
¨
 `
 ` starches, minimum dough torque during #
|`  

\$` cooling until 50 °C (C5), and gelling rate ('`

#### *2.4. Breadmaking Process*

Potato starch (16 g), pectin (4 g), and calcium citrate (5.8 g) were added to corn starch (60.3 g). The investigated protein powders (12 or 24 g) were added substituting the appropriate amount of corn starch. Subsequently, sugar (5 g), dried yeast (5 g), and salt (1.5 g) were dissolved separately in tap water (80 mL) and added to the dry ingredients together with oil (2.5 g). The batter was mixed for 12 min in a Brabender farinograph SEW (Brabender OHG, Duisburg, Germany). The resulting batter was proofed for 20 min in a proofing cabinet (35 °C/70% relative humidity). Then, the batter was divided into 200 g samples, placed in baking tins and proofed for another 20 min under the same conditions. The baking was carried out in convection oven at 200 °C for 25 min (Eurofours type 10AB20W2, Gommergnies, France). The obtained loaves were divided into two groups: the first was analyzed directly after cooling to room temperature (2 h); and the second was packed in polyethylene bags and stored 24 h at ambient temperature (22 ± 2 °C) for crumb structure analysis and sensory evaluation.

#### *2.5. Characteristic of Gluten-Free Bread*

#### 2.5.1. Chemical Composition and Nutritional Value

Bread moisture [20], proteins [21], ash [22], and fat [26] contents were determined following the standard methods. Total carbohydrates were determined by difference subtracting 100 g minus the sum of protein, ash, and fat expressed in g/100 g. The energy value was calculated by multiplying the amount of each macronutrient by the corresponding conversion factor (4, 9, and 4 for protein, fat, and carbohydrates, respectively) [27]. The percentage of energy delivered by protein was calculated.

#### 2.5.2. Technological Parameters

The bread loaf weight and volume (rapeseed displacement method) were determined. The specific volume and bake loss were calculated [18]. The height/width ratio of the central 10 mm slices was determined using Image J software (National Institutes of Health, Bethesda, MD, USA) [28]. The energy status of water in bread samples was measured as water activity (aw) using an Aqua Lab

Series 3 (Decagon Devices Pullman, USA) at 22 °C. The crust and crumb color was analyzed as previously described using a Minolta colorimeter. Values were the mean of nine replicates.

#### 2.5.3. Crumb Texture Properties

Texture profile analysis (TPA test) of the 24-h stored bread crumbs was performed using Texture Analyzer TA-XT2i (Stable Micro Systems, Surrey, UK) equipped with a 30 kg load cell and 25 mm aluminum cylindrical probe. Three middle slices of each loaf were evaluated. A 10 mm thick bread slice underwent a double compression test up to 50% deformation of its original height at a crosshead speed of 1 mm/s and a 30 s gap between compressions. From the two-bite texture profile curve the following texture parameters were obtained: hardness, adhesiveness, springiness, cohesiveness, chewiness, and resilience.

#### *2.6. Statistical Analysis*

One-way analysis of variance (ANOVA) and Fisher's least significant difference test (LSD) using Statistica 7.1 (StatSoft, Kraków, Poland) were applied to define significant differences (*p* < 0.05) between samples.

#### **3. Results and Discussion**

#### *3.1. Chemical, Physical and Functional Characteristic of Dairy Powders*

Detailed characteristics of selected dairy powders (NAS, CAS, ISO, OPT), including chemical composition, and physical and functional properties, are provided in order to evaluate the effect of each individual powder on the gluten-free batter and bread (Table 1). High concentration of proteins was confirmed for all dairy ingredients tested (Table 1). Total proteins content exceeded 85%, with distinguishing concentration in sodium caseinate, containing over 94% of proteins. According to the suppliers, all dairy ingredients tested were poor in fat (1.0%–1.5%) and lactose (0.5%–2%). Caseinates (CAS and NAS) were richer in mineral compounds (ash; near 4%) than whey proteins tested.

The measurement of particle size showed that the mean size of whey proteins particles was significantly higher in comparison with particles of caseinates tested, and ISO showed the highest particle size (Table 1). The color parameters of dairy powders indicated that they were very light, white, or creamy powders, characterized by high *L\** value, above 91, and in the case of NAS, near 94. The negative value of parameter *a*\* (below-` 
  # 
 # *b*\* values described the yellowish color, being significantly higher for the whey proteins.

