3.1. Chemical Composition
The contents of dry matter, ash, individual sugars, CF, NDF, and TDF and values of OMD and DMD are presented in
Table 1. The dry matter content ranged between 90.0% and 93.3%. The determination of this parameter is important in managing and marketing flakes, and also in correcting flake composition on dry weight basis. The ash contents of non-traditional flakes ranged from 1.00% to 2.10%. It could be assumed that quinoa and teff flakes might serve as a good source of minerals.
As can be seen in
Table 1, the predominant sugar in all flake samples was maltose with the concentration ranging from 4.91 to 42.0 mg/g. The highest maltose content was detected in red quinoa flakes (42.0 mg/g) followed by black quinoa flakes (39.4 mg/g). Regarding glucose and fructose amounts, their concentration ranged between 0.09 to 4.60 and 0.08 to 1.38 mg/g, respectively. Red and black quinoa flakes were not only rich in maltose, but also in glucose and fructose contents. Moreover, rhamnose was detected only in white rice flakes (below 0.02 mg/g) and xylose was found only in red rice flakes (0.03 mg/g). Saccharose was not found in any sample. A typical HPLC chromatogram can be seen in
Supplementary Figure S1. As far as our knowledge, no studies have been stated on the free sugar profile of hydrothermally treated non-tradition flakes with coloured layers. Nevertheless, Hu et al. (2017) provided an approximate maltose content in milled rice grains up to 1.34 mg/g, fructose content up to 0.11 mg/g, and glucose content up to 2.29 mg/g [
16]. Concentrations of sucrose, glucose, and fructose in quinoa seeds were the following: Up to 15.2, 8.0, and 1.6 mg/g, respectively [
23]. It might be assumed that the high maltose content in non-traditional flakes could point to degradation of starch during hydrothermal treatment of grains.
The crude fibre (CF, complex of lignin, and cellulose) and neutral-detergent fibre (NDF, complex of lignin, cellulose, and insoluble part of hemicelluloses) contents ranged from 1.27% to 1.93% and 2.39% to 8.95%, respectively. Total dietary fibre (TDF, complex of insoluble and soluble parts of fibre) was measured in the range of 7.30–24.4%, with the highest TDF content in red quinoa flakes. It has been shown that hydrothermal treatment of raw grains increased digestibility of both OMD and DMD value as well as effectively improved the nutritional value of grains by pioneering starch gelatinization. According to Qiao et al. (2015) the OMD values of intact rice grains increased by 5% (from 68.8% to 73.4%) when steam-flaked process was applied [
24]. In this study, the OMD values of non-traditional flakes ranged from 89.2% to 98.4%. It was found that rice flakes were more digestible than quinoa and teff flakes. When the CF values were high, DMD and OMD values exhibited as low, it could be considered that they were positively correlated as
r (Pearson´s correlation coefficient) values of 0.3349 and 0.4786, respectively (
Supplementary Table S1). In case of NDF and TDF values, it could not be seen any positive correlations with DMD or OMD values. Moreover, the positive correlations were also observed between high starch or resistant starch contents and DMD or OMD values, in which
r values were between 0.2911–0.7755.
3.2. Results of Total Phenolic and Flavonoid Contents and Antioxidant Activity Values
Results of total flavonoids and phenolics in free, soluble conjugated and insoluble bound fractions of non-traditional flakes are shown in
Table 2. The highest TFC content was observed in red quinoa flakes (1.06 mg RE/g), where flavonoids mostly concentrated in the free fraction (0.57 mg RE/g). Same situation was also seen in white and black quinoa flakes. The highest TFC content in soluble conjugated fraction was measured in white teff flakes (0.42 mg RE/g), in case of insoluble bound fraction, the highest TFC content was detected in brown teff flakes (0.32 mg RE/g). It may also be pointed out that the highest TFC content was found in insoluble bound fraction of red rice flakes, that value was 73% of total TFC.
