Technical note: optimization of lactose quantification based on coupled enzymatic reactions.
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abstract
A colorimetric microplate-adapted lactose assay was developed to quantify lactose in dairy products. The assay was based on the coupled enzymatic reaction of -galactosidase-glucose oxidase-horseradish peroxidase using Amplex red as detection probe. The assay showed good linearity in the range of 0.1 to 0.5mmol of lactose/L, with a limit of detection of 0.0433mmol/L and a limit of quantification of 0.1313mmol/L. The lactose assay at optimized conditions (5 U of -galactosidase/mL, 5 U of glucose oxidase/mL, 1 U of horseradish peroxidase/mL, and 100mol of Amplex red/L for 1h at 37C in the dark) showed good correlation with a commercial lactose enzymatic kit with intraassay variation below 10% and interassay variations below 7.6%. The developed lactose microplate assay can be adopted as routine analysis for lactose determination in dairy products due to its relatively low cost compared with a commercial kit, relatively short reaction time, and high sensitivity and reproducibility.
