*3.2. Textural and Viscoelastic Properties*

Texture is an important attribute regarding the quality of yogurt [37]. The firmness and cohesiveness of the different yogurts after 1 and 7 days of storage are shown in Table 2. The addition of the hydrolysate to the yogurts, both free and microencapsulated, resulted in a slight decrease in firmness compared to the YC sample (*p* < 0.05), representing approximately 11% (YH) and 10% (YHEn) after 1 day, and 30% (YH) and 19% (YHEn) after 7 days of storage. On the other hand, YH and YHEn showed increases in cohesiveness (*p* < 0.05) of approximately 12% and 15%, respectively, after 1 day and ~92% and 18%, respectively, after 7 days of storage compared with YC. After 7 days YH showed the highest cohesiveness values (*p* < 0.05). In general, the firmness and cohesiveness of the different yogurts were not significantly influenced by storage time, except for YC and YH, which showed greater firmness and greater cohesiveness, respectively, after 7 days of storage.

**Table 2.** Textural and viscoelastic parameters (Equations (2) and (3)) of yogurts during refrigerated storage.


YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate. Different lowercase letters (a,b,c) indicate a significant difference between samples for the same day (*p* < 0.05). Different uppercase letters (A,B) indicate a significant difference for the same sample on different days (*p* < 0.05).

> Barkallah et al. [4] studied the addition of different concentrations of *Spirulina platensis* (0.25%, 0.5%, 0.75% and 1.0%) to yogurt and observed greater firmness in the control yogurt and with the addition of 0.25% of *Spirulina platensis* (0.67 and 0.62 N, respectively). The addition of higher concentrations resulted in yogurts with lower firmness values which, according to the authors, was due to the interruption of gel formation as concentrations of

microalga were increased. In the same study, the authors found no differences in relation to cohesiveness for the samples. It is worth noting that both firmness and cohesiveness values were lower than those observed in the present study.

Additionally, Öztürk et al. [39], when studying the fortification of set-type yogurts with peeled or unpeeled oleaster (*Elaeagnus angustifolia* L.) flours (1% and 2%), reported a reduction in firmness and an increase in cohesiveness on the first day of storage compared to the control yogurt. According to the authors, the control yogurt showed a longer fermentation time resulting in greater firmness, while the increase in cohesiveness would occur due to the water retention capacity of the flours added into the protein matrix. During fermentation, the formation of lactic acid by the action of microorganisms occurs, and the pH drop induces the aggregation of casein and the formation of disulfide bonds between denatured whey proteins and k–casein, resulting in the characteristic gel formation, texture and properties of yogurt [37,40,41].

In the present study, the yogurts with the addition of the free and microencapsulated hydrolysate required less time to ferment (about 60–90 min) compared with the control yogurt or that with MD; this factor may have influenced the lower firmness values found. Moreover, the addition of these ingredients may have caused an interruption of gel formation, reducing firmness. Parallel to this, the greater cohesiveness may be associated with the water retention capacity, mainly due to the functional properties of protein hydrolysates [42].

The viscoelastic properties of the yogurt samples were evaluated by small-amplitude oscillatory shear (SAOS) tests. Mechanical spectra of the different yogurts after 1 and 7 days of storage showed typical gel-like behaviour, with *G* > *G* along the whole frequency range (Figure 2). The power law model may be used to fit both *G* and *G* with the angular frequency ω (Equations (2) and (3)):

$$G' = G\_0' \cdot \omega^{\mathbb{N}'} \tag{2}$$

$$G'' = G\_0'' \cdot \omega^{\text{n}''} \tag{3}$$

where *G*<sup>0</sup> and *G*<sup>0</sup> are the respective storage and loss moduli at 1 rad/s, and *n* and *n* exponents denote the viscoelastic response in terms of the time stability of both *G* and *G* at short time scales. The viscoelastic parameters resulting from Equations (2) and (3) (*G*<sup>0</sup> and *G*<sup>0</sup> ) are both a measurement of the gel strength of samples since they provide the complete (elastic and viscous) deformation resistance [43].

After day 1, the yogurts with the free hydrolysate (YH) and microencapsulated hydrolysate (YHEn) exhibited the lowest values for *G*<sup>0</sup> and *G*<sup>0</sup> (Table 2). This result indicates that the hydrolysate, in both forms, reduced the gel strength of the casein matrix, maintaining a similar degree of viscoelasticity as was evidenced by the similar values for the loss factor at 1 rad/s (*tanδ*) (Table 2). This result could be explained by the fact that the hydrolysate introduces a negative electric charge, consequently increasing the electrostatic repulsive forces within the micellar structure, which expand the network, enhancing the hydration of the gel network [44]. This result is consistent with the observed decrease in firmness in YH and YHEn as compared with YC from textural analysis.