Focusing on TPC values, the highest TPC values were observed in brown teff flakes (2.33 mg GAE/g) followed by red quinoa flakes (2.32 mg GAE/g). It could be concluded that insoluble bound fraction of all rice flakes was rich in TPC content, as in the range of 0.27–0.59 mg GAE/g, whereas free fractions of white and brown teff flakes were rich in TPC as 0.91 and 1.01 mg GAE/g, respectively. In case of quinoa samples, flakes with colour layers were rich in free TPC concentrations (0.87 and 0.85 mg GAE/g), while in case of white type quinoa flakes, the highest part of TPC was measured in the insoluble fraction (specifically 38% of total TPC). The concentrations of phenolics are naturally significant in pigmented types of grains [
3,
10,
23]. It must also be considered that currently available composition data on some pigmented grains and flakes are difficult to compare since researches used different extractions and each one was modified for one or more grain varieties. Moreover, the reactivity of Folin-Ciocaulteu reagent with other non-phenolic substances has to be taken into the consideration [
23]. In addition, not much research has been conducted to evaluate the contents of TPC and TFC values in all three fractions of non-traditional flakes.
In this study, two antioxidant assay procedures were applied using ABTS and DPPH free radicals in order to express the antioxidant property with two different mechanisms. The results of antioxidant properties are shown in
Table 3. The TE values of non-traditional pigmented flakes obtained using ABTS and DPPH radicals varied from 0.66 to 1.24 and from 1.18 to 1.59 mg TE/g, respectively. Non-pigmented flakes were poor in antioxidant properties compared to pigmented varieties. Similar results were also observed in some studies of antioxidant activity of pigmented and non-pigmented grains [
11,
23,
25]. It is clear that thermal processes lead to decomposition of phenolics and decrease in antioxidant activity values, for instance this fact was demonstrated in application of parboiling process on pigmented rice grains [
3,
12]. The results of ABTS and DPPH scavenging activity found in individual phenolic fractions were consistent mainly with TPC results (
Supplementary Table S1). The results showed a positive linear correlation between antioxidant activity assays and TPC contents in all fractions while applying ABTS (
r = 0.6487–0.9861) and DPPH (
r = 0.3795–0.9023) radicals. Conversely, non-linear correlation between TFC contents and antioxidant activities was confirmed for soluble conjugated fraction of flavonoids (
r = –0.0695) when DPPH free radical was applied.
3.3. Free, Soluble Conjugated and Insoluble Bound Phenolic Compounds Detected using HPLC
Determining the contents of free, soluble conjugated and insoluble bound phenolic fractions is actually very important from the nutritional point of view. Especially, insoluble bound phenolic fraction arrives to the colon as an intact form. After the fermentation of polysaccharides of cell wall to which they are bound to, they become accessible to colonic microflora and intestinal enzymes [
3]. Major individual phenolics in each fraction are presented in
Table 4,
Table 5 and
Table 6, and total individual phenolic concentrations are also exhibited in
Supplementary Table S2. Chromatograms of individual phenolics for black quinoa flakes can be seen in
Supplementary Figures S2–S4.
Regarding free phenolics, high contents of epigallocatechin (46.90 µg/g), rutin (129.0 µg/g), and sinapic acid (47.20 µg/g) were found in red quinoa flakes, while ferulic acid (150 µg/g) was the most abundant instead of sinapic acid in black quinoa flakes (
Table 4). Concerning the teff flakes, white teff was rich in epigallocatechin (45.50 µg/g) compared with brown variety where rutin (43.50 µg/g) and o-coumaric acid (49.70 µg/g) were predominant phenolics in free fractions. It is worth to notice that high contents of protocatechuic acid in black and red rice flakes (36.80 and 26.20 µg/g, respectively) were recorded. Moreover, quercetin was not detected in white and black rice flakes as well as in teff flakes, neochlorogenic acid was also not assessed in red and black rice flakes, similar to white quinoa flakes. Gallic,
p-hydroxybenzoic, ellagic,
p-coumaric, and cinnamic acids were not detected in red quinoa flakes.
The soluble conjugated phenolic contents of non-traditional flakes are shown in
Table 5. High concentrations of epigallocatechin were detected in flakes prepared from quinoa and teff grains (changed between 31.60 and 261.0 µg/g). In addition, white teff flakes significantly had the highest rutin content (81.0 µg/g). In case of soluble conjugated phenolic acids, high amounts of protocatechuic, vanillic, and ferulic acids were detected in quinoa flakes, while neochlorogenic and syringic acids dominated in teff flakes. The high concentration of sinapic acid (43.20 µg/g) was observed in soluble conjugated phenolic fraction of black rice flakes. Cinnamic acid was not detected in all rice and teff and red quinoa flake samples. Ferulic and
p-coumaric acids were not found in red and black rice flakes and ellagic,
o-coumaric acids and protocatechin ethyl ester were not detected only in red rice flakes. Similarly, kaempferol was not recorded in rice and teff flakes, quercetin was not measured in all rice and brown teff flakes.
