4.5. Lipid Oxidation Measurements

Lipid oxidation was assessed by measuring both primary (e.g., lipid hydroperoxides using ferrous oxidation-xylenol orange (FOX) reagent [44]; and secondary products (e.g., malonaldehyde using (TBARS) reagent [45,46]. Four replicates of meats from broilers fed with each diet were measured in triplicate in the FOX and TBARS, analyses, respectively. FOX and TBARS measurements for long term storage were done at monthly intervals from the beginning (0 months equated to nonfrozen) to the end of a 6-month storage period.

The FOX assay was used to measure concentration of lipid peroxides (ROOH) in broiler meats, a primary lipid oxidation product that oxidizes ferrous to ferric ion. To calibrate lipid peroxides, a hydrogen peroxide solution was used generate a calibration standard curve within a concentration range of 0–120 μM. We used a molar extinction coefficient of hydrogen peroxide at 240 nm of 43.6 M⁻¹cm⁻¹ [47]. One gram of minced meat sample was homogenized in 4 mL of propanol using a Polytron PT 3000 (Kinematica^®, Bohemia, NY, USA). Samples were centrifuged for 10 min at 12,000× g in a Model 5402 centrifuge (Eppendorf) at room temperature (24 °C). Fresh pre-FOX reagent was prepared from xylenol orange (1 mM; Sigma) and 2.5 mM ferrous ammonium (Sigma) in 250 mM sodium sulfate (Fisher Scientific). The FOX reagent was prepared from a mixture of pre-FOX solution and methanol containing 4.4 mM BHT (1:9 v/v). Samples were reacted with fresh FOX reagent, and left for 30 min before absorbance was determined at 560 nm using a UV-160 spectrophotometer (Shimadzu) and quartz cuvettes to avoid disturbances caused by plastic deterioration. Four replicates of minced samples from broilers fed with each diet were assayed in triplicate. Absorbance at 560 nm was converted to lipid peroxide concentration [ROOH] (μM) according to the standard equation below:

where:

Abs_560nm = absorbance measured at 560 nm

R² = coefficient of determination (e.g., the square of Pearson product moment calculation coefficient from known hydrogen peroxide concentrations and corresponding absorbance measured at 560 nm)

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.