The ability to bind and hold water without syneresis is critical in many foods, thus, hydration properties were determined in the dairy proteins. Results indicated that CAS followed by NAS showed the highest value of WAI at room temperature (Table 1), however, that trend was reversed when this parameter was determined after heating. Although caseins are relatively hydrophobic, they contain regions of high, medium, or low hydrophobicity [29] and they bind about 2 g water/g, which is typical of proteins [30]. Whey proteins in their native form exhibit little water-binding capacity [31]. Nevertheless, heat-denaturated whey proteins, although retaining most of their

secondary structure, are linked together and can have a perceived hydration of over 10 g of water/g protein, compared with 0.2 g water/g protein for whey protein in their native globular state [29]. Processing has also a considerable impact on solubility of dairy proteins (Table 1). At room temperature, whey proteins tested were soluble in very high degree, however the heat treatment impaired the solubility of these proteins. WSI of ISO and OPT decreased by over 80% and 70%, respectively. Whey proteins are susceptible to denaturation at temperature higher that 70 °C. When heated, the tertiary structure of protein globules are destroyed, then unfolding of the protein molecules and new protein-protein interactions occur [32]. In case of both caseinates tested the opposite situation was observed. Here, after boiling the WSI values for CAS and NAS increased by more than 15%. Analyzing the OAC, significant differences were found between the dairy proteins tested (Table 1). The highest OAC was observed for CAS, followed by ISO, and OPT, whereas NAS has the lowest oil absorption capacity, probably due to its highest protein concentration. According to Kinsella [33], the mechanism of fat absorption has been attributed mostly to the physical entrapment of oil, but as well may be influenced by lipophilicity of the protein concentrate. In protein powder foods fat binding can be influenced by the size of powder particles [34], however in the dairy proteins tested such correlation was not observed.

#### *3.2. Effect of Dairy Powders on the Gluten-Free Dough Characteristics*

The effect of the dairy powders at two different levels (12% and 24%) on the rheology of gluten-free dough was studied by using the Mixolab, where dough behavior subjected to shear and temperature constraints is recorded [25]. The Mixolab plots obtained in the presence of the dairy powders are shown in Figure 1.

Gluten-free doughs showed very low consistency during mixing, which only increased after heating when starches present in the recipe started to gelatinize. Consistency enhanced during heating till the rupture of the starch granules where the maximum consistency was detected. Further heating led to a decrease in the consistency derived from the starches stability during cooking and when temperature decreased the amylose retrogradation associated to cooling was observed as a consistency increase. This pattern agrees with previous description of the compounds changes reported by Rosell *et al*. [25]. The trends observed on the plots indicated that gluten-free dough rheology was clearly governed by starch changes during heating and cooling. The consistency of the gluten-free dough is greatly dependent on the amount of water or hydration, showing very low consistency during mixing when water adsorption is higher than 90% [35]. It must be remarked that during heating two consistency peaks were detected that were associated to the different gelatinization temperature of corn and potato starches. Matos and Rosell [36] also detected different peaks depending on the type of starch and their diverse pasting temperatures, being 65.4 °C for potato starch, 69.9 °C for corn starch and 70.2 °C for rice flour. In addition, Krupa-Kozak *et al*. [18] observed two different slopes during heating, the first one detected around 28–31 min corresponded to potato starch gelatinization, whereas the second one observed from 31 to 36 min was ascribed to corn starch gelatinization, which agrees with results of the present study. The patterns obtained during mixing, overmixing, pasting, and gelling greatly varied with the protein source and the level of proteins (Figure 1). It was not possible to record the consistency of the 24% NAS containing dough with the Mixolab due to its high consistency. The inclusion of the

dairy proteins decreased the dough consistency during the heating-cooling stages, with the exception of NAS added at 12% that showed higher consistency after cooling. Bonet *et al*. [37] found that the addition of protein sources (gelatin, egg, and lupine) to wheat flour significantly changed the Mixolab plot and the effect was attributable to the nature of the proteins. The presence of different proteins and starches modifies protein–protein interactions and also the starch gelatinization and the gelling processes [25,35,38]. Regarding the level of the proteins added, dough consistencies decreased with increasing level of proteins.

**Figure 1.** Effect of dairy proteins on the gluten-free dough consistency determined by Mixolab® device. Control: unfortified gluten-free dough; CAS 12: gluten-free dough with 12% of calcium caseinate; CAS 24: gluten-free dough with 24% of calcium caseinate; NAS 12: gluten-free dough with 12% of sodium caseinate; NAS 24: gluten-free dough with 24% of sodium caseinate: OPT 12: gluten-free dough with 12% of whey proteins hydrolysate; OPT 24: gluten-free dough with 24% of whey proteins hydrolysate; ISO 12: gluten-free dough with 12% of whey proteins isolate; ISO 24: gluten-free dough with 24% of whey proteins isolate. C3: maximum torque during heating of potato starch; C3³´ 
 torque during heating of corn starch; C4: minimum dough torque during heating; C5: final viscosity after cooling till 50 ¶|´ ¨
 
#´
¨
 
#\$´ stability rate.

Primary and secondary parameters were extracted from the Mixolab curves to quantify the effect of the different dairy proteins on dough empirical rheology (Table 2). Proteins added to experimental doughs retarded the initial pasting temperature and the temperatures at which maximum dough consistency (C3 and C3³` ^
 ^# # 