Insoluble bound phenolics are presented in
Table 6. Ferulic acid, which was detected in red and black rice flakes (228.0 and 257.0 µg/g, respectively), was the major insoluble bound phenolic acid as well as in teff samples. When the insoluble bound flavonoids were considered, high concentrations of epigallocatechin were detected in red and black quinoa and brown teff flakes (35.90, 40.20, and 45.50 µg/g, respectively). In addition, brown and white teff flakes were rich in catechin (31.80 and 62.30 µg/g, respectively) as well as in quercetin (28.60 and 43.60 µg/g, respectively). Similarly, high contents of quercetin were determined in red and black rice flakes. White and brown teff flakes further contained high amounts of sinapic acid (26.2 and 47.5 µg/g, respectively) and red rice flakes had the highest content of ellagic acid (15.40 µg/g). Insoluble bound cinnamic acid was detected only in black quinoa and brown teff flakes.
Literature data related to phenolics of flake products produced from non-traditional cereals and pseudocereals are not very frequent. It must also be highlighted that currently available composition data on some pigmented grains do not give concrete information, since researchers used different extraction methods for obtaining bioactive substances. Therefore, the polyphenol compound composition is much dependent on grain variety. Based on the above consideration, some results from thermally treated grain measurements were postulated. It was reported in many studies that heat treatment in the presence of moisture (e.g., steaming, boiling, parboiling) resulted in the reduction of the phenolic concentrations [
3,
12,
26]. The main reason responsible for decreasing in phenolic contents after hydrothermal treatment was reported as changing of chemical structure of grain [
12]. Additionally, other factors have an effect on the presence of phenolic compounds, e.g., environmental and agrotechnical conditions or the heat treatment processes. Scaglioni et al. (2014) detected chlorogenic and
p-coumaric acids as the main phenolic acids in free fractions of parboiled rice grains, while insoluble bound phenolic fraction of same grains included the high amounts of ferulic acid [
12]. The major free phenolics determined in hydrothermally treated white teff grains were epigallocatechin, rutin, ellagic,
p-coumaric and gallic acids, while epigallocatechin, rutin, gallic, protocatechuic,
p-coumaric and ferulic acids were the lead phenolics in brown teff grains. Concerning insoluble bound phenolics, white teff contained high values of catechin, epigallocatechin, ferulic, sinapic and ellagic acids, whereas catechin, gallic, vanillic, ferulic and sinapic acids were the main phenolics in brown teff grains [
26]. The principal polyphenolic acids of quinoa grains were reported as caffeic, ferulic,
p-coumaric, gallic and protocatechuic acids [
27].
To discover the main contributors to the antioxidant activity in each fraction the correlations between individual phenolic values and antioxidant activities were calculated. Corresponding correlation coefficients are displayed in
Supplementary Table S3. In terms of individual phenolics in free fractions, the main contributors to antioxidant activity seemed to be
p-coumaric acid >
o-coumaric acid > gallic acid > vanillic acid > ellagic acid > epigallocatechin > syringic and sinapic acids with the correlation coefficients
r ranging from 0.9493–0.3322. Regarding the soluble conjugated phenolic fractions, the main contributors to antioxidant activity were epigallocatechin > caffeic acid > vanillic acid > protocatechuic acid > epicatechin > rutin and gallic acid (
r in the range of 0.6906–0.3677, respectively). Considering the insoluble bound phenolic fractions, the main contributors to antioxidant activities were caffeic acid > protocatechuic acid > epigallocatechin > quercetin > epicatechin > sinapic acid > gallic and ferulic acids with the correlation coefficients between 0.8253 and 0.3759, respectively.
It is widely known that different kinds of interaction between phenolics and free radicals depend on their chemical structure, on specific antioxidant interactions in the tested system such as extraction procedures, etc. Moreover, there are also synergistic and antagonistic interactions between phenolic compounds and other antioxidants or substances included in the matrix of flakes. Clearly, theoretical correlations cannot be accurately reflected in the metabolic pathways in the gastrointestinal tract. In reality other factors as consumption of glycoside forms of phenolics, enzyme activity or digestive factors have influence on these interactions [
28,
29,
30].