title: Technical note: optimization of lactose quantification based on coupled enzymatic reactions.
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 β-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 β-galactosidase/mL, 5 U of glucose oxidase/mL, 1 U of horseradish peroxidase/mL, and 100μmol of Amplex red/L for 1h at 37°C 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.
authors.affiliation: School of Food Science, Washington State University, Pullman 99164.
authors.forename: Luis
authors.initials: L
authors.lastname: Condezo-Hoyos
authors.affiliation: School of Food Science, Washington State University, Pullman 99164.
authors.forename: Indira P
authors.initials: IP
authors.lastname: Mohanty
authors.affiliation: School of Food Science, Washington State University, Pullman 99164. Electronic address: giuliana.noratto@wsu.edu.
authors.forename: Giuliana D
authors.initials: GD
authors.lastname: Noratto
mesh_terms: D003124:Colorimetry; D003611:Dairy Products / Q000032:analysis*; D005949:Glucose Oxidase / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D007785:Lactose / Q000032:analysis*; D057230:Limit of Detection; D015203:Reproducibility of Results; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D001616:beta-Galactosidase; D007785:Lactose
keywords: enzymatic reaction; lactose; β-galactosidase