Assessment of milk pasteurisation in artisanal cheeses using colorimetric analysis: A novel approach

Background Alkaline phosphatase is an indigenous enzyme of bovine milk widely used to monitor pasteurisation efficiency due to its higher thermal resistance compared with nonspore‐forming pathogens. Accurate determination of its activity is particularly relevant for artisanal cheeses, where raw milk is frequently used. Conventional colorimetric and fluorometric methods present limitations related to cost, sustainability and applicability in small‐scale production. Aim This study aimed to propose and validate a quantitative approach for alkaline phosphatase activity assessment using the CIELAB color coordinate b * and to compare three enzyme extraction methods adapted for artisanal cheese analysis. Methods Artisanal cheeses produced from raw and pasteurised milk were ripened for 70 days. Alkaline phosphatase was extracted using three protocols: Extractions I and II with 8.3% neutralised n‐butyl alcohol (with and without magnesium acetate) and Extraction III using distilled water. Enzyme activity was evaluated by absorbance at 650 nm and b * values. Method validation included linearity, limits of detection and quantification, precision, and accuracy. Statistical analyses were performed using ANOVA, MANOVA and Pearson's correlation ( P < 0.05). Major Findings Pasteurised‐milk cheeses consistently showed enzyme activity below detection limits. In raw milk cheeses, alkaline phosphatase activity decreased during maturation, independently of extraction and instrumental analysis applied. Organic solvent extractions resulted in higher activity values, whereas aqueous extraction effectively differentiated raw from pasteurised cheeses. The b * coordinate showed an inverse correlation with enzyme activity and met acceptable precision and accuracy criteria (≤ 20%). Compared with the fluorometric reference method, the approach reduced by 99.5% reagent costs per sample. Scientific and Industrial Implications This study presents a practical, sustainable and accessible method for verifying milk pasteurisation in artisanal cheeses. The use of the CIELAB b * color coordinate enables enzyme quantification without specialised equipment or toxic solvents, supporting routine quality control and regulatory inspection.