**2.3. Preparation of bread crude extracts for measurement of antioxidant capacity by ABTS and DPPH assays, and reducing capacity by cyclic voltammetry (CV)**

Bread samples (0.25 g) were extracted in triplicate at 25°C with 5 mL of 67% aqueous methanol using Thermomixer comfort (Eppendorf, Germany) by shaking at 1400 rpm for 60 min. Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R centrifuge, Eppendorf, Germany). After that, the 67% methanol extracts were directly used to determine the antioxidant capacity.

### **2.4. Preparation of hydrophilic and lipophilic bread extracts for measurement of antioxidant capacity by photochemiluminescence assay**

Hydrophilic extracts: About 0.1 g of bread samples were extracted in triplicate for 3 min with 1 mL of deionized water using Genie-2 type vortex (Scientific Industries, USA). Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R, Eppendorf, Germany) and the fresh supernatants were used to determination of antioxidant activity formed by water-soluble antioxidants (ACW). Lipophilic extracts: About 0.1 g of bread samples were extracted in triplicate for 3 min with an n-hexane and methanol (1:4 v/v) using Genie-2 type vortex (Scientific Industries, USA). Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R, Eppendorf, Germany) and the fresh supernatants were used to determine the antioxidant capacity of lipid-soluble antioxidants (ACL).

## **2.5. Antioxidant capacity measured by ABTS, DPPH and PCL assays**

#### *2.5.1. ABTS assay*

buckwheat-enhanced dark wheat breads formulation and baking conditions. Three pieces of each type of bread was baked. Samples were freeze-dried, milled and sieved through of 0.6

Dark wheat flour (g) 350 315 280 245 175 Buckwheat flour (g) – 35 70 105 175 Roasted buckwheat flour (g) – 35 70 105 175 Water (mL) 228 228 228 228 228 Salt (g) 3.5 3.5 3.5 3.5 3.5 Yeast (g) 10.5 10.5 10.5 10.5 10.5

Temperature (°C)/time (min) 37/90 37/90 37/90 37/90 37/90 Pieces of dough (g) 250 250 250 250 250

Temperature (°C)/time (min) 37/25 37/25 37/25 37/25 37/25

Temperature (°C)/time (min) 250/30 250/30 250/30 250/30 250/30

**2.3. Preparation of bread crude extracts for measurement of antioxidant capacity by ABTS**

Bread samples (0.25 g) were extracted in triplicate at 25°C with 5 mL of 67% aqueous methanol using Thermomixer comfort (Eppendorf, Germany) by shaking at 1400 rpm for 60 min. Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R centrifuge, Eppendorf, Germany). After that, the 67% methanol extracts were directly used to determine the antioxidant capacity.

**2.4. Preparation of hydrophilic and lipophilic bread extracts for measurement of antioxidant**

Hydrophilic extracts: About 0.1 g of bread samples were extracted in triplicate for 3 min with 1 mL of deionized water using Genie-2 type vortex (Scientific Industries, USA). Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R, Eppendorf, Germany) and the fresh supernatants were used to determination of antioxidant activity formed by water-soluble antioxidants (ACW). Lipophilic extracts: About 0.1 g of bread samples were extracted in triplicate for 3 min with an n-hexane and methanol (1:4 v/v) using Genie-2 type vortex (Scientific Industries, USA). Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R, Eppendorf, Germany) and the fresh supernatants were used to determine the antioxidant

**Table 1.** Buckwheat and reference dark wheat breads formulation and baking conditions.

**and DPPH assays, and reducing capacity by cyclic voltammetry (CV)**

**capacity by photochemiluminescence assay**

capacity of lipid-soluble antioxidants (ACL).

0 10 20 30 50

mm, and then were stored at –20°C before using for analysis.

276 Superfood and Functional Food - An Overview of Their Processing and Utilization

Fermentation

Proofing (75% rh)

Baking

**Ingredient and conditions Addition of buckwheat flours (%)**

For the determination of the antioxidant activity was using the method described by Re et al. [28]. The ABTS•+ stock solution was diluted with 67% methanol to the absorbance of 0.70 ± 0.02 at 734 nm. Appropriate, solvent blank was used in each assay. The Trolox standard curve was determined in the range of 0.1–2.5 mM. The measurements were performed by a spectrophotometer UV-160 1PC with CPS-controller (Shimadzu, Japan). The antioxidant capacity was expressed in µmol Trolox/g of bread dry matter (DM).

### *2.5.2. DPPH assay*

DPPH• scavenging activity was determined as described previously in details [29]. The Trolox standard solutions were prepared in 67% methanol in the range of 0.1–2.5 mM. The measurements were performed by a spectrophotometer UV-160 1PC with CPS-Controller (Shimadzu, Japan). The antioxidant capacity was expressed in µmol Trolox/g of bread dry matter.

