As a necessary step for the precipitation of proteins, three samples
containing 10 g of yogurt or milk drink was blended with
0.5 mL of K4Fe(CN)63H2O (0.35 mol L1) and 0.5 mL ZnSO47H2O
(1 mol L1) in a 50 mL Falcon tube. The solution was agitated with
a tube shaker (PA162 PACHANE) immediately after each addition
and left to stand for 10 min. After this time interval, 10 mL of solution
methanol:ammonium hydroxide (2 mol L1) (80:20) was
added, the solution was stirred and centrifuged using a centrifuge
(Cientec CT-5000DR) at 6000 rpm and 4 C for 10 min. The centrifugal
step was repeated twice more, each time with an addition
of 10 mL methanol:ammonium hydroxide, in order to extract the
maximum possible colors from the samples.
After each centrifugation, the fractions were collected and
transferred to the same 50 mL volumetric flask and its volume
was topped up with purified water. An aliquot of the final solution
(1 mL) was filtered through 0.45 lm membrane filters from
Millipore (Brazil) using a plastic syringe with a capacity of 10 mL
of Plascalpand and transferred to a vial with capacity of 1.5 mL of
Uniglas and then injected into the chromatograph. The concentration
of each sample was determined by HPLC–PAD.