2.5.1. Cathepsin D Activity

Cathepsin D activity was determined by the HPLC method proposed by O'Drissol et al. [22] and modified by Hurley et al. [23] using the synthetic peptide Pro-Thr—Glu-Phe-[p-nitro-Phe]-Arg-Leu (Bachem, Switzerland) as substrate. The Waters HPLC system described previously was used. Sample preparation, HPLC conditions and results interpretation were according to Moatsou et al. [24].

#### 2.5.2. Alkaline Phosphatase Activity

Alkaline Phosphatase (ALP) activity was determined according to the spectrophotometric method of the International Dairy Federation Standard [25]. The milk sample was diluted with a buffer, pH 10.6, containing disodium phenyl phosphate (substrate) and was incubated at 37 ◦C for 1 h. After incubation, any active ALP had liberated phenol from the substrate. The phenol was detected by adding 2,6-dibromoquinonechlorimide to detect blue color (dibromoindophenol), which was measured on a spectrophotometer (model Lambda 20, Perkin Elmer, Waltham, MA, USA) at 610 nm. The ALP activity was expressed in μg of phenol per mL of milk.
