4.5.2. Thiobarbituric Acid Reactive Substances (TBARS) Assay

This assay measures the TBA-reactive malondialdehyde (MDA), a secondary lipid oxidation chromogen with a maximal absorbance wavelength of 532 nm. The assay was calibrated using a standard of 1,1,3,3-tetraethoxypropane (TEP; Sigma) in aqueous solution. Standard curves for TBARS were plotted in a straight line over the range of 0–10 μM. The TBARS concentration (μM) was calculated as:

where:

Abs_532nm = Absorbance measured at 532 nm

R² = Coefficient of determination (e.g., the square of Pearson product moment correlation coefficient through data points in known-TEP concentrations and corresponding absorbance measured at 532 nm)

For TBARS analysis in meats, minced samples (2.5 g) were homogenized with 25 mL of 1.6% phosphoric acid (Fisher Scientific) in 20% trichloroacetic acid (TCA; Fisher Scientific) using a Polytron PT 3000 (Kinematica^®). Filtrated samples were vortex-mixed with fresh TBA reagent (0.5% 2-thiobarbituric acid and 0.02% butylated hydroxytoluene in 0.025 M sodium hydroxide). The tubes were heated in boiling water bath for 15 min, cooled to room temperature and then read at an absorbance of 532 nm. Four replicates of meat samples from broilers fed with each diet were assayed in triplicate. Absorbance was converted into a TBA value (mg MDA/kg meat) using the following equations [48]:

where:

Abs_532nm(Sample) = absorbance of sample at 532 nm

K = constant coefficient

Standard = number of moles TBAR standard (μmol in 2 mL)

Abs_532nm(Standard) = absorbance of standard at 532 nm

MW = molecular weight (72.063 g/mol)

E = sample equivalent

A standard containing 2 × 10⁻⁸ mol of TEP/2mL (10 μM) was 1.074. The sample equivalent (E) was 0.1 g, in which 2.5 g of sample was diluted to 50.0 mL and 2.0 mL was analyzed. These constants resulted in a K value of 13.4. The TBA values were calculated by multiplying the absorbance by 13.4.