Optimization of microbiological assay of folic acid and determination of folate content in spinach

The microbiological method for the determination of folate in plant foods uses the growth response of folate-dependent Lactobacillus rhamnosus in extracts that have been enzymatically treated to release the bound vitamin. However, the use of cryoprotected cultures is hampered by low recovery of the microorganism after extended frozen storage times. In this study, growth of L. rhamnosus was enhanced using a microaerophilic enrichment procedure and optimal pH conditions and enzyme reaction times were determined for the release of bound folate in spinach. Optimum pH values for the release of bound folate in spinach samples treated with alpha-amylase or protease were 3.0 and 4.0, respectively. Although treatment with alpha-amylase had no significant (P > 0.05) effect on measured folate, addition of protease at pH 4.0 significantly (P less than or equal to 0.05) increased the release of folate at an optimum incubation time of 8 h. Therefore, a dual-enzyme treatment (protease and conjugase) is sufficient to determine folate content in spinach.