The extraction of antioxidant compounds was carried out by blending 150 mg of sample (flour, pre- and post-fermented dough, and bread) with 1.5 mL (1:10) of ethanol (96%)/HCl (1N) (85:15, v/v) and stirred for 30 min at room temperature. Subsequently, the samples were centrifuged (Thermo Fisher Scientific, Sorval ST40R, Madrid, Spain) at 8000× g for 10 min, and the supernatant was recovered. The extraction was repeated 3 times, and the supernatants were pooled.
Total anthocyanin content (TAC) was determined by a differential pH method, according to Lee et al. [15 (link)], using the calculation formula:
where A = DF = dilution factor; using a molar extinction coefficient ( ) of 26,900 L/mol/cm and a molecular weight (MW) of 449.2 g/mol for cyanidin-3-glucoside. The absorbance at 520 and 700 nm was measured with a spectrophotometer (UV-vis JascoV-730, Jasco Corporation, Tokio, Japan). The results were expressed in mg of cyanidin-3-glucoside equivalent per 100 g of sample (mg c3-GE/100 g). Total polyphenol content (TPC) was measured using the Folin-Ciocalteu method, as adapted by Lopez-Martinez et al. [16 (link)]. The results were expressed in mg of gallic acid per each 100 g of sample (mg AG/100 g), using a standard curve from 0 to 0.5 mg AG/mL. Ferulic acid content (FA) was measured according to Podio et al. [17 (link)] with a modification proposed by Navarro et al. [18 (link)]. Briefly, after alkaline hydrolysis and acidification treatment, the samples were centrifuged at 16,000× g for 20 min at 4 °C. Three milliliters of ethyl acetate were added to the supernatant and mixed at 6000 g for 15 min. The ethyl acetate phase was recovered from the resulting multilayer system formed. This procedure was repeated to complete three ethyl acetate washes. Then, sodium sulfate was added to the organic phase, stirred, and kept for 1 h in the dark, and the absorbance at 320 nm was measured with a spectrophotometer (UV-vis JascoV-730, Jasco Corporation, Japan). The results were calculated with linear regression, using acid ferulic as standard and expressed in μg of acid ferulic per g of sample (μg FA/g).
The antioxidant activity was quantified by free radical scavenging capacity through a TEAC (Trolox Equivalent Antioxidant Capacity) assay, according to Re et al. [19 (link)]. The reducing power was determined with a FRAP (Ferric Reducing Antioxidant Power) assay, according to Benzie and Strain [20 (link)]. The results were quantified from a trolox calibration curve and expressed in µmol of trolox per g of sample (µmol tr/g). All determinations were analyzed at least in quadruplicate.
Total anthocyanin content (TAC) was determined by a differential pH method, according to Lee et al. [15 (link)], using the calculation formula:
where A = DF = dilution factor; using a molar extinction coefficient ( ) of 26,900 L/mol/cm and a molecular weight (MW) of 449.2 g/mol for cyanidin-3-glucoside. The absorbance at 520 and 700 nm was measured with a spectrophotometer (UV-vis JascoV-730, Jasco Corporation, Tokio, Japan). The results were expressed in mg of cyanidin-3-glucoside equivalent per 100 g of sample (mg c3-GE/100 g). Total polyphenol content (TPC) was measured using the Folin-Ciocalteu method, as adapted by Lopez-Martinez et al. [16 (link)]. The results were expressed in mg of gallic acid per each 100 g of sample (mg AG/100 g), using a standard curve from 0 to 0.5 mg AG/mL. Ferulic acid content (FA) was measured according to Podio et al. [17 (link)] with a modification proposed by Navarro et al. [18 (link)]. Briefly, after alkaline hydrolysis and acidification treatment, the samples were centrifuged at 16,000× g for 20 min at 4 °C. Three milliliters of ethyl acetate were added to the supernatant and mixed at 6000 g for 15 min. The ethyl acetate phase was recovered from the resulting multilayer system formed. This procedure was repeated to complete three ethyl acetate washes. Then, sodium sulfate was added to the organic phase, stirred, and kept for 1 h in the dark, and the absorbance at 320 nm was measured with a spectrophotometer (UV-vis JascoV-730, Jasco Corporation, Japan). The results were calculated with linear regression, using acid ferulic as standard and expressed in μg of acid ferulic per g of sample (μg FA/g).
The antioxidant activity was quantified by free radical scavenging capacity through a TEAC (Trolox Equivalent Antioxidant Capacity) assay, according to Re et al. [19 (link)]. The reducing power was determined with a FRAP (Ferric Reducing Antioxidant Power) assay, according to Benzie and Strain [20 (link)]. The results were quantified from a trolox calibration curve and expressed in µmol of trolox per g of sample (µmol tr/g). All determinations were analyzed at least in quadruplicate.
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