*3.3. Chemical Composition and Nutritional Value of the Gluten-Free Breads Containing Different Dairy Powders*

The addition of dairy proteins to gluten-free bread formulations is a common practice for increasing nutritional value, as in general, the commercial gluten-free formulas are poor in proteins [35,40]. Similarly to the results of our previous study [41], the control gluten-free bread, mainly composed of corn and potato starches, was poor in proteins (Table 3).

As was expected, the proteins content in all experimental gluten-free breads with dairy powders increased significantly (*p* < 0.05). Breads supplemented with 12 g of milk powders were more than five-times richer in protein than the control. The increase of the level of supplementation with dairy proteins (up to 24 g) caused the further increase of protein content in the bread samples, especially in NAS 24 with sodium caseinate, which was ten-times richer in proteins than the control. Milk proteins have a high nutritional value [10,13] and the addition of milk proteins and essential amino acids, such as lysine, methionine and tryptophan also increases the nutritional value of the bakery products [14,15]. Within bread samples with dairy proteins addition, a fat content was low ranging from 0.39 to 2.08 (Table 3) as all dairy powders added were relatively poor in fat (Table 1). However, comparing with the control, experimental breads were more affluent in minerals, especially when 24 g of calcium caseinate was included to formulation (CAS 24). There are inconsistencies about

CD patients being overweight at diagnosis and gaining weight while on GFD [42,43]. Taking this into account, the calorie content of gluten-free products is of importance [44]. Energetic value (in kcal) of experimental gluten-free bread with dairy proteins ranged from 228 to 246, and was comparable to unfortified control (239 kcal). Hager *et al*. [45] indicated that although the calorie content was higher in white and wholemeal wheat bread than in oat, buckwheat, maize, sorghum, teff, and rice, these breads were of inferior quality at the same time. In the present study the two levels of dairy proteins supplementation to experimental gluten-free bread were tested regarding the energy delivered by proteins (Table 3). In the case of experimental breads with lower amount (12 g) of caseinates and whey proteins powders, the proteins delivered around 15% or above 13% of energy, respectively. The higher threshold of proteins supplementation influenced further significant growth of energy delivered by proteins up to 26% in NAS 24. According to the European Parliament regulation on nutrition and health claims made on foods, a claim that a food is a source of protein may only be applied to food product where at least 12% of the energy value of the food is provided by protein, whereas a claim that a food is high in protein may only be made where at least 20% of the energy value of the food is provided by protein [19]. Based on that regulation, all experimental gluten-free breads with 12 g dairy proteins addition can be recognized as a source of proteins, whereas all experimental gluten-free breads with 24 g dairy proteins addition are high in protein.

#### *3.4. Technological Parameters of Gluten-Free Breads with Dairy Powders*

In general, milk products have been described as volume-depressing contributory factors of wheat bread [46,47]. In this study, the addition of 12 g dairy proteins to the experimental gluten-free formulations increased significantly the specific volume of all breads, comparing with the control bread, with distinguishing results obtained in bread NAS 12 (Table 4). Similarly, Gallagher *et al*. [17] indicated that addition of high protein/low lactose dairy powders resulted in gluten-free breads of improved overall shape and volume. Additionally, in the case of increase of sodium caseinate concentration in gluten-free formulation, the further increase of specific volume of bread NAS 24 was observed. In all remaining samples, the used of increased amount of dairy proteins (up to 24 g) affected specific volume in a different manner. Compared with the control, increased level of hydrolyzed whey proteins decreased significantly the volume of bread OPT 24, whereas a higher concentration of whey proteins isolate increased the specific value of bread ISO 24. Specific volume of bread CAS 24 was similar to the control. The volume of bread with dairy powders depends on the powder type and level of addition. Breads with addition of dairy supplements showed higher height/width ratio of central slices in comparison with the unfortified control (Table 4). However, only in the case of breads with the 12 g of proteins in formulation, the increase in the height/width ratio of slice was significant. Analyzing relationship between specific volume and H/W ratio a linear positively correlation (Pearson's *r* = 0.49) was found.



**Table 3.** Chemical composition and nutritional value of the gluten-free breads containing different dairy powders.

Control: unfortified gluten-free bread; CAS 12: gluten-free bread with 12% of calcium caseinate; CAS 24: gluten-free bread with 24% of calcium caseinate; NAS 12: gluten-free bread with 12% of sodium caseinate; NAS 24: gluten-free bread with 24% of sodium caseinate: OPT 12: gluten-free bread with 12% of whey proteins hydrolysate; OPT 24: gluten-free bread with 24% of whey proteins hydrolysate; ISO 12: gluten-free bread with 12% of whey proteins isolate; ISO 24: gluten-free bread with 24% of whey proteins isolate; Mean values labeled with different letters in the same column are significantly different (*p* < 0.05). Lower case letters were used with the low-level protein addition and upper case letters are referred to the highest level of protein addition.

