Rheological characteristics of dough
The analysis of viscoelastic properties of the dough allows evaluating the influence of additives, and other modifications of recipe, on rheological properties of the dough. Figure 1a demonstrates mechanical spectra of control dough and samples in which part of starch was replaced with acorn flour. Figure 1b shows the respective changes in phase shift tangent at varied angular frequency. All dough samples exhibited the domination of elastic properties over viscous ones (G′ > G″) in the whole applied range of angular frequencies. It was confirmed by the values of phase shift tangent tan δ < 1 (Fig. 1b). Relative position of the curves representing moduli and the values of tan δ > 0.1 indicate properties typical for weak gels. It confirms earlier observations made for gluten-free dough based on starch [23, 28]. Partial replacement of starch with acorn flour resulted in a significant increase in both moduli (Fig. 1a), which was proportional to the extent of this replacement. This was accompanied by the drop in the values of phase shift tangent (Fig. 1b), which was also corresponding to the amount of acorn flour used for starch replacement, and reflected by the constants of power-law equations used to describe experimental data (Table 1). The values of K′ and K″ parameters increased significantly with rising level of acorn flour in the dough, which corresponds to the increase in G′ and G″. A parallel drop observed in the case of n′ and n” parameters, which diminished with added acorn flour, indicates lesser dependence of moduli on angular frequency. The values of phase shift tangent at δ = 1 rad/s collected in Table 1 were significantly lower for samples with acorn flour in comparison with control dough. Although they indicate a lack of statistical variance at 1 Hz, at the same time, their values at higher frequencies significantly differ, indicating that growing share of acorn flour in the dough results in its greater elasticity and smaller susceptibility to applied stress. One of the factors causing such changes in rheological characteristics of the dough is water absorption capacity, which is significantly different for starch and acorn flour. The flour contains significant levels of fiber, which is characterized by high water absorption. The increase in an amount of absorbed water results in a greater volume of hydrated molecules, which are more likely to interact under the applied stress. Another factor which could influence rheological characteristics of the dough with acorn flour is the presence of other non-starch structure-forming components. The applied flour contains approximately 5 % protein and other organic molecules classified as soluble dietary fiber. All these components may interact with hydrocolloids present in a dough mix (guar gum and pectin) strengthening structure of the dough and further bread crumb.
Chemical and physical characteristics of bread
Basic parameters characterizing analyzed bread are collected in Table 2. Partial replacement of starch with acorn flour caused a significant increase in the content of protein, fat and dietary fiber, and a decrease in total carbohydrate content in comparison with control bread. The extent of these changes was parallel with the level of replacement which seems obvious. The application of acorn flour resulted in an increase in protein content by 37–105 %, which seems to be especially important, taking into account protein deficiency in gluten-free products. The increase in fat level varied between 24 and 72 %, and in dietary fiber—76–220 %, while a decrease in carbohydrate content ranged between 5 and 13 %. The changes were the consequence of chemical composition of acorn flour, which contained more analyzed components than starch, with the exception of total carbohydrates. In comparison with wheat flour, acorn flour contains less protein (54 vs. 80–134 g kg−1) and starch (458 vs. 618–739 g kg−1), but more fat (52 vs. 13–22) and total fiber (181 vs. 38 g kg−1), including its soluble (36 vs. 9 g kg−1) and insoluble (145 vs. 29 g kg−1) fractions [29–32]. The results could hardly be compared with wheat bread, as its composition significantly depends on applied formulations which differ significantly among authors. However, it could be observed that gluten-free bread with acorn flour contained less protein (16,7–25 vs. 73,7–78,9 g kg−1) and total carbohydrates (400–439 vs. 532–735 g kg−1) than wheat bread, while the level of fat (23,33–32,40 vs. 5,6–7,9 g kg−1) and dietary fiber (47,14–85,49 vs. 11,5–17,6 g kg−1) was elevated in this case [33–35].
