Determination of Total Flavonoid Content in Kefir

The total flavonoid contents (TFCs) of kefir water samples were measured following a method described by Saeed et al. [36 (link)] with some adjustments. Nine microlitres of 5% NaNO₃ was mixed with 150 µL of kefir water samples (50 mg/mL) or catechin standard and incubated for 6 min in the dark. Then, 9 µL of 10% AlCl₃ was added and incubated for 5 min. After that, 60 µL of 1M NaOH and 72 µL of distilled water were added and mixed well by vortexing. The absorbance was then read at 430 nm using an ELISA plate reader (Bio-Tek Instruments, Winooski, VT, USA). The TFCs of the samples were expressed as µg catechin equivalents (µg CAT/µL). This experiment was performed in triplicates.