Applied dairy supplements, regardless of the amount, influenced the bake loss, defined as the amount of water and organic material lost during baking (Table 4). In comparison with the control, bake loss of all breads tested increased significantly. The opposite effect was observed only in bread OPT 24, where the value of bake loss was significantly reduced by higher concentration of hydrolyzed whey proteins. Crumb characteristic affects the rate of water transport [48]. Small size of crumb pore slowed down moisture migration [49], whereas a larger number of connections between gas cells would give a faster transport of water. Additionally, the number of connections of each gas cell increased with increased size of gas cell [50].



Incorporation of dairy powders affected the color of both, crust and crumb of experimental gluten-free breads (Table 4). Crust of the control bread was characterized by the highest lightness (*L*\* = 46.37), whereas the inclusion of dairy proteins resulted in crust darkening, influenced by the level of dairy proteins addition rather than the protein type. All gluten-free breads containing dairy supplements showed the significantly lower *L*\* value. Additionally, in the case of experimental gluten-free breads containing 12 g of dairy proteins, the further crust color diversification was observed. Crust of bread CAS 12 and NAS 12, containing calcium or sodium caseinate, respectively, was significantly lighter in comparison with dark crust of breads containing whey proteins, with distinguishing OPT12 where *L*\* value reached 27.56. The value of parameter *a\** (red hue) was positive for crust of all experimental breads (Table 4). Comparing with the control, the incorporation of dairy powders to gluten-free formulation affected the significant decrease in redness, especially in breads containing sodium caseinate at both levels tested (NAS 12 and NAS 24). Crust of the control gluten-free bread obtained the highest *b*\* value (yellow hue). Whereas, the addition of dairy proteins to formulation produced a significant decrease of crust yellowness of breads obtained, however all the values were still positive. The crust yellowness was especially low in the case of breads with higher dairy proteins concentration. Observed darkening of crust color resulted probably from the Maillard browning, a chemical reaction between amino groups and reducing sugars. In the case of milk derivatives undergoing a high temperature treatment, lactose as a reducing sugar interacts mainly with lysine residues, resulting in the formation of brown melanoidins [51]. These non-enzymatic reactions are responsible for numerous changes on food properties. From the technological point of view, the brown crust formation on gluten-free bread is desirable and the resulting color, taste and flavor characteristics are generally experienced as pleasant. Crumb color was influenced by a level of dairy proteins addition. Lower concentration of proteins tested in the formulations resulted in bread of similar to control crumb lightness, with slightly distinguishing NAS 12 (Table 4). Only in CAS 12 the *L\** was significantly reduced. Whereas, the lightness of bread crumb supplemented with 24 g of dairy proteins was higher in comparison with crumb of breads with 12 g dairy proteins addition, except for crumb of bread NAS 24. The *a*\* values for the crumbs were all negative, with the lowest redness detected in the control crumb.

Comparing with the control, increasing concentration of dairy powders increased significantly the value of *a*\* parameter of tested crumbs. This effect was especially visible in the case of breads containing calcium (CAS 24) and sodium (NAS 24) caseinate, where the *a*\* value reached -! and - · ^ *b*\*) of all crumb samples was positive. Similarly to redness, also the value of *b*\* increased significantly with increased dairy proteins concentration. Gluten-free breads containing dairy powders had an appealing dark crust and white crumb appearance, and received good acceptability scores in sensory tests [14,17]. As a wide variety of dairy supplement are available, their application in baked product development need to be determined adequately. In addition to the type and the amount of dairy supplement, the choice must be based on their physicochemical and functional properties, which varies remarkably.

#### *3.5. Textural Parameters of Gluten-Free Breads with Dairy Powders*

The values obtained for the textural parameters of the bread crumbs are shown in Table 5. Wide variations in the crumb hardness (3.66 to 25.28 N) were observed among the gluten-free bread samples. These results reflect large differences depending on type of proteins used. Dairy proteins incorporated at 12% level significantly (*p* < 0.05) decreased the hardness, with the exception of NAS 12. Nevertheless, the addition of increasing amounts of proteins led to the opposite effect and only ISO 24 remained softer than the control crumb. NAS at any level of addition led to harder crumbs and the same effect was observed in chewiness. Nunes *et al*. [14] analyzed the influence of low lactose dairy powders on gluten-free bread quality indicated that sodium caseinate had a negative impact on crumb hardness, whereas whey proteins demonstrated the ability to increase significantly the specific volume of the breads and decrease its the hardness.


