3.2.3. Microstructure of Doughs and Breads

Environment scanning electron microscopy was used to evaluate the microstructure of the doughs, obtained with different levels of yoghurt and curd cheese (30 g and 50 g), after 60 min of fermentation at 30 ◦C (Figure 5A1–A5). The microstructure of the breads crumb was also observed (Figure 5B1–B5). Figure 5A1–A5 show that dough is made up of the gluten network with small and large starch granules characteristic of the wheat dough.

**Figure 5.** Scanning electron micrographs (400 ×, scale bar = 200μm for dough; 150 ×, scale bar = 500 μm for bread crumb): A—fermented dough (60 min/37 ◦C); **A1**—Control dough; **A2**—30 g Yg, A3—50 g Yg; A4—30 g Cc, A5—50 g Cc; B—breads: B1—control bread, B2—30 g Yg, B3—50 g Yg; B4—30 g Cc, B5—50 g Cc, at first day of storage time.

Comparing the Yg doughs, the dough with 30 g and 50 g (Figure 5A2,A3) displayed a remarkable gluten film, with defined glutenins strands when compared to control dough, which is more evident at 30 g of Yg addition. These results are in agreement with those obtained on the extension properties, supporting the improvement of the flexibility of the net responsible for the increase on the extensibility and deformation energy values (Figure 1B,C). With Cc additions, the gluten film became less notable, as shown in Figure 5 (A4—30 g and A5—50 g of Cc addition), revealing that denatured whey protein interfered with the development of the gluten network, reducing the extensibility of the net (Figure 1B) and thus affecting the gas retention. Therefore, with the structure morphology of these doughs, the reduction of the specific volume of the breads was expected, as observed (Figure 4B).

Figure 5 (B1–B5) show the changes of crumb bread structure of the Yg bread (B2–B3) and Cc bread (B4–B5) compared to the control bread (B1). A more complex cell structure with higher number of gas cells between starch granules and denatured gluten in bread crumb with 30 g of Yg compared to the control is also noticed. This microstructural feature is associated with a lower staling rate, a low degree of firmness, and better bread volume [8]. For breads with 50 g of Yg addition, a more sheet-like structure was observed, probably due to the higher caseins interactions and most probably by the presence of exopolysaccharides that interact with the gluten network, leading to a more

homogeneous structure [21]. For the Cc addition, Figure 5B4,B5, a heterogeneous and disaggregated bread crumb is observed, which is a consequence of the interference of the denatured whey protein in the gluten network.

The scanning electron microscopy images obtained for breads with Yg and Cc revealed that the interactions between dairy proteins with different nature and structures with gluten and starch matrix could partially explain and support the results obtained in dough extensibility and bread quality properties.

## *3.3. Nutritional Composition of the Breads*

The nutritional composition, including the mineral contents, was determined for control and experimental breads obtained with 30 g and 50 g of Yg and Cc addition. A positive impact on protein and ash content was observed for both levels of dairy products tested. However, a remarkable effect was obtained for 50 g of addition in both cases, representing an increase of 7% and 30%, respectively, for Yg breads and 31% and 66% for Cc bread (Table 4). In terms of lipids, there was an increase of 28.0% for 50 g Yg addition and 163.0% of 30 g of Cc addition (the highest level to be considered in terms of bread quality). This increasing in fat is not high for the Yg breads but is considerable in the case of the Cc breads, as these are mainly saturated fatty acids from milk. This can be considered an additional restriction related with the curd cheese incorporation.

**Table 4.** Nutritional composition \* and mineral content of breads produced with yoghurt and curd cheese: 30 g and 50 g of yoghurt (Yg) and curd cheese (Cc).


**\*** Different letters (a, b, c, d) within the same row indicate statistically significant differences at p < 0.05 (Tukey test), compared with the bread control parameters.

Milk and dairy products are valuable sources of minerals with a good assimilability [11] and exert several essential physiological functions in the human body. As they contain major minerals (Ca, K, Mg, and P) and trace elements (including Cu, Fe, Mn, Zn), incorporation of both dairy products promotes a significant improvement on mineral composition (p < 0.05) in general (Table 4).

In the breads with 50 g of addition, a significant increase was observed in Ca (Yg–51%, Cc–145%), K (Yg–20%), P (Yg–21%, Cc–54%), S (Yg–6%, Cc–43%), and Mg (Yg–24%, Cc–12%) compared to control bread. The fortification of the Yg and Cc breads with major and trace minerals is clearly noticeable, representing, in general, more than 15% of the recommended daily dosage for Ca (Yg–16%; Cc–24%), K (Yg–15%), and P (Yg–16%; Cc–18%).

For trace elements, interesting values were also noticed, especially for Cu (Yg–58%; Cc–46%) and Mn (Yg–32%; Cc–34%) (Reg. (CE), No 1924/2006; Dir. N<sup>o</sup> 90/494 (CE)). These significant improvements will contribute to increase the minerals intake in not only daily diet of children's but also in adulthood and the elderly, where the aging process is associated with a gradual and progressive bone demineralization, along with lowered strength and physical endurance [4].

The results are supported by other researchers [11,22], which have demonstrated that the mineral and protein content of bread samples increased by the addition of different levels of whey protein, milk products, and dairy by-products.
