Technical note: optimization of lactose quantification based on coupled enzymatic reactions.

Area of Science:

• Biochemistry
• Analytical Chemistry

Background:

• Accurate lactose quantification is crucial for dairy product quality control and for individuals with lactose intolerance.
• Existing methods for lactose determination can be costly or time-consuming.

Purpose of the Study:

• To develop and validate a novel colorimetric microplate assay for the sensitive and reproducible quantification of lactose in dairy products.

Main Methods:

• The assay utilizes a coupled enzymatic reaction involving β-galactosidase, glucose oxidase, and horseradish peroxidase with Amplex red as the detection probe.
• Optimization of reaction conditions including enzyme concentrations, Amplex red concentration, incubation time, and temperature was performed.
• Assay performance was evaluated for linearity, limit of detection (LOD), limit of quantification (LOQ), and compared against a commercial enzymatic kit.

Main Results:

• The developed assay demonstrated good linearity between 0.1 and 0.5 mmol/L lactose.
• The limit of detection was 0.0433 mmol/L and the limit of quantification was 0.1313 mmol/L.
• Optimized assay conditions showed high sensitivity and reproducibility, with intra-assay variation <10% and inter-assay variation <7.6%, correlating well with a commercial kit.

Conclusions:

• The developed microplate assay provides a sensitive, reproducible, and cost-effective method for routine lactose determination in dairy products.
• Its advantages include a relatively short reaction time and high accuracy, making it suitable for widespread adoption.