The extraction of betalains was achieved using trichloroacetic
acid (4% TCA in water) solution. For the sample preparation,
4.0 mL of milk was poured into a 15 mL glass tube; 4 mL of TCA
solution were added, and the mixture was vortexed for 3 min
and centrifuged (Hettich-EBA 20, Germany) for 10 min at
6000 rpm at 25 C. The clear liquid phase was filtered through a
0.45-lm PTFE filter, and 15 lL of filtrate was directly injected into
the Agilent 1260 HPLC system (Agilent Technologies Inc., Folsom,
CA). The recovery of betalains for this extraction was 95.94%. All
samples were prepared in a dark room to eliminate the effects of
light. Degradation of betalains in milk during heating was followed
by determining the content of betalains (betanin + isobetanin) by
HPLC method of