The yogurt with maltodextrin (YMD) exhibited slightly higher values for *G*<sup>0</sup> and *G*<sup>0</sup> than those in YC after day 1. The electrostatic neutral character of these glucose polymers would favor the mutual associations between maltodextrin and casein by hydrogen bonding and dipole–dipole interactions, showing a certain binder role for MD in the yogurt matrix. This result is consistent with the lower *tanδ* value for YMD vs. YC (Table 2), which suggests greater strength in the intermolecular interactions and consequently a longer bond lifetime in the gel matrix [45].

**Figure 2.** Mechanical spectra of yogurts during storage at 5 ◦C. Storage modulus −*G* at 1 day (**a**); loss modulus −*G* at 1 day (**b**); storage modulus −*G* at 7 days (**c**); loss modulus −*G* at 7 days (**d**). YC: control yogurt; YMD: yogurt with addition of maltodextrin; YH: yogurt with addition of free protein hydrolysate; YHEn: yogurt with addition of microencapsulated protein hydrolysate.

As regards *n* and *n* exponents, it might be observed that for all samples, *n* > *n* , this means that the rate of decrease of *G* with decreasing ω, was higher than that for *G* , resulting in a shear-induced gelation at lower frequencies (higher oscillation times) [46]. This fact shows a shear-induced increase in the energy stabilization of network bonds at lower frequencies, compatible with the decrease in the gel strength at higher oscillation times [47].

After 7 days of storage, *G*<sup>0</sup> and *G*<sup>0</sup> values increased in YC and YMD compared with those found after day 1. This result was consistent with the observed increase in firmness from textural analysis, indicating considerable gel reinforcement during storage, which would be explained by the natural strengthening of the dipolar interactions and hydrogen bonds in the casein matrix induced by cool storage [44]. This effect was partially mitigated by the presence of maltodextrin, which would stabilize the structural rearrangements in the casein matrix during storage. In addition, both *G*<sup>0</sup> and *G*<sup>0</sup> were scarcely modified in YH and YHEn during the storage period, showing the stabilizing role of the hydrolysate in the casein matrix, especially in microencapsulated form.

The increase in both *G*<sup>0</sup> and *G*<sup>0</sup> after 7 days was consistent with the observed increase in L\* (Table 1). L\* shows the light scattered by various structural elements (casein aggregates, molecular fragments, etc.). After 7 days, the gel strength of the different networks increased moderately, so that denser matrices were formed, enhancing the diffuse reflection of electromagnetic waves and consequently increasing L\* [45].

In general, *n* and *n* values decreased after day 7 of storage, maintaining a similar positive difference between *n* and *n* (Table 2). Therefore, the lower exponents (*n* and *n*) indicate an improvement in stability in the four yogurt networks over time, maintaining a similar stabilization energy at lower frequencies than on day 1.

In order to gain insight into the rheological differences among the three ingredients (H, MD and HEn) in model systems, i.e., outside the protein gel matrix environment at the same concentration, the flow behaviour and the viscoelastic characteristics of diluted aqueous solutions (1.5%, *w*/*v*) were also analyzed (Figure 3). Such a low concentration was selected to resemble the low concentration of the hydrolysate in the yogurt matrix. The dissolved maltodextrin (MD) and the microencapsulated hydrolysate (HEn) presented a similar trend (shear thinning flow) and similar viscosity values during the three-step flow, while the opposite was observed in the free hydrolysate, which exhibited the lowest viscosity and virtually Newtonian behaviour, as was evidenced by the stationary values of viscosity in the three steps (Figure 3a). The dynamic oscillatory test showed an evident fluid-like response in the three samples based on noticeably higher values for *G* compared with those for *G* (Figure 3b). Both MD and HEn solutions showed considerably higher *G* values compared with the H aqueous solution, while showing no significant differences between each other. This result was consistent with the decrease in viscosity in the second step, and their regeneration ability in the third step, attributed to the contribution of maltodextrin (Figure 3a). In contrast, *G* decreased in H, and more evidently in HEn, with respect to plain MD (Figure 3b). These findings reflect a lower level of intermolecular association at small oscillatory shear in HEn, and suggest that microcapsules may be less prone to interacting with each other and also with the surrounding medium, explaining why this preparation in yogurt resulted in more stable samples, which behaved differently compared with plain MD yogurts.
