Chen Liwen, Eitenmiller Ronald R
Department of Food Science and Technology, University of Georgia, Athens, Georgia 30602, USA.
J Agric Food Chem. 2007 May 16;55(10):3884-8. doi: 10.1021/jf0634350. Epub 2007 Apr 17.
Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.
采用响应面法优化用于从蔬菜中提取叶酸的三酶消化法。三酶提取法是一种通过蛋白酶、α-淀粉酶和结合酶（γ-谷氨酰水解酶）进行的联合酶消化法，用于从食物基质中释放与碳水化合物和蛋白质结合的叶酸，以进行总叶酸分析。它是AOAC官方方法2004.05中用于测定谷物制品中总叶酸的提取方法。使用有证标准物质（CRM）485混合蔬菜来代表蔬菜基质。通过统计分析软件进行回归和岭分析。预测的二阶多项式模型是合适的（R2 = 0.947），不存在显著的失拟（p > 0.1）。蛋白酶和α-淀粉酶对提取均有显著影响。岭分析得出了最佳的三酶消化时间：链霉蛋白酶，1.5小时；α-淀粉酶，1.5小时；结合酶，3小时。在此最佳消化条件下CRM 485的实验值接近模型预测值，证实了模型的有效性和适用性。将优化后的三酶消化条件应用于五种蔬菜，得到的叶酸水平高于AOAC官方方法2004.05中使用的三酶消化参数。
