1. Introduction
The food industry, including the plant oil industry, generates a great amount of food waste and by-products, which in many cases are not fully valorized, thus generating a major environmental problem [
1]. The need to transform food processing by-products into useful ingredients is part of the circular economy and the zero-waste concept [
2]. After processing, the agro-industrial waste can be used in the food, cosmetic, textile, and pharmaceutical industries. Moreover, some of these waste products and by-products can be considered a source of bioactive compounds, such as phenolic compounds, vitamins, pigments, oils, and various biopolymers such as polysaccharides (including dietary fiber) and proteins [
3]. Press cakes, deriving from oilseeds extraction, represent interesting co-products due to their nutritional value, high biopolymers (proteins and polysaccharides) content, and the presence of bioactive phytochemicals, such as phenolic acids, flavonoids, lignans, and other antioxidant compounds [
4,
5,
6]. The use of oilseed press cakes could be a sustainable alternative to reduce waste disposal and also contributes to the development of new, low-cost products rich in nutrients [
1,
7]. In fact, the application of various oilseed press cakes as a potential source for value addition in conventional food products is already reported [
8,
9]. Moreover, it should be emphasized that these by-products have improved nutritional value (elevated protein, fiber, macro- and microelements, polyphenols contents, and higher antioxidant capacities), as well as functional properties of gluten-free products [
10].
Flaxseed (
Linum usitatissimum L.) has emerged as an ample nutritional and functional food due to its appreciable amounts of high-quality proteins and minerals and exceptionally high content of α-linolenic acid, omega-3 fatty acid, lignans, and dietary fiber [
11,
12,
13]. Numerous studies reported the production of high-quality flaxseed-enriched cereal products with the desired health attributes, exhibiting similar or improved shelf life compared to equivalent products [
12]. For instance, Kaur et al. analyzed the effect of wheat flour replacement with flaxseed flour on the nutritional, functional, and antioxidant properties of cookies and reported that cookies produced with composite flour mixes were higher in protein, fat, ash, and fiber contents than control products [
14]. Moreover, flaxseed-enriched cookies showed higher total polyphenolic content, antioxidant activity, as well as highly acceptable sensory scores. Similarly, Khouryieh and Aramouni evaluated the effect of flaxseed flour addition on the physical and sensory characteristics of cereal bars and indicated that flaxseed flour incorporation substantially enhanced the nutritional qualities of the cereal bars without affecting their sensory and quality properties [
15]. After cold screw-pressed oil extraction, a flaxseed oil cake (FOC) is obtained [
7]. This valuable and cheap by-product is underutilized in terms of food science and human food systems [
16]. Only a few examples of FOC applications for the production of conventional foods are reported. For instance, Sanmartin et al. evaluated FOC as a tool for the improvement of the nutraceutical and sensorial features of sourdough bread [
3]. Their results demonstrated that flaxseed cake-enriched sourdough bread could represent a potential vehicle for bioactive compounds with the possibility of obtaining high-quality products with improved nutritional profiles and desired health attributes. Similarly, Taglieri et al. reported that bread fortified with FOC showed a significant improvement in the nutraceutical profile and high antioxidant activity [
7]. It can therefore be assumed that FOC has a high potential for use as a valuable additive in other food products.
Bakery products, including bread, are one of the most common staple foods willingly consumed all over the world every day [
17]. However, some consumers cannot eat conventional wheat or wheat-rye products due to wheat/gluten-related disorders [
18] or lifestyle, opting for gluten-free (GF) counterparts. Thus, the demand for high-quality GF products is continuously growing, and their market has become one of the most profitable segments of the food industry [
19]. Many commercially available gluten-free breads (GFBs) have some disadvantages, such as unsatisfactory texture, low nutritional value, and short shelf life [
20]. In addition, they are expensive and difficult to access. A characteristic feature of GFBs is their inability to develop a complex three-dimensional network due to a lack of gluten proteins (lack of gliadin and glutenin) [
21]. Thus, to build up a network similar to that formed by gluten and mimic its viscoelastic properties (consequently, the appearance, quality, and sensory properties of bread-like products), the inclusion of other polymeric improvers is a critical factor [
22]. Moreover, the additives should increase the water binding capacity at the dough/batter level resulting in increased loaf volume at the bread level [
23]. Flours and/or starches of various origins (such as rice, corn, potato, and cassava), which are usually included in GFB formulations have a low structure-building ability and are often combined with binding agents such as proteins and hydrocolloids [
24].