In most cases the application of acorn flour significantly influenced color and other physical characteristics of the loaves. Significant increase in bread volume could be observed, when the level of starch replacement with acorn flour was the lowest, while larger amounts of the flour caused a decrease in bread volume. This suggests that limited amounts of water-binding substances (e.g., protein and fiber) present in acorn flour positively influence structure and gas retention capacity of rising dough, which results in greater volume. However, too strong dough, obtained at higher levels of those substances, due to increased water binding and/or changes in density, could restrain proofing process. The sample with 20 % addition of acorn flour was characterized with the highest porosity and largest number of pores larger than 5 mm (Table 2; Online Resource 1). Although porosity of bread with higher amounts of acorn flour was smaller, it still over-passed the value for control sample. Substantial (40 and 60 %) share of acorn flour caused decrease in large pores (larger than 5 mm) to values comparable or lower to as those observed for control. The smallest number of pores per volume was found for the sample with 20 % addition of acorn flour, which was due to its highest volume and porosity. The values of this parameter measured for two other samples with acorn flour were also smaller in comparison with control. Improvement of structure of gluten-free bread, reflected also by its greater volume, was earlier found after addition of soy flour, which was a source of structure-forming protein and active enzymes . Also Miñarro et al.  received gluten-free bread with improved quality (including sensory parameters) after application of chickpea flour.
Color of a product significantly affects its consumer acceptance. Spectrophotometric measurement of color may be precisely expressed in a three-dimensional color space, which corresponds to its perception by human eye. The results acquired for bread crumb of control sample and products supplemented with acorn flour are represented in Table 2. The highest value of L* corresponding to lightness was obtained for control sample. Such a high lightness of gluten-free bread is especially typical for bakery products based on starch . Application of acorn flour significantly changed this value, decreasing L* in a way proportional to the level of supplementation. The involvement of acorn flour in bread formulation had also an influence on other color parameters. In the case of a*, which was negative for control, the addition of acorn flour resulted in its change to positive values, signifying predominance of red over green in bread color. However, the differences between samples with varying levels of acorn flour were small. In all cases, the value of b* was positive, which corresponds to that yellow was more intensive than blue both in control bread and acorn flour supplemented samples. 20 % replacement of starch with acorn flour caused an increase in crumb yellowness, while further additions caused a slight drop in this value. Instrumental analysis of crumb color revealed a significant influence of applied acorn flour on bread crumb appearance. It seems that a reduction of lightness could be most beneficial in terms of sensory attractiveness.
Bread texture is an important quality parameter. Because of relatively neutral taste of such bakery products, it could often be taken as a key factor for bread acceptance. It is also the most obvious indicator of the changes, which occur in crumb structure during storage due to the loss of moisture, starch retrogradation and other physicochemical interactions between bread constituents. Supplementation of gluten-free bread with various types of plant flour could modify these phenomena and in this way affect crumb texture. The changes in texture of analyzed samples during storage are shown in Fig. 2. In most cases bread hardness increased significantly over the analyzed period. However, an addition of acorn flour modified the extent of these changes. Two-way analysis of variance proved statistically significant influence of supplementation level (p < 0.001), time of storage (p < 0.001) and interaction of these two factors (p < 0.001). The replacement of starch with acorn flour at 20 % level had a beneficial effect on hardness over storage. On the day of baking, the loaves with 20 and 40 % were comparably firm as a control sample. On the following days, the sample with 20 % addition of acorn flour was significantly softer than all other samples. It was also observed that in this case, the changes in crumb hardness between 2 and 3 days of storage were not statistically significant. In the case of bread with 40 % acorn flour, the increase in hardness over storage was comparable to control. This type of bread revealed best consumer acceptance in terms of structure and porosity (Fig. 3). Bread with 60 % was much harder than all other samples on the day of baking, and staled so quickly, that after two days of storage was more than twice as hard as other samples. The influence of non-cereal flours on gluten-free bread was earlier reported by Alvarez-Jubete et al. , who observed a decrease after supplementation of the dough with buckwheat and quinoa flours. According to these authors, the reduction of crumb hardness is caused by the incorporation of natural emulsifiers, present in such flours. It could be also observed that there is a correlation between bread volume and crumb hardness , which has been confirmed by the above data, as the bread with the highest volume, which contained 20 % acorn flour (Table 2) had the softest crumb (Fig. 2), and one with the lowest volume, containing 60 % acorn flour, was the hardest (Table 2). Springiness (data not shown) was not variable in a statistically significant way, and two-way ANOVA proved lack of the influence of addition level and storage time, as well as interaction of these two factors, on its values.
