**3. Results**

tometer UV-160 1PC with CPS-Controller (Shimadzu, Japan). Results were expressed as µmol

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

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

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

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

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

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

EDTA equivalents/g DM.

**cyclic voltammetry**

expressed as µmol ascorbic acid equivalents/g DM.

expressed in terms of µmol Trolox/g DM.

**2.9. Statistical analysis**

Free radical scavenging activity of food extracts should be determined by using different techniques to evaluate the antioxidant capacity of food *in vitro* to cover all aspects of antioxidant effectiveness. Recently, we provided evidences for the main differences in bioactive compounds content as well as in antioxidant properties of two types of buckwheat flours, e.g. BF and BFR when compared to DWF [34]. The estimated values of antioxidant capacity of flours based on the relative abilities of 67% methanol crude extracts to scavenge the ABTS•+ and DPPH• radicals in comparison to Trolox showed the following order: BF > BFR > DWF. Moreover, chelating and reducing power of two types of buckwheat flour showed a comparable level, being higher than determined for DWF. A well-illustrated difference in the reducing capacity of buckwheat flours and dark wheat flour is presented on **Figure 1** when cyclic voltammetry technique was applied.

**Figure 1.** Cyclic voltammograms of buckwheat flour (BF), flour from roasted buckwheat groats (BFR) and dark wheat flour (DWF). Measurements were performed with 67% methanol extracts (100 mg/mL) mixed with 0.1 M sodium acetate-acetic buffer (pH 4.5) at ratio 1:1 (v/v); scan rate 100 mV s−1. The higher total charge under anodic current wave indicates a higher reducing capacity of the investigated flour extracts.

#### **3.1. Antioxidant capacity of buckwheat-enhanced dark wheat breads as measured against free radicals**

The antioxidant activity of buckwheat-enhanced dark wheat breads determined by ABTS, DPPH and PCL assays is shown in **Table 2**. The PCL values show the sum of antioxidant capacity of the hydrophilic (ACW) and lipophilic (ACL) fractions of bread (**Figure 2**). The rank of scavenging effect of reference DWB extract was 5.24 ± 0.24 µmol Trolox/g DM (DPPH assay) > 4.31 ± 0.07 µmol Trolox/g DM (ABTS assay) > 1.48 ± 0.01 µmol Trolox/g DM (PCL assay). The addition of BF or BFR in the range of 10, 20, 30 and 50% in the bread formula caused a significant (*p* < 0.05) increase in antioxidant capacity as compared to the reference DWB. The highest scavenging activity was found in BEDWBs with addition of 50% of BF (for ABTS assay 15.02 ± 0.90 µmol Trolox/g DM, for DPPH assay 8.36 ± 0.12 µmol Trolox/g DM and 3.18 ± 0.07 µmol Trolox/g DM for PCL assay). A similar rank of values was noted in BEDWBs after substitution of DWF by BFR at 50% level (13.99 ± 0.05 µmol Trolox/g DM, 9.20 ± 0.17 µmol Trolox/g DM and 4.43 ± 0.06 µmol Trolox/g DM, respectively). The increased substitution level of DWF by BF or BFR resulted in higher ACL values as compared to ACW (**Figure 2**).


Values are means of three determinations ± standard deviation. Values within column followed by the same letter are not significantly different at 95% confidence level. PCL values show the sum of ACW and ACL values.

