2.3. Determination of Total Phenolic Content (TPC)

The total phenolic content (TPC) in feedstuffs were determined using the Folin–Ciocalteu method as described by Makkar et al. [17]. To this end, 200 mg of the sample was extracted twice with a 5-mL mixture of ultrapure water: acetone: formic acid (47.5:47.5:5, v:v:v). Subsequently, 20 µL of both extracts, 0.98 mL of ultrapure water, 0.5 mL of the Folin–Ciocalteu reagent, and 2.5 mL of 20% (w/v) Na₂CO₃ in ultrapure water were mixed in a 10-mL tube with a vortex stirrer for 35 min at room temperature. Finally, the absorbance was read at 725 nm using a Heλiosβ spectrophotometer (Thermo Electron Corporation, Waltham, MA, USA).

To determine the total phenolic content (TPC) in meat, the method of Vázquez-Vázquez et al. [18] was used with some modifications. Briefly, 1 g of lyophilized meat and 9 mL of milli-Q water (Ultramatic GR Wasserlab) were mixed in a 50-mL polypropylene tube and 10 mL of aqueous solution of methanol (50:50, v:v) was added. The obtained solution was vortexed for 5 min before 500 μL of Carrez I solution (Scharlau) and 500 μL of Carrez II solution were added and shaken for 1 min. Next, 5 mL of acetonitrile was added and shaken for 5 min. The samples were centrifuged at 4000 rpm for 15 min at 4 °C. The upper layer was filtered into a 15-mL polypropylene tube using a 0.22-μm polytetrafluoroethylene (PTFE) filter. Finally, 15 µL of extract, 147 µL of milli-Q water, 13 µL of Folin–Ciocalteu reagent, and 7% Na₂CO₃ were mixed in a microplate well and left to react for 30 min before the absorbance was read at 725 nm using an Epoch microplate spectrophotometer (BioTek, Winooski, VT, USA).