91A-4 |
Optimization of microbiological assay of folic acid and determination of folate content in spinach |
S. PANDRANGI and L. F. LaBorde. Department of Food Science, Pennsylvania State University, 119 Borland Lab, University Park, PA 16802 A widely used microbiological method for determination of total folate in plant foods uses the growth response of folate-dependent L. rhamnosus (ATCC 7469) in extracts that have been enzymatically treated to release the bound vitamin. However, inconsistent growth of the organism hampers assay repeatability. Folate analysis may be improved by optimizing growth of L. rhamnosus as well as conditions for enzymatic extraction of folate. The objectives of this study were to improve the folate analysis method by altering the growth atmosphere of L. rhamnosus and to then determine optimal extract pH and time of enzyme treatments for spinach. L. rhamnosus was serially transferred in aerobic and microaerophilic environments and growth was compared. Assays used the 96-well microplate technique with 5-formyl tetrahydrofolate (standard) and assayed plasma (internal standard). Assay validity was confirmed using a NIST standard. To release bound folate, alpha-amylase, protease and conjugase were added to extracts. Optimum pH for amylase and protease activity was tested over a pH range of 3-7. Treated extracts were then incubated with conjugase before determining the folate content. Optimum incubation time was determined by incubating spinach extracts with amylase / protease at 37ºC in buffer at the optimum pH (amylase, pH 3.0; protease, pH 4.0) for 0-12hrs. Samples were removed every 3hrs, heat-treated, and then conjugase-treated prior to microbiological assay. L. rhamnosus growth was more consistent when cultured in microaerophilic atmosphere. Addition of protease at pH 4 significantly increased measured folate although amylase had no effect. Optimum incubation time for protease was 8hr. Therefore, a dual enzyme treatment (protease and conjugase) is sufficient to determine folate in spinach. Consistent growth of L. rhamnosus in a microaerophilic atmosphere will offer researchers a more precise method for measuring folate in food products. Eliminating the amylase incubation step reduces the assay time.
Session 91A, Food Chemistry: Enzymes, vitamins and plant pigments
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