In this context, increasing attention is being paid to by-products and co-products deriving from flaxseed [
3,
12,
13]. FOC is gluten-free; therefore, it could be used as an alternative raw material in GF-products, enriching them with a significant amount of proteins and minerals [
25]. Flaxseed oil cake extract (FOCE) is a liquid matrix with unique properties due to its simultaneous flaxseed protein (FP) and flaxseed gum (FG) content, which is also abundant in antioxidants [
26]. In previous works, it was demonstrated that strong FG and FP synergistic water holding and oil binding abilities make FOCE very a promising ingredient to stabilize food systems due to its high emulsifying [
16,
26,
27] and encapsulating ability [
28,
29]. FG resembles functionally Arabic or guar gums more closely than other common gums, and it can be used to replace most non-gelling gums for food and non-food applications due to its ‘weak gel’-like property and remarkable water-holding capacity [
26]. Flaxseed proteins are also investigated for their emulsifying properties [
30]. FP and FG are also responsible for the formation of multiform structure and improved resistance to environmental stresses, causing FOCE to meet the requirements for both proteins and hydrocolloids in the context of GFB enhancement. Thus, based on the technological and nutritional features of FOCE, it could be potentially applied as a gluten replacer in GF breadmaking.
To the best of our knowledge, no studies have been carried out on GFB fortified with FOCE. We hypothesized that supplementation level could change the extent of influence exerted on GFB properties. Thus, the aim of the presented study was to produce, for the first time, FOCE-enriched GFBs and examine the influence of water replacement levels on their nutritional value, antioxidant properties, and sensory features.
2. Results and Discussion
The proximal chemical composition and energy value of experimental GFBs with FOCE replacing water in the gluten-free blend are presented in
Table 1. The control GFB, composed mainly of starchy ingredients, was characterized by a low nutritional value, mainly due to the low proteins content (
Table 1). A similar characteristic was noticed in other experimental GFBs composed of basic ingredients [
31,
32]. However, it should be emphasized that reduced nutritional adequacy is a worrisome trend observed in commercially available GFBs worldwide [
20]. Comparing packaged gluten-free products to their regular gluten-containing counterparts, similar conclusions could be drawn [
33,
34]. These disadvantages negatively affect the nutrient status and health of patients on a strict gluten-free diet and therefore represent an urgent need to further efforts aimed to improve the nutritional quality of GFB.
In the presented research, a laboratory-produced FOCE was used [
26], which is an extract from the post-production of flaxseed oil side-product, in order to improve the technical quality and nutritional value of GFB. Our research follows the sustainable food trend that is focused on the application of food processing side-products as a potential source of value addition to foods, resulting in novel foods of improved nutritional and nutraceutical value [
9,
31]. In fact, Taglieri et al. reported a significant improvement in the nutraceutical profile of the bread fortified with flaxseed cake in a dose-dependent manner [
7]. Water substitution by FOCE in the experimental GFB formula caused a significant (
p < 0.05) increase in protein content in the obtained products (
Table 1), in particular, FOCE100% was 60% richer in proteins than the control. In addition, in breads enriched with the highest FOCE percentage (FOCE75% and FOCE100%), a significant (
p < 0.05) increase in fat content was determined; however, in practice, this increase was relatively small and amounted to about 0.4 g/100 g (
Table 1). On the other hand, the carbohydrate content in the samples containing FOCE was significantly reduced (
p < 0.05).
