Abstract

Nutritional folate deficiencies in Southern African communities necessitated mandatory fortification. Current microbiological assays (MA) used to measure food folates, essential for quality control and regulatory purposes, are time-consuming. This study describes an alternative extraction and detection method for folates in dairy products and Propionibacterium freudenreichii cultures. Folates were extracted by heating with a phosphate buffer (pH 6.0). Polyglutamates were deconjugated with chicken pancreas and hog kidney deconjugases. Samples were purified using strong anion exchange solid phase extraction. Reversed-phase high-performance liquid chromatography (HPLC) using an acetonitrile-phosphate buffer (pH 2.2) gradient effectively separated four vitamers. Fluorescence (tetrahydrofolate (THF), 5-CH₃-THF and 5-CHO-THF), and UV detection (folic acid) were used, calibration curves were linear (R²>0.0997), and detection and quantification limits were 0.0006 to 0.015 and 0.002 to 0.05 mg/ml, respectively. Accuracy was 80 to 108% and intra- and inter-day precision [%relative standard deviation (%RSD)] were lower than 4%. The method, validated against the standard MA, is a selective, sensitive, reliable and rapid alternative.

Key words: Propionibacterium freudenreichii, microbiological folate assay, high-performance liquid chromatography (HPLC), folate.

Abbreviation

AOAC, Association of Official Analytical Chemists; HPLC, high-performance liquid chromatography; MA, microbiological assay; NTDs, neural tube defects; PAB, propionibacteria; PGA, pteroylglutamic acid; THF, tetrahydrofolate;RSD, relative standard deviation.