High-Performance Liquid Chromatographic Determination of Naturally Occurring Folates during Tempe Preparation

A trienzyme treatment (protease, α-amylase, and human plasma conjugase), followed by purification using SPE with SAX cartridges and reversed-phase HPLC with UV-PDA detection, was performed for determination of the distribution of various folate forms and content at various stages of tempe preparation. The major folate form in soybean identified was 5-formyl tetrahydrofolate (5-CHO-H₄folate), followed by 10-formyl tetrahydrofolate (10-CHO-PGA), and 5-methyl tetrahydrofolate (5-CH₃-H₄folate), whereas folic acid was not detected and tetrahydrofolic acid (H₄folate) was not detectable. The most predominant form in tempe was also 5-CHO-H₄folate, followed by 10-CHO-PGA, whereas the quantities of 5-CH₃-H₄folate and folic acid were negligible. Quantities and retention of folate significantly decreased during the first boiling, dehulling, soaking, and second boiling procedures, yielding folate retention of 32%. A remarkable increase in folate content was found after fermentation, 5.2-fold higher than that of the boiled soybean. This may be due to de novo formation of folate by Rhizopus oligosporus, the principal mold in tempe fermentation. HPLC results were 38−55% lower than the values obtained from the microbiological assay using Lactobacillus casei.

Keywords: Folates; tempe; trienzyme treatment; HPLC; microbiological assay