Flaxseed proteins are known for their valuable amino acids composition. They are a source of arginine, aspartic acid, glutamic acid [
35], cysteine, and methionine that were shown to improve the antioxidant status; thus, may have health-beneficial effects [
36]. Changes determined in macronutrients content in experimental GFBs, in particular in proteins and fat, resulted directly from their relatively high content in FOCE (14 mg/mL of protein, 6.5 mg/mL carbohydrate, and 9.5 mg/mL of other extractable compounds) [
27]. This had a direct impact on the energy value of the obtained baked goods (expressed in KJ and Kcal), which was lower compared to the control.
The mineral composition of FOCE and the experimental GFBs is presented in
Figure 1 and
Figure 2, respectively. As expected, FOCE was a very rich source of potassium (K = 1843.34 µg/g) and contained a high amount of phosphorous (P = 88.387 µg/g) and magnesium (Mg = 52.86 µg/g) (
Figure 1). Zinc (Zn) was the dominant microelement in FOCE, followed by copper (Cu) and small amounts of iron (Fe) and manganese (Mn) (
Figure 1). The qualitative and quantitative minerals profile determined in FOCE reflects the characteristics of the raw material it derives from. According to the literature, flaxseed has a high amount of K (5600–9200 mg/kg) and is a good source of P (650 mg/100 g), Mg (350–431 mg/100 g), and Ca (Ca = 236–250 mg/100 g); however, it has a low amount of sodium (Na; 27 mg/100 g) [
37,
38].
The use of FOCE as a liquid component of the gluten-free blend resulted in the significant enrichment of GFBs in K. Its amount rose with the increasing FOCE to water ratio, and in FOCE100% K content was 60% higher than in the control (
Figure 2A). High K intake is linked to improvements in cardiovascular diseases and is inversely related to blood platelet aggregation, free radicals in the blood, and stroke incidence [
38]. Recently, Stone et al. [
39] conducted a short-term clinical trial assessing the effect of increased K intake from different sources on blood pressure and microvascular outcomes in pre-hypertensive-to-hypertensive (systolic blood pressure > 120 mmHg) men and women. They observed a greater change in systolic blood pressure over time between the group on the K-rich diet (whose source were baked/cooked potatoes) compared with controls (−6.0 mmHg vs. −2.6 mmHg;
p = 0.011) and concluded that increasing K intake might be beneficial for individuals with a higher risk of cardiometabolic diseases. Experimental GFBs with FOCE were characterized by a significantly (
p < 0.05) higher content of Mg and P than the control bread (
Figure 2A). On the other hand, the Na content decreased significantly with the increasing amount of FOCE in the experimental GFBs.
As shown by the analysis of atomic absorption spectrometry, FOCE was not abundant in the microelements (
Figure 1). Among the analyzed microelements, only Zn content exceeded 1 µg/g, while the content of Cu, Fe, and Mn was very low. For this reason, the experimental GFBs with FOCE were characterized by a significantly (
p < 0.05) lower amount of all micronutrients compared to the control bread (
Figure 2B). Therefore, it is possible that other components of the bread formula and water were the main source of micronutrients in the GFB rather than FOCE itself.
