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AACC International Approved Methods -AACC Method 86-47.01. Total Folate in Cereal Products—Microbiological Assay Using Trienzyme Extraction

AACC International Approved Methods


AACCI Method 86-47.01
Total Folate in Cereal Products—Microbiological Assay Using Trienzyme Extraction


This is a microbiological method employing the organism Lactobacillus casei subsp. rhamnosus (ATCC no. 7469) to determine the amount of folate present in foods and in vitamin concentrates. This method is semiautomated through the use of automated dilution and turbidity reading instruments or, optionally, the 96-well microtiter plate and reader system. See Note 1. Samples, with water added, are autoclaved to break up particles, gelatinize starch, and denature proteins to enhance enzymatic attack and make folate more extractable. Folate (pteroylglutamic acid in various forms) occurs naturally in foods bound to glutamic acid residues of varying chain lengths. Most naturally occurring folates cannot be utilized by the assay organism. Folic acid (pteroylglutamic acid) is extracted from the sample using a triple enzyme system. A protease and an amylase are used to digest the food matrix and aid in the release of folates. Desiccated chicken pancreas conjugase is used to hydrolyze folylpolyglutamates to folyldiglutamates, which, along with folic acid, can be utilized by the assay organism. The freed folates are extracted and diluted with basal medium containing all required growth nutrients except folate, and the turbidity of the L. casei subsp. rhamnosus growth response for the samples is compared quantitatively to that of known standard solutions. The method is applicable to cereal grains and cereal grain foods containing added folate (folic acid) or naturally occurring folates with levels from 5 µg/100 g to 100% folate.