**Table 2.** Antioxidant capacity of bread samples determined against ABTS, DPPH and PCL assays.

**3.1. Antioxidant capacity of buckwheat-enhanced dark wheat breads as measured against**

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

The antioxidant activity of buckwheat-enhanced dark wheat breads determined by ABTS, DPPH and PCL assays is shown in **Table 2**. The PCL values show the sum of antioxidant capacity of the hydrophilic (ACW) and lipophilic (ACL) fractions of bread (**Figure 2**). The rank of scavenging effect of reference DWB extract was 5.24 ± 0.24 µmol Trolox/g DM (DPPH assay) > 4.31 ± 0.07 µmol Trolox/g DM (ABTS assay) > 1.48 ± 0.01 µmol Trolox/g DM (PCL assay). The addition of BF or BFR in the range of 10, 20, 30 and 50% in the bread formula caused a significant (*p* < 0.05) increase in antioxidant capacity as compared to the reference DWB. The highest scavenging activity was found in BEDWBs with addition of 50% of BF (for ABTS assay 15.02 ± 0.90 µmol Trolox/g DM, for DPPH assay 8.36 ± 0.12 µmol Trolox/g DM and 3.18 ± 0.07 µmol Trolox/g DM for PCL assay). A similar rank of values was noted in BEDWBs after substitution of DWF by BFR at 50% level (13.99 ± 0.05 µmol Trolox/g DM, 9.20 ± 0.17 µmol Trolox/g DM and 4.43 ± 0.06 µmol Trolox/g DM, respectively). The increased substitution level of DWF by

**Type of bread % of buckwheat flours Antioxidant capacity (μmol Trolox/g d.m.)** 

Dark wheat bread (DWB) 0 4.31 ± 0.07a 5.24 ± 0.24a 1.48 ± 0.01a

**ABTS DPPH PCL**

5.82 ± 0.07b

1.55 ± 0.01b

1.72 ± 0.03b

2.54 ± 0.16d

3.18 ± 0.07e

1.92 ± 0.04b

2.07 ± 0.11c

2.94 ± 0.04d

4.43 ± 0.06e

7.48 ± 0.15c

8.24 ± 0.04d

8.36 ± 0.12d

5.99 ± 0.08b

7.68 ± 0.12c

9.00 ± 0.17d

9.20 ± 0.17d

8.07 ± 0.15b

10.06 ± 0.24c

10.82 ± 0.78c

15.02 ± 0.90e

7.13 ± 0.22b

8.83 ± 0.05c

10.03 ± 0.23c

13.99 ± 0.05d

BF or BFR resulted in higher ACL values as compared to ACW (**Figure 2**).

10

20

30

50

10

20

30

50

not significantly different at 95% confidence level. PCL values show the sum of ACW and ACL values.

**Table 2.** Antioxidant capacity of bread samples determined against ABTS, DPPH and PCL assays.

Values are means of three determinations ± standard deviation. Values within column followed by the same letter are

**free radicals**

Buckwheat-enhanced dark wheat breads

Buckwheat-enhanced dark wheat breads

(BEDWBs) with BF

(BEDWBs) with BFR

**Substitution level of dark wheat flour by buckwheat flour (BF) (%)** 

**Substitution level of dark wheat flour by roasted buckwheat flour (BFR) (%)** 

**Figure 2.** Antioxidant capacity of buckwheat-enriched dark wheat breads formed by hydrophilic (ACW) and lipophilic (ACL) antioxidants. (A) DWF was substituted by BF. (B) DWF was substituted by BFR.

#### **3.2. Reducing power and capacity of buckwheat-enhanced dark wheat breads**

**Table 3** illustrates the reducing power of BEDWBs as determined by the potassium ferricyanide method. The reducing power of BEDWBs was higher (*p* < 0.05) than noted for DWB. It was found that substitution of DWF by BF or BFR at levels of 10, 20, 30 and 50% w/w on the total flour basis caused an increase of the reducing power of BEDWBs. The highest level of DWF substitution (50%) by BF of BFR resulted in 2.5-fold increase of the reducing power of breads as compared to the reference DWB.


Values are means of three determinations ± standard deviation. Values within column followed by the same letter are not significantly different at 95% confidence level.

**Table 3.** Reducing power (µmol ascorbic acid equivalents/g DM), reducing capacity (µmol Trolox/g DM) and Fe(II) chelating capacity (µmol EDTA equivalents/g DM) of buckwheat-enhanced dark wheat breads.

A special focus was put on the cyclic voltammetry (CV) experiments as a novel technique. The cyclic voltammograms of 67% MeOH extracts from breads were recorded as it is shown on **Figure 3**. The reducing capacity of BEDWBs was higher (*p* < 0.05) than noted for DWB. The reducing capacity of BEDWBs provided by CV assay was comparable to their reducing power determined by the potassium ferricyanide method (**Table 3**), and antioxidant capacity provided by photochemiluminescence (**Table 2**). In contrast, reducing capacity of BEDWBs was about threefold lower than antioxidant capacity determined against ABTS•+ and DPPH• radicals.