Technological parameters and the appearance of experimental GFBs are presented in
Table 2 and
Figure 3, respectively. The specific volume of the control bread was similar to results reported previously [
31]; however, in comparison with a regular wheat bread, whose specific volume ranges from 3.5 to 5.5 cm
3/g [
40,
41], the value of this parameter determined in the present study (2.39 cm
3/g) was much lower. The use of FOCE in the experimental GFB formula resulted in a significant (
p < 0.05) increase in the specific volume and height/width ratio, while the density of the obtained breads was reduced (
Table 2). These changes were especially distinct in the case of the FOCE100%, whose specific volume was nearly 30% higher compared to the control, while its density was reduced by about 20%. The volume of bread representing the ability of the dough to expand without losing gas retention affects its specific volume, which is the primary determinant of the technological quality of the bread. The evidence obtained in the present study (
Table 2) indicated that the increasing percentage of FOCE in the experimental formula promotes the quality of the obtained GFBs. The beneficial impact of FOCE on the technological features of GFBs could result from the chemical characteristics of FOCE. FOCE is derived from flaxseed, which besides being ample in nutrients, contains dietary fiber, in particular cellulose, mucilage gums, and lignin [
37,
38]. On the one side, the enrichment of regular bread with nutrient-dense and fiber-containing material due to possible gluten dilution, competitive water-binding [
42] or physical disruption of the gas cells and gluten network [
43] was shown to have a detrimental effect on the dough viscoelastic properties and bread volume. On the other side, soluble fibers of flaxseed had positive effects on the wheat dough structure and loaf volume [
44,
45]. The high value of specific volume, together with the proper aeration of GFBs crumb resulting from relatively small pores regularly distributed across the crumb (
Figure 3), are required to obtain products of satisfactory sensorial quality [
3]. In fact, FOCE increased the number of cells in GFBs. In addition, compared to the control sample, there was a significant reduction in the average cells area and perimeter but an increase in their circularity in FOCE-enriched GFBs. This observation is in line with the results reported by Aranibar et al. [
9], who analyzed the influence of chia by-products on the structure of wheat muffins. Sabanis and Tzia [
46] indicated that soluble fibers of a high water-binding capacity improve the retention of water during dough mixing, which evaporates during baking, increases the internal pressure, and consequently increases the volume of the loaf. Therefore, an improvement in the technological parameters of experimental GFBs could result from the functional ingredients of FOCE. Drozłowska et al. reported that FOCE has high stabilizing potential due to the different water-holding and oil-binding capacities of flaxseed gum and protein and the effective decreasing of interfacial tension [
26]. This probably facilitated the creation and entrapping of carbon dioxide in the pores during baking. Moreover, as the GFBs formulations also contained oil, their enhanced technological features can also be presumably linked with the emulsifying activity of FP, as proteins preferentially adsorb to the oil–water interfaces and form a viscoelastic film, which provides physical stability to the emulsions during their subsequent processing and storage [
26,
27]. Furthermore, the baking process takes place at elevated temperatures, during which the thermal partial or complete denaturation of proteins can occur, depending on the temperature level and exposure time. The main process causing denaturation is the exposure of previously unexposed hydrophilic molecules and sulfhydryl groups to water. These groups are responsible for hydrophobic interactions with oil phases, while hydrophilic amino acid residues located on the surface can absorb water. Due to this structure, which is the result of controlled heat treatment, FP can interact with both oil and water phases and can act as an effective emulsifying agent to stabilize the phase interfaces, similar to some other heat-modified proteins [
27]. In fact, it was shown that denatured FOCE has a higher ability to stabilize oil-containing systems than the native one [
47].
The results of the instrumental color analysis conducted in the control and GFBs enriched with FOCE are presented in
Table 2. Due to the very uneven and inhomogeneous crust surface of the experimental breads (
Figure 3), only the crumb color was analyzed. The crumb of the control bread was light (
L* = 71.29) and of creamy-to-beige color. This result corresponds well with the results of our earlier study (
L* = 71.58) [
31]. The starch-based GFBs usually have a whitish crumb and light-colored crust perceived as pale and unattractive when compared with regular wheat bread [
48]. According to the instrumental color analysis, FOCE had a significant impact on the color of the crumb of experimental GFBs. As reported in
Table 2, FOCE100% showed the highest values of both lightness (
L*), as well as the blue–yellow components (
b*) and red–green components (
a*). All these parameters increased as a function of the concentration of FOCE in the bread formula. This observation is in agreement with the results of Tavarini et al. [
36], who also reported that the color of the crumb of bread was significantly affected by FOC addition. The shade of the crumb depends mainly on the ingredients used in the formulation. FOCE was of a slightly creamy-to-beige liquid (data not shown); therefore, apparent differences in crumb color were not easily distinguished by the human eye (
Figure 3). However, when the metric distances among the coordinates were calculated (ΔE) [
49], the hue of the crumb of the control bread was evidently different (1 < ΔE < 3 or ΔE > 3) from the hue of the FOCE-enriched GFBs crumbs (
Table 2).