#### *2.5.3. Antioxidant capacity measured by photochemiluminescence (PCL) assay*

The PCL assay was carried out using the method according to Popov and Lewin [30]. This method consists in determining the superoxide anion radicals generated by luminol (under UV light) in the presence of antioxidants. The antioxidant capacity of buckwheat bread extract was determined using the analytical kits, which are designed to determine the antioxidant activity of the hydrophilic (ACW) and lipophilic (ACL) compounds, as reported previously by Zielińska et al. [29]. Measurements were performed with a Photochem® apparatus (Analytik Jena, Leipzig, Germany). PCL values are showed as a sum of ACW and ACL. The antioxidant capacity was expressed in µmol Trolox/g of bread dry matter.

#### **2.6. Metal chelating activity of buckwheat-enhanced dark wheat breads**

Bread samples (0.1 g) were extracted in triplicate with 1 mL of 0.2 M phosphate buffered saline (PBS, pH 7.4), which contains 1% (m/v) SDS for 30 s using VC 750 type sonicator (SONICS, USA) followed by vigorously shaking for 30 s using Genie-2 type vortex (Scientific Industries, USA). That stage was repeated three times. Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R, Eppendorf, Germany). The supernatants were directly used to determine Fe(II) chelating power of breads. Chelating power was measured using the method of Wang et al. [31]. To the reaction tube was added 0.25 mL of 1 mM FeSO4, 0.25 mL of fresh bread extract, 1 mL of PBS (pH 7.4) with 1% (m/v) SDS, 1 mL of 2,2'-bipyridyl solution (0.1% in 0.2 M HCl), 0.4 mL of 10% NH2OH HCl and 2.1 mL of ethanol. The mixture was shaken and left at room temperature for 20 min. The absorbance at 522 nm was determined and used to evaluate Fe+2 chelating activity using ethylenediaminetetraacetate (EDTA) as a standard. The standard curve was constructed within the range of 0.125–2.0 mM of EDTA. The Fe(II) chelating capacity of samples was measured in triplicate using a temperature-controlled spectrophotometer UV-160 1PC with CPS-Controller (Shimadzu, Japan). Results were expressed as µmol EDTA equivalents/g DM.

#### **2.7. Measurement of reducing power of buckwheat-enhanced dark wheat breads**

Bread samples (0.25 g) were extracted in triplicate at 25°C with 5 mL of 67% aqueous methanol using Thermomixer comfort (Eppendorf, Germany) by shaking at 1400 rpm for 60 min. Next, samples were centrifuged for 5 min (16,100 × g, 4°C) (5415 R centrifuge, Eppendorf, Germany). After that, extracts were dried at 40°C using a rotary evaporator. Then samples were dissolved in 5 mL of phosphate buffer (0.2 M, pH 6.6) and were used immediately for the measurement of reducing power of bread extracts. The reducing power was determined by Oyaizu [32] with minor modification according to Liyana-Pathirana and Shahidi [33]. The assay mixture contained 1 mL of sample, 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide, incubated at 50°C for 20 min. Then, 2.5 mL of 10% TCA was added to the mixture and centrifuged for 5 min (2000 × g, 4°C). Exactly 2.5 mL of the extract of sample was mixed with 2.5 mL water and 0.5 mL of 0.1% FeCL3 and was measured at 700 nm using a spectrophotometer UV-160 1PC with CPS-Controller (Shimadzu, Japan). A standard curve was prepared using ascorbic acid within the range of 0.015–0.5 mM and the reducing power was expressed as µmol ascorbic acid equivalents/g DM.

#### **2.8. Measurement of reducing capacity of buckwheat-enhanced dark wheat breads by cyclic voltammetry**

The cyclic voltammetry experiments were performed in 67% methanol bread extracts mixed with 0.1 M sodium acetate–acetic buffer (pH 4.5) at ratio 1:1 (v/v) according to Zielińska et al. [29]. The sodium acetate–acetic buffer acted as a supporting electrolyte for cyclic voltammetry measurements. A micro-electrochemical cell (with the total volume of 200 µL), made all of Teflon, was used during the course of this experiment. Three electrodes: a glassy carbon (GC) working electrode (BAS MF-2012, 3 mM diameter), an Ag/AgCl (3.5 M KCl) reference and a Pt (0.5 mM diameter coiled Pt wire) counter electrode constituted the cell. Working electrode was hand-polished with 0.05 µm alumina-water paste (BAS CF-1050), using BAS (MF-1040) polishing cloth and then rinsed with ultra-pure water and methanol. The cyclic voltammetry experiment was performed in the range of 100–1100 mV at a potential sweep-rate of 100 mV s−1 at room temperature using a potentiostat/galvanostat G 750 (Gamry Ins., USA). The total charge below the anodic wave curve of the voltammogram was calculated. The cyclic voltammograms of Trolox solutions over the concentration range of 0.05–2.5 mM was determined. The reducing capacity of buckwheat-rich wheat breads was expressed in terms of µmol Trolox/g DM.

#### **2.9. Statistical analysis**

Results of the chemical analyses are illustrated as mean values and the standard deviation of three independent measurements. The obtained results were analysed with one-way ANOVA. Fisher Least Significant Difference (LSD) test at a significance level of *p* < 0.05 was performed for post-hoc comparison. The Statistica ver. 5.0 software was used (General Convention and Statistica, StatSoft, USA, 1995).
