Determination of citrate using immobilized citrate lyase and malate dehydrogenase in a flow system and its application to analyze citrate content of beverages

Abstract

The quantity of citrate was determined using apparatus comprised of a reactor with immobilized citrate lyase and malate dehydrogenase in the flow line. The decrease of β-nicotinamide adenine dinucleotide, reduced form by enzymatic reactions was spectropho-tometrically detected as a negative peak. The maximum peak area was obtained at pH 7.6 when the pH of the carrier consisting of phosphate buffer ranged from 6.6 to 8.0. Various buffer types were also examined as carrier media at pH 7.6 and phosphate buffer showed the maximum peak area. When the carrier composed of phosphate buffer (0.1 M, pH 7.6) was used, the calibration curve for citrate was linear in the range 0.5-100 μM(r = 1.000). The detection limit (S/N = 3) was 0.2 μM. The relative standard deviation of the peak area at 10 μM was 2.2% (n = 7). This method was applied to analyze citrate in beverages, and citrate content determined by this method agreed with that determined by a commercially available test kit.