2.8. Tiobarbituric Acid Reactive Substances (TBARS) in Milk

Analysis of TBARS was performed as described by Vyncke [31], with few adaptations. A 500 µL aliquot of milk was transferred into a 15-mL falcon tube (Cellstar, Greiner Bio-One, Americana, SP, Brazil) containing 2.0 mL of thiobarbituric acid solution (TBA 1%, TCA 15%, and 562.5 mM HCl). The samples were heated in a boiling water bath (100 °C) for 15 min, cooled in ice water for 5 min and then centrifuged at 1080× g for 10 min. The supernatant was transferred to a cuvette for later reading at 532nm on a UV–vis spectrophotometer (Spectrum SP2000, Thermo Fisher, Waltham, MA, USA). The results were expressed in concentration (mmol/kg fat).