2.9. Quantification of Sialic Acid

The thiobarbituric acid assay was applied to quantify the sialic acid content [29]. Samples were hydrolyzed with 8 N HCl at 80 °C for 2 h, then neutralized with 8 N NaOH. The hydrolyzed samples were mixed with 0.2 M NaIO4 (dissolved in 9 M H3PO4) and left to react at room temperature for 20 min to oxidize sialic acid. The reaction was stopped with an arsenite solution. The thiobarbituric acid reagent was added and heated at 100 °C for 15 min to form the sialic acid–thiobarbituric acid complex. After cooling and phase separation using cyclohexanone and centrifugation, absorbance at 549 nm was measured to determine sialic acid concentrations according to the standard curve with N-acetylneuraminic acid.