*3.4. Antioxidant Activity of the Breads*

The total phenol contents and antioxidant activity of quinoa, sorghum, millet and rice breads are reported in Table 4. The total phenol content of the breads varied from 70.34 to 398.42 mg FAE/100 g d.w. The highest total phenol content was registered in case of bread prepared with quinoa flour, while the lowest content was obtained for bread prepared with rice flour.

**Table 4.** Total phenol contents and antioxidant activity of quinoa, sorghum, millet and rice breads.


The antioxidant properties of breads were evaluated by measuring the DPPH-radical scavenging activity, and by TEAC and ABTS and FRAP methods. Re et al. [43] mentioned that the TEAC method quantifies both lipophilic and hydrophilic antioxidants, comprising carotenoids, hydroxycinnamates, and flavonoids, while the DPPH-radical scavenging activity is mainly due to the phenolic compounds [25]. In order to directly measure the antioxidants or reductants in samples the FRAP assay was used.

All methods applied for assessing the antioxidant properties indicated that the antioxidant ability of the bread samples decreased in the following order: sorghum > quinoa > millet > rice (Table 4). Sorghum and quinoa breads had higher value of FRAP, indicating the presence of Fe2<sup>+</sup> chelating agents in these samples.

The results reported in the literature for the antioxidant activity of different types of flours and breads are very different due to many factors related both to the investigated samples, such as the genotype of the grains and environmental conditions, and to the techniques used for preparing the extracts and quantification of the antioxidant activity [13]. Therefore, is rather difficult to compare our results with those of other authors. Xu et al. [3] studied the antioxidant properties of bread prepared with blends of wheat flour and quinoa flour and found that the phenolic content, ABTS and DPPH-RSA increased with increase of the levels of quinoa flours. They reported a loss of total polyphenol after baking. Similarly, when testing the possibility of using amaranth, quinoa, buckwheat and wheat for obtaining nutritionally enhanced gluten-free formulations, Alvarez-Jubete et al. [44] reported the overall decrease of the antioxidant activity and total phenol content following bread making. Anyway, new substance with antioxidant properties can be synthesized during baking, such as Maillard reaction products, which can be mainly found in the bread crust. Jan et al. [45] observed the increase of the antioxidant activity of cookies during baking process with increasing the sugar level, temperature and time, most probably due to the formation of melanoidins. The antioxidant activity of cookies was stable after baking at 180 ◦C suggesting the completion of the reactions resulting in molecules with radical scavenging ability. Furthermore, the phenolics breakdown or other degradation reactions might result in products responsible for the increase antioxidant activity. Lindenmeier and Hofmann [46] reported the increase of the antioxidant activity up to five times with the increase of baking temperature and time. They explained this increase by the formation of the antioxidant compound pronyl-L-lysine. Moreover, they found higher antioxidant activity in crust in comparison to the crumb and untreated flour.

Khan et al. [14] reported for the free phenolic acids of red and white sorghum flours values of 81.19 and 150.67 μg GAE/g d.w., respectively. Yousif et al. [17] found that the breads prepared from blends of wheat flour and 40% sorghum flour (red and white, respectively) had the free phenolic contents of 1.09–0.49 mg GAE/g d.w. Additionally, Yousif et al. [17] reported that the addition of red or white sorghum flour to the wheat flour increased the polyphenolic content and the antioxidant capacity of the bread. Khan et al. [14] noted that the phenolic acids prevailing in the extract were ferulic acid, p-hydroxibenzoic acid and salicylic acid. Besides these phenolic acids, the red sorghum flour contained flavonoids such as anthocyanins (luteolinidin and apigeninidin). Thermal processing of cereals usually causes the reduction of the flavonoid contents to different extents, depending on the food matrix and on the intensity of the thermal treatment [44]. In particular, Khan et al. [14] noted that anthocyanins were less stable than phenolic acids during cooking. There are other phytochemicals, such as carotenoids, that contribute to the antioxidant activity of the samples [14]. Lopez-Contreras et al. [47] studied the antioxidant activity of ten sorghum genotypes and reported variation of the FRAP, ABTS and DPPH values from 3.94 to 63.34, from 44.21 to 121.73 and from 1.83 to 65.73 μmol Trolox/g, respectively, and of total phenol content values from 1.408 to 12.348 μg catechin equivalents/g.

Chandrasekara and Shahidi [15] found higher free phenolic contents in finger millet (411–610 mg FAE/100 g), compared to the pearl and proso millets (168 mg FAE/100 g and 140 mg FAE/100 g, respectively). They registered different flavonoid contents of 203–228, 49 and 140 mg/100 g catechin equivalent in the phenolic extracts obtained from finger, pearl and proso millet, respectively. Chethan and Malleshi [48] showed that the main phenolic acids in millets are ferulic acid, gallic acid, p-hydroxibenzoic acid, cumaric acid and proto-catechuic acid. Sreeramulu et al. [49] found higher antioxidant activity (FRAP of 471.71 μmol/g, DPPH-RSA and total phenol content of 1.73 and 373.15 mg Trolox/g, respectively) in finger millet compared to the rice (FRAP of 60.93–67.48 μmol/g, DPPH-RSA and total phenol content of 0.49–1.23 and 47.64–56.61 mg Trolox/g, respectively).

Previous studies mentioned a significant correlation between phenolics and antioxidant activity in quinoa [50]. Large variation of the total phenol content of quinoa seed from 28.49 to 1.59 mg gallic acid/100 g d.w. was reported by Miranda et al. [12]. Later study of Pellegrini et al. [13] revealed the phenolic profile of quinoa flour; the most abundant phenolic acids were 4-hydroxybenzoic, syringic acid, vanillic acid, gallic acid, ferulic acid and p-cumaric acid, while the most dominant flavonoids were neohesperidin, kaempfenol and isoquercitin. In addition to the polyphenols and flavonoids which are know the possess antioxidant properties, Aloisi et al. [50] also mentioned the contribution of the proteins, including the 11S fraction, to the antioxidant activity of the quinoa seeds. Other bioactive compounds like carotene, vitamins, tocopherols etc., might also contribute to the antioxidant properties of quinoa flour [50].

The total phenol content and antioxidant properties varied significantly between breads prepared with quinoa, sorghum, millet and rice flour. Moreover, between the total phenol content and DPPH, FRAP and TEAC were registered strong correlations (*p* < 0.05) of 0.99, 0.88 and 0.71, respectively. These results indicate that the antioxidant properties of breads are mainly due to presence of phenols and flavonoids.
