# Retention of Antioxidants from Dried Carrot Pomace in Wheat Bread
## 2 Materials and Methods
### Methods  
Fresh carrot pomace after juice extraction (juicer Zelmer 177.T, with a yield around 60%) was placed on the sheets of filter-paper (approx. 0.5 mm height) and dried in a sterilizer (Memmert SF-55, Memmert GmbH + Co. KG, Schwabach, Germany) at 60 \({}^{\circ}\)C for 24 h. The dried pomace was finely ground using a laboratory mill (Perten LM 3100, Perten Instruments, Hagersten, Sweden) and stored in glass jars. Farinographic evaluation of the flour type 650 and its mix with 15% dried carrot pomace was made using Brabender farinograph-E (Brabender Technologie GmbH & Co. KG, Duisburg, Germany), according to the AACC method 54-21 [24]. The bread was prepared from the dough consisting of wheat flour mix (in the case of the CP sample, 15% of flour was replaced with dried carrot pomace), yeast (2% on flour basis) and water (depending on water absorption). Dough pieces (approx. 300 g) was proofed for 40 min at 35 \({}^{\circ}\)C and baked for 40 min at 200 \({}^{\circ}\)C (MIWE Condo; MIWE Michael Wenz GmbH, Arnstein, Germany). After baking, the loaves were removed from baking pans, cooled to ambient temperature, cut into slices (5 mm. thick), dried overnight under ambient conditions, ground into crumbs and stored in glass jars for further analyzes. * Chemical evaluation Determination of dry matter, ash and protein was carried out in accordance with the AACC method 08-01 [24]. The nitrogen content was determined by the semi-micro-Kjeldahl method. A conventional factor of 5.7 for bread and 6.25 for carrot pomace was used to convert nitrogen into protein. Fat content was assessed with the Ancom XT15 Fat Extractor (USA). To remove moisture before extraction, the sample (1.5 g, W1) was weighed into a special filter bag (XT4, Ankom Technology, Macedon, NY, USA)) and dried in an oven (WTB, Binder GmbH, Tuttlingen, Germany) at 105 \({}^{\circ}\)C for 180 min, after which it was cooled in a desiccant pouch for 15 min and weighed again (W2). Petroleum ether extraction was carried out at 90 \({}^{\circ}\)C for 60 min. The defatted samples were dried in an oven at 105 \({}^{\circ}\)C for 30 min, cooled in a desiccant pouch and weighed again (W3). The following formula was used to calculate the fat content (%): [(W2 \(-\) W3)/W1] \(\times\) 100. Ethanol extracts used for the determination of antioxidant constituents were obtained by dissolving 0.6 g of the sample in 30 mL of ethanol (80 g/100 g). The mixture was shaken for 120 min (electric shaker: type WB22, Memmert, Schwabach, Germany), and centrifuged (15 min, 4500 rpm. 1050\(\times\)\(g\)) in a centrifuge type MPW-350, (MPW MED. Instruments, Warsaw, Poland). The supernatant was decanted and stored at \(-\)20 \({}^{\circ}\)C for further analyses. * Total content of polyphenols was determined with Folin-Ciocalteau reagent, using a spectrophotometer, according to [PERSON] et al. [25]. 5 mL of the extract was diluted to a volume of 50 mL with distilled water. 5 mL of the diluted extract was combined with 0.25 mL of Folin-Ciocalteau reagent (previously diluted with distilled water in the proportion 1:1 \(v/v\)), 0.5 mL 7% Na\({}_{2}\)CO\({}_{3}\). The contents were vortexed (WF2, Janke & Kunkel, Staufen, Germany) and stored for 30 min in a dark place. The absorbance was measured using Helios Gamma 100-240 (Thermo Fisher Scientific, Runcorn, UK), at the wavelength \(\lambda\) = 760 nm. The results were converted to mg catechin/100 g d.m. * Determination of flavonoids was performed by the spectrophotometric method, according to [PERSON] et al. [26], 0.5 mL of the extract was combined with 1.8 mL distilled water and 0.2 mL 2-aminoethyldiphenylborate reagent in a test tube. The contents were vortexed, and the absorbance was measured at the wavelength \(\lambda\) = 404 nm. Flavonoid content was expressed as mg of rutin/100 g d.m. * Contents of total polyphenols (TPC-NFC, without F-C reagent), phenolic acids, flavonoids and anthocyanins were determined by spectrophotometric method, according to [PERSON] et al. [27], with the modification of [PERSON] et al. [28].