Crumb cohesiveness was the next analyzed texture parameter (Fig. 2). Although two-way analysis of variance proved a significant influence of addition level (p < 0.001) and storage time on this parameter, the values did not differ much between samples. Supplementation of bread with 20 % acorn flour had no statistically significant influence on its cohesiveness in comparison with control. Only at larger quantities of the additive, its value was decreased in a statistically significant way, especially on the second and third day of storage.
Also chewiness was influenced by the addition level and storage time as well as their interaction in a statistically significant way (p < 0.001). In general, on the day of baking, the values of chewiness for bread containing 20 and 40 % share of acorn flour were on the same level as control. In case of 20 % addition, the increase in chewiness after storage was not statistically significant, while for bread with 40 % acorn flour and control it could be observed only on the second day of storage. In the case of a sample with 60 % acorn flour, the pattern was slightly different. The initial value of chewiness was in this case significantly higher than for other samples on the day of baking and on the second day of storage. The drop was observed on the third day of storage, which could be connected to a decrease in crumb springiness observed for this sample at the end of storage period.
Bread crumb thermograms registered during heating exhibit a peak referring to swelling of retrograded amylopectin. The range of temperatures for this transition varied between 49.2 and 55.0 °C for onset, 59.6–63.2 °C for peak maximum and from 75.0 to 80.0 °C for its end (data not shown). Basing on the results of applied two-way ANOVA, it was, however, found that neither the level, time of storage, nor interaction of both these factors had statistically important effects on these values. The influence was only observed for transition enthalpy (p < 0.001), which could be seen on Fig. 4. For fresh bread (on the day of baking), the addition of acorn flour, at all applied levels, caused a decrease in enthalpy value in comparison to control, although the exact level of this addition was not statistically important. Significant reduction of melting enthalpy in comparison with control could also be observed on the second day of storage for samples with 40 and 60 % share of acorn flour; however, the difference between these values was not statistically important. On the third day of baking, the differences between enthalpies measured for samples with varying levels of acorn flour were observed, and a decrease in these values was proportional to applied replacement level. The reduction in recrystallisation enthalpy of amylopectin could be caused by two factors. Partial replacement of starch with acorn flour causes a decrease of total amount of amylopectin in a system, and an addition of acorn flour results in an introduction of new structure-forming substances, which could interact with amylopectin and retard its recrystallisation. However, staling is a complex process, which involves recrystallization of amylopectin only as one of many factors; thus, crumb hardening should not be identified with amylopectin retrogradation.
Sensory assessment of bread
The results of sensory assessment of analyzed bread are represented on Fig. 4. Most parameters were positively influenced by the application of acorn flour, showing greater consumer acceptance than the control sample. The replacement of 20–40 % of starch in bread recipe with acorn flour had a beneficial influence on bread appearance. Further addition of the flour (60 %) caused a decrease in this parameter, but the sample received better scores than control. Taking into account structure and porosity, the worst scores were given to control sample, while bread with 40 % share of acorn flour was most accepted. It revealed medium porosity, pore density and percentage of pores larger than 5 mm (Table 2). Color of crumb was much more acceptable in the case of bread supplemented with acorn flour in comparison with control, most probably due to reduced paleness (Table 2), which is commonly regarded as an indicator of highly refined, less healthy products. Smell was also more acceptable for supplemented loaves than for control, but in this case, the acceptability observed at 20 % addition level was much higher than for all other samples. Bread with 60 % acorn flour and control were the least acceptable in terms of their taste, which is probably due to easily identified, odd flavor typical either for hydrocolloids, or acorns, but not bread. Both other samples were much better accepted, which proves that limited addition (up to 40 %) of acorn flour positively impacts sensory parameters and overall acceptance of gluten-free bread.