Use of a microbiological assay with tri-enzyme extraction for measurement of pre-fortification levels of folates in enriched cereal-grain products

January 1, 1998 was the effective date for PDA regulations that mandated the fortification in the USA of a wide range of enriched cereal-grain products with folic acid at levels specified in federal regulations. Because data on prefortification levels of folate in such products are limited, we measured folate in 56 enriched foods, including enriched breads and rolls, flours, corn grits and meals, rices, and macaroni and noodle products. Folate was measured by a modification of the Association of Official Analytical Chemistsʹ microbiological method 992.05 using Lactobacillus casei. Foods were composited, suspended in 0. 1 m phosphate buffer containing 1% ascorbic acid (pH 7.8), autoclaved and cooled. Chicken pancreas conjugase was added and the suspensions were incubated for 16h at 37 °C. Values for folate in enriched products were (μg/lOOg): bread and rolls, 24–40; flours, 19–24; corn grits and meals, 22–32; macaroni and noodle products, 27–42; rice, 19–32. Because the single-enzyme method is usually insufficient to liberate food-bound folates, suspensions of foods were also incubated with α-amylase and conjugase followed by treatment with protease to determine the effects of the tri-enzyme digestion on release of folates. For many foods, total folate was 20–30% higher after the tri-enzyme digestion than after incubation with conjugase alone. The modifications of AOAC method 992.05 described here provide a microbiological assay method for the determination of folates in cereal-grain products that may be appropriate for collaborative testing.