Measurable human milk folate is increased by treatment with α-amylase and protease in addition to folate conjugase

The application of trienzyme treatment prior to microbiological assay using Lactobacillus casei for the measurement of folate in human milk was evaluated. Trienzyme treatment consisted of α-amylase, protease, and folate conjugase. Human milk samples (n = 84) were heat treated for 5 min at 100 °C in 0.1 m potassium phosphate buffer containing 0.05 mol liter−1 ascorbic acid, pH 4.1, then incubated with the enzymes by stepwise addition. Following trienzyme treatment, folate was measured microbiologically using a 96-well microplate method. The results show that treatment at 37 °C with α-amylase for 4 h followed by protease for 8 h, and finally by folate conjugase for 3 h significantly increased the measurable folate in human milk by an average of 85% compared with values after folate conjugase treatment alone (p < 0.001). Neither the pH of the extraction buffer, the source of folate conjugase, nor the method/length of heat treatment of the samples were significant variables influencing the values of human milk folate. Data indicate that folate in human milk can be underestimated unless samples are treated with α-amylase, protease, and folate conjugase prior to microbiological assay using L. casei.