Microassay for the quantitation of protein precipitable polyphenols: use of bovine serum albumin–benzidine conjugate as a protein probe

A simple, sensitive and indirect spectrophotometric method for the determination of protein precipitable polyphenols (tannins) has been developed, based on the ability of the polyphenols to precipitate the synthetic, brown coloured azo-protein, bovine serum albumin–benzidine conjugate (BSA–benzidine, mole ratio 1:7), which shows an absorption maxima at 405 nm. The amount of unprecipitated BSA–benzidine is measured directly at 405 nm, which is inversely related to the polyphenol concentration. Tannic acid was used as a reference standard. The microassay was performed in citrate/phosphate buffer (0.1 m), pH 4.8. The method was found to be linear in the range of 5–150 μg (3–88 nmol) of tannic acid (y=1.0+(−0.007)x; r=−0.989). Spiking studies carried out with various levels of tannic acid (0.01, 0.1 and 1.0%) indicated a recovery in the range of 94–101% and 94–98% in rice and sorghum samples, respectively. Free phenolics, when added in the range of 50–150 μg (catechin, chlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) had no influence on the protein precipitation in the microassay. Also spectral analysis of free phenolics and acid-methanolic sorghum extracts showed no interference in the present method. The conjugate was found to be stable over a period of 24 weeks in a freeze-dried condition and at 4°C, with <5% deterioration in aqueous condition. The microassay method developed has been used for the quantitation of protein precipitable polyphenols in various sorghum (Sorghum bicolor L. Moench) genotypes and compared with the widely used Folin–Denis chemical method of analysis.