The content of the total phenolic content (TPC) and the antioxidant power were analyzed in both FOCE and GFBs with different percentages of FOCE, and the obtained results are reported in
Table 3. It was not surprising that FOCE, due to the production method, was characterized by a relatively low content of TPC (0.16 mg GAE/g DM) when compared to flaxseed cake [
7] and whole flaxseed flour [
14]. Nevertheless, FOCE was characterized by the antioxidant capacity that was confirmed by ABTS, DPPH, and FRAP assays, while not by PCL-ACW (
Table 3). On the other hand, FOCE showed the PCL-ACL activity which is associated with the lipophilic antioxidants (
Table 3), possibly fat-soluble vitamins (A and E). γ-tocopherol is an antioxidant providing protection to cell proteins and fat from oxidation and is related to the reduced risk of Alzheimer’s disease [
50].
The TPC content of GFBs containing FOCE doubled compared to the control (
Table 3), but the increase noted was not directly related to the amount of FOCE replacing water in the gluten-free blend. While the increase of the antioxidative activity, determined by ABTS, DPPH, FRAP, and PCL-ACW assays, was proportional to the level of FOCE in the GFB formulas. In particular, FOCE100% showed the highest antioxidant power in comparison to the control (
Table 3). Results obtained in the present study are in accordance with observations by Sanmartin et al. [
3], who investigated the nutraceutical properties of wheat bread fortified with FOC. They found that the antioxidant power significantly increased (
p < 0.001) with the growing percentage of flaxseed cake added to the flour mix. Additionally, Man et al. [
51], in their study, aimed to examine the effects of partial replacement of wheat flour with roasted flaxseed flour and found that the total phenolic content and the antioxidant activity increased with increasing amounts of the roasted flaxseed flour in the biscuits. Taglieri et al. [
7] also reported an increased level of total phenols and anti-radical activity in FOC-enriched bread.
The FOCE used in the present study was characterized by moderate antioxidant activity, but it turned out to be a valuable material influencing the bioactive characteristics of GFBs. The enhanced antioxidant capacity detected in FOCE-enriched GFBs could potentially result from FOCE composition [
27,
29]. On the other side, the whole flaxseed is known to be an excellent source of lignans (predominantly secoisolariciresinol diglucoside) and other phenolic compounds (ferulic acid, syringic acid, cinnamic acid, vanillic acid,
p-coumaric acid, and gallic acid) of antioxidative properties [
52,
53,
54]. Secoisolariciresinol diglucoside lignan-enriched flaxseed powder was demonstrated to reduce body weight and fat accumulation, improve the lipid profile, and lower blood pressure in the animal model [
55]. Moreover, the population-based case–control observational study [
56] reported that the consumption of rich in lignans flaxseed was associated with reduced breast cancer risk. On the other hand, substracts delivered by FOCE to the GFB matrix became the reagents of the Maillard reaction. In general, this non-enzymatic browning reaction involves two main types of reactants, reducing sugars and amino acids; however, the condensation reactions between amino acids and lipid oxidation products may also form Maillard reaction products (MRPs), and the role of lipids in the Maillard reaction is similar to the role of reducing sugars [
57]. MRPs, especially melanoidins, are reported to have antioxidant activity [
58]. Therefore, FOCE used as liquid replacing water in the GFB formula can promote the formation of MRPs during baking. In fact, the elevated temperature is reported to promote MRPs formation in FOCE during spray-drying [
27,
29]. The bread crust present in the analyzed samples, while making up only a small fraction of the total bread weight, was the major contributor to the observed increase in the antioxidant capacity of GFBs with FOCE.
