Improve the way folate content is measured.

As you may know, folate and folic acid are forms of a water-soluble B vitamin. Folate occurs naturally in food. Folic acid is the synthetic form of this vitamin that is found in supplements and fortified foods. Folate is necessary for the production and maintenance of new cells. This is especially important during periods of rapid cell division and growth, such as during infancy and pregnancy.

A widely used microbiological technique for determining the folate content of plant foods utilizes the growth response of folate-dependent L. rhamnosus (ATCC 7469) in extracts. These extracts have been enzymatically treated to release the bound vitamin. However, the inconsistent growth of the organism hampers the repeatability of the assay.

It might be possible to improve folate analysis by optimizing the growth of L. rhamnosus as well as the conditions for enzymatically extracting folate. Scientists at Pennsylvania State University wanted to do that by altering the growth atmosphere of L. rhamnosus and determining the optimal extract pH and time of enzyme treatments for spinach.

L. rhamnosus was serially transferred in aerobic and microaerophilic environments, as the researchers examined its growth. Assays involved the use of the 96-well microplate technique with standard 5-formyl tetrahydrofolate and assayed plasma. The researchers confirmed the validity of the assay.

To release bound folate, alpha-amylase, protease and conjugase were added to the extracts. The investigators incubated the treated extracts with conjugase before determining their folate content. Optimum incubation time was determined by incubating spinach extracts with amylase or protease at 37 C in a buffer at the optimum pH (amylase, pH 3; protease, pH 4) for up to 12 hours.

Samples were removed every three hours, heat-treated and treated with conjugase before the microbiological assay was performed. The growth of L. rhamnosus was more consistent when the organism was cultured in a microaerophilic atmosphere. Adding protease at pH 4 significantly increased the amount of measured folate, although amylase had no effect. Optimum incubation time for protease was eight hours. So, it appears that a dual enzyme treatment with protease and conjugase is sufficient to determine the folate content of spinach.

Consistent growth of L. rhamnosus in a microaerophilic atmosphere will offer researchers a more precise way to measure the amount of folate in foods. Eliminating the amylase incubation step reduces the assay time.

Further information. Srilatha Pandrangi, Department of Food Science, Pennsylvania State University, 119 Borland Lab, University Park, PA 16802; phone: [phone]; email: [e-mail].