Optimization of Enzyme Extractions for Total Folate in Cereals Using Response Surface Methodology

The trienzyme digestion including protease, alpha-amylase, and conjugase gamma-glutamyl hydrolase) for the extraction of total folate from cereal grain products used in AOAC Official Method 2004.05 was optimized using response surface methodology. Digestion times of alpha-amylase (p < 0.05) and conjugase (p < 0.01) showed significant effects on the response (total folate). Digestion time for Pronase digestion was not significant (p > 0.1). Ridge analysis showed that total times required for the trienzyme digestions ranged from 6.1 h for buckwheat to 8.7 h for CAM 121 (wholemeal flour). The predicted maximal value for CAM 121 (51 mu g/100 g) was close to the certified value (50 +/- 7 mu g/100 g). The optimized extractions for the four cereals suggest that a generalized trienzyme extraction for cereals of 1, 2.5, and 6 h for Pronase, alpha-amylase, and conjugase, respectively, could be used to approximate total folate in cereals and cereal products. These conditions provide data comparable to AOAC Official Method 2004.05 for the cereals included in this study and would be suitable to approximate total folate in most cereals and cereal products. Compared to the official method, the shortened digestion provides cost savings and minimizes the folate loss possible with prolonged digestions.