Experimental GFBs were subjected to quantitative descriptive analysis (QDA), the results of which are presented in
Table 4 and
Figure 4. The FOCE used in the bread blend significantly influenced (
p < 0.05) the appearance of the analyzed breads. Regardless of their amount, all GFBs containing FOCE were characterized by a regularly distributed pore collocation (from 7.17 to 7.73 AU) in comparison to the control (2.57) (
Table 4). Moreover, the color of the crumb of GFBs with FOCE was more beige. For the pore dimension, no significant difference was encountered between the control sample and GFBs with lower FOCE percentages (FOCE25% and FOCE50%); however, in samples with a higher FOCE to water ratio (FOCE75% and FOCE100%) the dimension of the pores was 40–60% bigger than in the control. Although crumb cellular structure has a significant effect on the bread quality, its mechanical properties are weakly dependent on cell size while being influenced by the distribution of cells [
59]. As indicated in
Table 4, the control bread was of lower quality (lower specific volume and denser crumb) than the GFBs with FOCE. The beneficial effect of FOCE used in the experimental formula was proven by an enhancement in the technological properties (
Table 2) and was additionally reflected in the visual structure of the crumb (
Figure 3) with evenly collocated pores (
Table 4).
When assessing the odor features of the experimental GFBs, panelists identified four odor attributes: acid, oily, wheat bread, and sweet. All GFBs were perceived as similarly slightly sweet (ranging from 1.33–1.49 AU) and oily (approximately 2 AU), and their odor resembled wheat bread (
Table 4). The only difference between the samples resulted from an acid odor that was significantly less intensive (
p < 0.05) in the GFBs containing higher levels of FOCE (FOCE75% and FOCE100%) in comparison to the control. The fresh flaxseed had a unique, slightly nutty flavor [
60], which could be pleasant for the consumers; however, this odor was not detected in the FOCE-enriched GFBs. Nevertheless, the odor of the obtained baked goods improved when FOCE was used in the formula. Assessing the taste, panelists indicated that GFBs containing FOCE were perceived as significantly less salty than the control (
Table 4). This results directly from the low sodium content of FOCE and its decreasing content in GFBs with increasing FOCE content (
Figure 2). In contrast, the acid taste, which was barely perceptible in the control (1.54 AU), was more distinct in GFBs with FOCE. In particular, the higher the ratio of FOCE to water in the gluten-free mixture, the more intense the acid taste was perceived. This observation is in line with the results of Taglieri et al. [
7], who reported increased acidity in FOC-fortified breads due to the presence of unsaturated free fatty acids.
The texture plays a key role in the consumers’ preferences for foods. In the present study, GFBs were evaluated through their behavior in the mouth while eaten and the elasticity while pressing by finger. The texture parameters evaluated in the mouth (chewiness, adhesiveness, moisture) did not differ meaningfully for all experimental breads, while when bread crumb was pressed by a finger, the GFBs with FOCE were more elastic than the control (
Table 4). This can be related to the relatively high proteins content in FOCE (
Table 1) and probably to the soluble fibers that may bind water, thus influencing the textural attributes of the obtained product. Although compared to the control, some sensory attributes of the GFBs with FOCE deteriorated (acid taste), the appearance, aroma, and texture resulted in a high overall appreciation of GFBs with FOCE (
Figure 4). Among the experimental GFBs with FOCE, the highest scores in overall quality obtained FOCE75% (5.66 AU). This result was almost twice as high in comparison to the control bread (2.88 AU). Taking into account the above results of the sensory analysis, it can be concluded that the higher level of FOCE in the GFB formula allows us to obtain a product with a favorable appearance and higher sensory quality compared to both the control containing only water and the bread with the lower content of FOCE (FOCE 25% and FOCE 50%).