*3.6. Cathepsin D Activity*

Cathepsin D, a lysosomal aspartic proteinase, is found in somatic cells and acts on κ-, αs1- and β-casein similarly to how it acts on chymosin [36]. The assay used for its determination was based on the presence of peak P (Figure 3), which is the product that resulted from the action of cathepsin D on the substrate. The quantitative determination (Table 4) was based on the chromatographic area of this peak. Peak 1 is another aspartic proteinase, whereas peaks 2 and 3 are possible cysteine- proteinases [24]. Cathepsin D was determined in the acid whey fraction of the feed milks, MF permeates and retentates,

and there was no significant difference among them, since whey is easily filtered through a membrane of 1.4 μm pore size. However, it is noteworthy that although somatic cells were completely removed from permeates, the acid proteinases associated with them were present. It seems that the shear forces induced by circulation removes cathepsin from somatic cells and, thus, it remains in the permeate. The higher flux in the case of bovine milk might cause higher dissociation from somatic cells and, therefore, the product resulted from cathepsin activity was higher than that of ovine milk.

**Figure 3.** Proteinase activities in the acid whey fractions of feed bovine milk (B), bovine permeate (BP) and bovine retentate (BR), or feed ovine milk (O), ovine permeate (OP) and ovine retentate (OR), after incubation with the substrate Pro-Thr-Glu-Phe-[p-nitro-Phe]-Arg-Leu at pH 3.2 for 12 h at 37 ◦C. S: residual substrate; P: cathepsin D-like product; peaks 1, 2 and 3: other proteinases.
