**4. Discussion**

The obtained results show that substitution of DWF by two types of buckwheat flour, especially by BF, enhanced the antioxidant properties of BEDWBs. This clear beneficial effect may be due to the enrichment of DWB in bioactive compounds, including rutin with well-recognized antioxidant properties. These results are consistent with the results obtained by Zielińska et al. [14] and Zieliński et al. [35]. Similarly, Lin et al. [21] showed that supplementation of wholegrain buckwheat flour in wheat bread resulted in increase of the antioxidant properties more than the application of light buckwheat flour. Whereas Yoo et al. [15] and Błaszczak et al. [16] found that rutin content in buckwheat groats is greatly reduced by thermal processing (by approximately 60%). This finding may explain the lower antioxidant capacity of buckwheat-enhanced dark wheat bread based on flour from roasted groats as compared to bread formulated with buckwheat flour as it was shown in this study. Many researchers also argue that phenolic compounds as well as compounds formed in Maillard reaction (e.g. HMF, furfural and acrylamide) play a significant role in scavenging of free radicals [36, 37]. However, the formation of Maillard reaction compounds can disguise actual reduction of phenolic contents and antioxidant capacity as well as loss of antioxidant activity in bread samples throughout the heat treatment [38].

Furthermore, the present study showed that BEDWBs were more effective scavengers of radical cation (ABTS•+) than DPPH• radicals and superoxide anion radical (O2 −•). These differences were statistically significant (*p* < 0.05) (**Table 2**). An inverse relationship was found in the reference DWB. This trend in the rank of the radical scavenging activity was demonstrated by Floegel et al. [39] and Xu et al. [40]. Similarly, Sakač et al. [38] observed clear differences in antioxidant capacity between the light buckwheat enriched bread and wholegrain buckwheat enriched bread.

One of the significant mechanisms to defend against oxidative damage and lipid peroxidation is to chelate metal ions. In this study, we observed significant differences in metal chelating activity and reducing power between the reference DWB and BEDWBs (**Table 3**). Especially, supplementation of BFR has contributed to an increased metal chelating activity of BEDWBs. Whereas, Sakač et al. [38] found significant differences in antioxidant capacity measured by metal chelating activity and reducing power between the light and wholegrain buckwheat enriched breads. Enhancement of the antioxidant activity of bread after application of buckwheat flour from milled roasted groats can be related to the modification and/or degradation of phenolic compounds and formation of Maillard reaction products such as melanoidins, which may also act as antioxidants [36, 37]. It is possible that the enhancement of bread

with BF or BFR contributed to metal chelating activity due to the rutin content, since rutin is well-known as a potent metal chelator. Symonowicz and Kolanek [27] reported that the interactions of phenolic compounds with metal ions caused the formation of chelates. Metal chelation may be important to limit the formation of free radicals, thus reduce oxidative stress. Several studies have confirmed that flavonoids possess antioxidant properties due to their ability to chelate metal ions [26, 27]. Filipčev et al. [18] observed clear differences in metal chelating activity between the buckwheat and rye cookies. They also noticed that buckwheat enriched cookies (in amount 30, 40 and 50%) show a higher antioxidative properties than cookies enriched in rye. In this study a special focus was put on the cyclic voltammetry methodology, which allowed rapid screening of the electrochemical profile of buckwheatenhanced dark wheat bread samples. This reducing capacity of BEDWBs was based on the electrochemical behaviour and chemical properties of the electroactive compounds being in bread [41]. In this study was found that substitution of DWF by BF or BFR at levels of 10, 20, 30 and 50% w/w on total flour basis caused almost a linear increase of the reducing capacity of BEWBs (**Table 3**). It should be mentioned that practical limitation of CV methodology was that the working electrode had to be frequently cleaned to remove residues of sample from its surface and to maintain its sensitivity. However, the advantage of CV was related not to do requiring the use of reactive chemicals.
