To investigate the spoilage effect of the purified peptidase on
UHT milk, UHT milk containing 0.1% (w/v) sodium azide was
supplemented with various enzyme activities from
0.001 pkat mL1 to 10 pkat mL1 and stored at 20 C in the dark
for four weeks. The peptidase activity was determined with milk
as a substrate and subsequent derivatisation of the newlyformed
amino groups, as described above (see Section 2.7.2).
To show the coagulation of the milk protein, 15 mL samples were
centrifuged (8000 g, 4 C, 15 min), and the resulting pellet was
lyophilised, weighed and expressed as % (w/v). The pH 4.6-
soluble peptides released (free peptides) were measured after
derivatisation with o-phthaldialdehyde (see Section 2.7.2). For
this, the milk samples were brought to pH 4.6 with 0.1 M HCl and
centrifuged (20,000 g, 4 C, 10 min). SDS-PAGE analysis of the
milk samples at the end of storage was performed as described
in Section 2.4.