Analytical Methods for the Evaluation of α-l -Fucosidase Activity in Sheep Milk

The lack of analytical methods for measuring the activity of highly thermolable endogenous enzymes in sheep milk is a factor that hampers the protection of typical Protected Designation of Origin (PDO) dairy products made from raw milk. In order to provide a solution, this study assesses, tests, and fully validates analytical procedures for the determination of α-L-fucosidase activity in sheep milk. While the UV–VIS method has been optimized for this matrix in order to solve clarification problems before the spectrophotometric reading, a reversed phase high-performance liquid chromatography literature method proposed for bovine milk has been successfully applied also to sheep milk. Both methods have been fully validated in terms of sensitivity, linearity, precision, and accuracy, displaying low detection and quantification limits, excellent linearity over a wide enzymatic activity interval, very good repeatability and reproducibility, and the lack of any bias. The analysis of a number of real samples of whole sheep milk has allowed the evaluation of an average value (46.78±5.49 U mL−1) and range (from 29.27±2.60 to 72.64±1.17 U mL−1) of α-L-fucosidase activity. Such activity does not seem to differ substantially from those measured for bovine milk. Finally, marked seasonal variability has been observed in this preliminary dataset.