Antioxidant Properties of Dark Wheat Bread with Exogenous Addition of Buckwheat Flour http://dx.doi.org/10.5772/65411 283

**3.2. Reducing power and capacity of buckwheat-enhanced dark wheat breads**

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

**tution**

10

20

30

50

10

20

30

50

chelating capacity (µmol EDTA equivalents/g DM) of buckwheat-enhanced dark wheat breads.

**level (%)** 

Dark wheat bread (DWB) 0 9.89 ± 0.01a 1.86 ± 0.11ab 1.76 ± 0.00a

as compared to the reference DWB.

Buckwheat-enhanced dark

Buckwheat-enhanced dark

radicals.

wheat breads (BEDWBs) with BFR

not significantly different at 95% confidence level.

wheat breads (BEDWBs) with BF

**Type of bread Substi**

**Table 3** illustrates the reducing power of BEDWBs as determined by the potassium ferricyanide method. The reducing power of BEDWBs was higher (*p* < 0.05) than noted for DWB. It was found that substitution of DWF by BF or BFR at levels of 10, 20, 30 and 50% w/w on the total flour basis caused an increase of the reducing power of BEDWBs. The highest level of DWF substitution (50%) by BF of BFR resulted in 2.5-fold increase of the reducing power of breads

**Fe(II)**

**chelating**

**Reducing**

**Reducing power by**

**Fe (III) reduction**

2.56 ± 0.03b

3.34 ± 0.09c

3.59 ± 0.05c

4.40 ± 0.26d

3.37 ± 0.29c

3.93 ± 0.01c

4.62 ± 0.01d

5.34 ± 0.06e

**capacity**

**CV method**

1.79 ± 0.12a

3.24 ± 0.21c

3.35 ± 0.22c

4.05 ± 0.27d

2.43 ± 0.16b

2.40 ± 0.16b

3.57 ± 0.24c

4.92 ± 0.31d

**by**

**capacity**

10.81 ± 0.12b

10.86 ± 0.09b

13.14 ± 0.32c

13.71 ± 0.05d

12.32 ± 0.15bc

13.48 ± 0.09c

13.78 ± 0.34d

13.97 ± 0.20d

Values are means of three determinations ± standard deviation. Values within column followed by the same letter are

**Table 3.** Reducing power (µmol ascorbic acid equivalents/g DM), reducing capacity (µmol Trolox/g DM) and Fe(II)

A special focus was put on the cyclic voltammetry (CV) experiments as a novel technique. The cyclic voltammograms of 67% MeOH extracts from breads were recorded as it is shown on **Figure 3**. The reducing capacity of BEDWBs was higher (*p* < 0.05) than noted for DWB. The reducing capacity of BEDWBs provided by CV assay was comparable to their reducing power determined by the potassium ferricyanide method (**Table 3**), and antioxidant capacity provided by photochemiluminescence (**Table 2**). In contrast, reducing capacity of BEDWBs was about threefold lower than antioxidant capacity determined against ABTS•+ and DPPH•

**Figure 3.** Cyclic voltammograms of buckwheat-enriched dark wheat breads. (A) DWF was substituted by BF. (B) DWF was substituted by BFR. Measurements were performed with 67% methanol extracts (100 mg/mL) mixed with 0.1 M sodium acetate-acetic buffer (pH 4.5) at ratio 1:1 (v/v); scan rate 100 mV s−1. The higher total charge under anodic current wave indicates a higher reducing capacity of the investigated bread extracts.

#### **3.3. Fe(II) chelating capacity (ChC) of buckwheat-enhanced dark wheat breads**

The results of Fe(II) chelating capacity of BEDWBs are summarized in **Table 3**. It was found that DWB as well as all types of BEDWBs contained compounds with Fe(II) chelating capacity. Both buckwheat types of flour were a good source of these compounds since substitution of DWF by BF or BFR at levels of 10, 20, 30 and 50% w/w on the total flour basis resulted in increased chelating capacity of breads. The highest Fe(II) chelating capacity was noted for BEDWBs with 50% substitution of DWF by BF or by BFR. This level of DWF substitution resulted in 40% increase in the chelating capacity of bread as compared to the reference DWB (9.89 ± 0.01 µmol EDTA/g d.m.).
