the nutritional factor and hazard substances. To comprehensive evaluate the quality of soymilk, a directed method without any derivatization was developed via HPLC-ESI-Q-Orbitrap, which could simultaneously analyze 20 amino acids and 3 Maillard reaction products. The limit of detection (LOD) and limit of quantification (LOQ) in amino acids was 0.4 and 5.0 µM, respectively; 4-aminobutanoic acid was used as the internal standard. For Maillard reaction products, furosine was used as the internal standard, and the LOD and LOQ was 1.1 and 5.0 µM, respectively. The matrix-match calibration curve is good for quantification (R > 0.99). The method is validated, according to the accuracy and precision is agreed with the validation requirements of food safety tests. In addition, the isoflavones of soymilk were analyzed using HPLC-DAD. The long-term and accelerated shelf-life test were performed in 5, 25, 35, or 45℃ to evaluate the variation in nutritional quality of soymilk. The quality indicators of soymilk during the storage are isoflavones, amino acids and the Maillard reaction products. Results showed that the content of lysine was increased first, and then decrease during the storage period. While lysine was decreasing, the content of furosine was increased and the CML was generated in the same trend. Those results indicated the Maillard reaction happened during the storage of soymilk, leading to the decrease in nutritional quality. The results of univariate reaction kinetics showed that amino acids, Maillard reaction products and isoflavones followed a first-order reaction in the early stage of storage. The Q10 and activation energy of amino acids are 0.3-2.3 and 9.4-62.9 kJ/mol, respectively. The Q10 and activation energy of Maillard reaction products are 1.7-2.1 and 41.8-55.5 kJ/mol, respectively. The Q10 and activation energy of isoflavones are 1.2-2.8 and 13.8-78.3 kJ/mol, respectively. This study established a non-derivatization method for simultaneously determination of amino acids and Maillard reaction products in soymilk using HPLC-ESI-Q-Orbitrap. This analytical platform may useful on the evaluating the quality of soymilk for assessment of the shelf life.