# Retention of Antioxidants from Dried Carrot Pomace in Wheat Bread
## 2 Materials and Methods
### Methods
0.1 mL of the extract was mixed with 2.4 mL of 2% HCl in 75% in a test tube. The contents were vortexed and the absorbance was measured in at the wavelength \(\lambda\) = 280 nm (TPC), \(\lambda\) = 320 nm (phenolic acids), \(\lambda\) = 360 nm (flavonols) and \(\lambda\) = 520 nm (anthocyanins). TPC was expressed in mg of catechin/100 g d.m., phenolic acids in mg of ferulic acid/100 g d.m., flavonoids in mg quercetin/100 g d.m., and anthocyanins in mg glycoside-3-cyanidin/ 100 g d.m. * Free-radical scavenging activity  
Free-radical scavenging activity of samples was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) by method of [PERSON] et al. [29]. The ethanol extract of carrot pomace (0.4 mL) was mixed with 3.6 mL of DPPH solution (0.025 g DPPH in 100 mL ethanol). In the case of bread samples, the extracts (1 mL) were mixed with 4 mL of DPPH solution (0.025 g DPPH in 100 mL ethanol). Absorbance of the reaction mixture was determined at 515 nm using the spectrophotometer Jenway (6405 UV/Vis, Jenway Ltd., Dunmow, UK). Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (10-100 mg/L; R\({}^{2}\) = 0.989) was used as the standard, and the results were expressed in mg/g Trolox equivalents. * Antiradical activity  
The radical scavenging activity was measured by the ABTS [2,2\({}^{\prime}\)-azino-bis (3-ethylbenzothazoline-6-sulfonic acid)] radical cation discoloration assay, as described by [PERSON] et al. [30]. ABTS was dissolved in water to a 7 mM concentration. ABTS radical cation (ABTS\({}^{\star\star}\)) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature (12-16 h) before use. The bleaching rate of ABTS\({}^{\star\star}\) in the presence of the sample was monitored at 734 nm using a Helios Gamma 100-240 (Thermo Fisher Scientific, Runcorn, UK) spectrophotometer. The ABTS\({}^{\star\star}\) solution was diluted in PBS buffer (pH 7.4) to give absorption value of 0.700 \(\pm\) 0.05 for the analysis of extracts. Volumes of 2.00 mL of ABTS\({}^{\star\star}\) and respective extracts in PBS buffer solution were used. The ABTS\({}^{\star\star}\) bleaching was monitored at 30 \({}^{\circ}\)C, and the discoloration after 6 min was used as the measure of anti-radical activity. Radical scavenging activity was measured as Trolox equivalent antioxidant capacity (mg of Trolox per g of sample). Trolox solutions used for calibration curve were used in the concentration range 10-100 mg/L (R\({}^{2}\) = 0.9957). * Ferric reducing antioxidant power (FRAP)  
The reducing power of samples was determined by the method of Oyaizu [31]. One milliliter of sample extract was mixed with 5 mL PBS (phosphate buffer with pH 6.6) and 5 mL of 1% potassium ferricyanide in the test tube. The mixture was stirred thoroughly and heated in water bath for 20 min at 50 \({}^{\circ}\)C. After cooling, 5 mL of 10% trichloroacetic acid was added. A volume of 5 mL of mixture was pipetted into the test tube and mixed with 5 mL of distilled water and 1 mL of 0.1% ferric chloride solution. Absorbance at 700 nm was measured using the spectrophotometer Jenway (6405 UV/VIS, Jenway Ltd., Dunmow, UK). Trolox (10-100 mg/L; R\({}^{2}\) = 0.9974) was used as the standard, and the results were expressed in mg/g Trolox equivalents. * Molybdenum reducing antioxidant power (FOMO)  
The molybdenum reducing antioxidant power of samples was determined by the method of [PERSON] et al. [32], with slight modifications. The mixture of sample (1 mL), monopotassium phosphate (2.8 mL, 0.1 M), sulfuric acid (6 mL, 1 M), ammonium heptamolybdate (0.4 mL, 0.1 M) and distilled water (0.8 mL) was incubated at 90 \({}^{\circ}\)C for 120 min, then rapidly cooled and detected by monitoring absorbance at 700 nm using the spectrophotometer Jenway (6405 UV/VIS, Jenway Ltd., Dunmow, UK). Trolox (10-1000 mg/L; R\({}^{2}\) = 0.998) was used as the standard, and the results were expressed in mg/g Trolox equivalents.