Enzymatic reactions for the determination of sugars in food samples using the differential pH technique

Abstract

All ATP coupled reactions, when performed at neutral or moderately alkaline pH, produce an acidification of the reaction mixture. The detection of small pH changes — 0.1 mpH (1 mpH = 10⁻³ pH)—in a constant buffering capacity solution makes it possible to quantify, over a wide concentration range (1–1500 mmol L⁻¹), various analytes with very high precision and accuracy. Glucose, fructose, glycerol and gluconic acid can be analysed in less than 1 min with a single step reaction. Wine samples were analysed using the hexokinase reaction for glucose + fructose (sugars undergoing fermentation) and compared against an established method, showing excellent performance over the whole range of concentrations (R = 0.9994). Increased sensitivity in some applications can be obtained by cycling reactions, e.g. a kinase reaction followed by a phosphatase reaction, in a one step analysis, as required for lactulose assay in milk, a useful indicator of heat treatment damage. A sensitivity well below 0.1 mmol L⁻¹ in the original milk sample has been demonstrated.