**Table 5.** Texture profile analysis of the gluten-free bread crumbs containing different dairy powders.

Control: unfortified gluten-free bread; CAS 12: gluten-free bread with 12% of calcium caseinate; CAS 24: gluten-free bread with 24% of calcium caseinate; NAS 12: gluten-free bread with 12% of sodium caseinate; NAS 24: gluten-free bread with 24% of sodium caseinate: OPT 12: gluten-free bread with 12% of whey proteins hydrolysate; OPT 24: gluten-free bread with 24% of whey proteins hydrolysate; ISO 12: gluten-free bread with 12% of whey proteins isolate; ISO 24: gluten-free bread with 24% of whey proteins isolate; Mean values labeled with different letters in the same column are significantly different (*p* < 0.05). Lower case letters were used with the low-level protein addition and upper case letters are referred to the highest level of protein addition.

Springiness is associated with a fresh, aerated and elastic product, thus high springiness is desirable in this type of products. Low springiness value is indicative of brittleness and this reflects the tendency of the bread to crumble when is sliced. Although the proteins addition decreased the springiness, the effect was only significant in the presence of the protein isolates (ISO and OPT) at both levels tested. Marco and Rosell [35] found springiness values that ranged from 0.77 to 0.94 when study the protein enrichment of rice based gluten-free breads, and later on Matos and Rosell [8] reported springiness values from 0.76 to 1.00 in commercial gluten free breads. Therefore, springiness values obtained in the present study agree with reported ones.

Cohesiveness characterizes the extent to which a material can be deformed before it ruptures, reflecting the internal cohesion of the material. Bread with high cohesiveness is desirable because it forms a bolus rather than disintegrates during mastication, whereas low cohesiveness indicates increased susceptibility of the bread to fracture or crumble [8]. In case of breads containing caseinates (CAS and NAS) values obtained for cohesiveness were similar to control, while whey

proteins (OPT and ISO) decreased, significantly, the value of this parameter (*p* < 0.05). Very low resilience values were obtained for experimental gluten-free breads, especially for breads with whey proteins, indicating a low elasticity. Values obtained agreed with results reported for commercial gluten-free bread where resilience ranged from 0.09 to 0.84 [8].

#### **4. Conclusions**

The present study has shown that the application of low-lactose dairy proteins in a gluten-free formulation influenced considerably the characteristic of experimental doughs and breads. Gluten-free doughs containing dairy proteins tested showed very low consistency during mixing stage and decreased consistency during the heating-cooling stages. Experimental breads were significantly richer in proteins and more affluent in minerals than the control one. Energetic value of experimental gluten-free bread with dairy proteins was comparable to unfortified control, however regarding the energy delivered by proteins they can be recognized as a source of proteins or as high in protein. Addition of dairy proteins to the experimental gluten-free formulations increased significantly (*p* < 0.05) the specific volume of all breads, with distinguishing results obtained in bread NAS 24. Inclusion of dairy proteins resulted in crust darkening and crumb lightness, influenced by the level of dairy proteins addition rather than the protein type. Dairy proteins incorporated at a 12% level significantly (*p* < 0.05) decreased the hardness, with the exception of NAS 12. Nevertheless, the addition of increasing amounts of proteins led to the opposite effect. Obtained results suggest that dairy proteins tested in this study could be used successfully in gluten-free recipes in order to obtain gluten-free bread of a pleasant color, taste, and flavor characteristics, and improved technological and nutritional properties.

#### **Acknowledgments**

Authors acknowledge the financial support of the Spanish Ministry of Economy and Competitiveness (Project AGL2011-23802), the European Regional Development Fund (FEDER) and Generalitat Valenciana (Project Prometeo 2012/064) and Project REFRESH—Unlocking the potential of the Institute of Animal Reproduction and Food Research for strengthening integration with the European Research Area and regional development of the 7th Framework Programme EU-Research Potential-Capacities-FP7-REGPOT-2010-1-264103. The authors would like to thank to Józef Warechowski from the Faculty of Food Sciences of the University of Warmia and Mazury in Olsztyn for the particle size distribution analysis.

#### **Conflicts of Interest**

The authors declare no conflict of interest.

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¦^# -a valuable component of gluten-free formulations. *Pol*. *J*. *Food Nutr*. *Sci*. **2008**, *58*, 59–63.
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Reprinted from *Nutrients*. Cite as: Penagini, F.; Dilillo, D.; Meneghin, F.; Mameli, C.; Fabiano, V.; Zuccotti, G.V. Gluten-Free Diet in Children: An Approach to a Nutritionally Adequate and Balanced Diet. *Nutrients* **2013**, *5*, 4553-4565.

*Review* 
