*3.3. Microbiological Analysis*

To confirm that the process of obtaining the yogurts was properly carried out, specific counts of the two major bacterial species present in yogurt were determined. The counts of *S. thermophilus* were YC: 8.72 ± 0.10 log CFU/g; YMD: 8.71 ± 0.10 log CFU/g; YH: 8.82 ± 0.04 log CFU/g; YHEn: 9.20 ± 0.01 log CFU/g (*p* < 0.05). Thus, the addition of the microencapsulated hydrolysate seems to have favored the growth of *S. thermophilus*, which could explain the lower pH and higher acidity compared with the YC sample. Regarding *L. bulgaricus*, counts were slightly higher in YH (YC: 6.42 ± 0.11; YMD: 6.92 ± 0.06; YH: 7.25 ± 0.09; YHEn: 6.94 ± 0.09 log UFC/g) (*p* < 0.05), indicating that the incorporation of the hydrolysate favored the growth of the starter culture in some way. YHEn registered higher values than the control yogurt, and similar values to those of YMD (*p* < 0.05), which indicated that maltodextrin, as a glucose supply, could also promote in some way the growth of this group of microorganisms. The viable counts of *S. thermophilus* were significantly more numerous than those of *L. bulgaricus* in all samples (*p* < 0.05). Zhao et al. [48] verified the same tendency in yogurts elaborated with α-lactalbumin hydrolysatecalcium (α-LAH-Ca) complexes.

The relatively low pH of yogurt and other fermented dairy products is a result of the fermentation of milk lactose into lactic acid, caused by the activity of lactic acid bacteria usually added as starter cultures [49,50]. In this study, as shown in Table 1, the pH of the yogurts evaluated ranged from 4.35 to 4.61. Furthermore, the yogurt starter cultures contained *Streptococcus thermophilus* and *Lactobacillus delbrueckii* ssp. *bulgaricus*, which largely out-compete other bacteria present in milk due the inhibitory effect of lactic acid and the utilization of the primary carbohydrate source (lactose), as well as the production of other inhibitory compounds [49,50].

**Figure 3.** Evolution of viscosity values for the three-step flow (**a**) and viscoelastic parameters (**b**) for aqueous solutions at 1.5% concentration of maltodextrin (MD), hydrolysate (H) and microencapsulated hydrolysate (HEn) at 20 ◦C. Different letters (a,b) or (A,B) indicate a significant difference between samples (*p* < 0.05).

In this study, the presence of *Enterobacteriaceae*, molds and yeasts was not found after 1 and 7 days of storage (data not shown). There is growing evidence that the *Enterobacteriaceae* family can accurately reflect the hygienic conditions of processing milk and its derivatives, even during storage, despite the widespread use of coliforms as an indicator of hygienic– sanitary conditions. In addition, the presence of molds and yeasts in yogurt is indicative of unsatisfactory sanitary practices in manufacturing or packaging [49]. Accordingly, Barkallah et al. [4] did not observe the presence of the mentioned microorganisms in yogurt with the addition of *Spirulina platensis*.

#### *3.4. Bioactivity*

## 3.4.1. Antioxidant Activity

Antioxidant compounds can contribute to health promotion by eliminating free radicals, such as reactive oxygen species, as well as to increase the shelf life of foods, slowing down the process of lipid oxidation [36]. Yogurt per se, in general, may present antioxidant activity in view of its content of bioactive peptides, as reported in previous studies [6,9,36,39].

The antioxidant activity of the different yogurts after 1 and 7 days of storage was evaluated by assessing the ABTS radical scavenging activity and reducing power (Figures 4A and 4B, respectively). The addition of the free and microencapsulated hydrolysate to yogurts (YH

and YHEn, respectively) increased the ABTS radicals scavenging activity (*p* < 0.05) in comparison with the YC and YMD samples, which showed a similar behavior (*p* > 0.05). The increase in antioxidant activity was probably due to peptides with antioxidant activity present in the protein hydrolysate of stripped weakfish, as previously reported by Lima et al. [25]. The activity of the hydrolysates was maintained, both in the free and microencapsulated forms in the yogurt matrix. These findings are in line with those reported by Abdel-Hamid et al. [9] and Demirci et al. [6], who found an increase in the antioxidant activity of yogurts with the addition of the fruit extracts *Siraitia grosvenorii* and rice bran, respectively. According to the authors, this was due to the composition of added phytochemicals with antioxidant activity. Additionally, Silva et al. [5] evaluated the antioxidant activity of yogurts functionalized with *Spirulina platensis* in free form and microencapsulated with MD, or with MD cross-linked with citric acid obtained by spray drying, reporting greater antioxidant activity (DPPH assay) for both functionalized yogurts compared with the control yogurt (especially in the case of the sample with MD cross-linked with citric acid). According to the authors, the addition of *Spirulina platensis* in the encapsulated form presents advantages such as masking the unpleasant fishy odor. Similarly, in the mentioned study, no differences in antioxidant activity were observed in terms of storage time (4 and 7 days).

Francisco et al. [7] reported a decrease in antioxidant activity during storage (0–7 days) for yogurts with added mushroom extracts (*Agaricus bisporus*) in free form and encapsulated by spray drying with MD cross-linked with citric acid (thermally untreated forms), while the opposite behavior was observed in thermally treated microencapsulated extracts after atomization. This decrease throughout storage time may be associated with the degradation of the extract when incorporated in free form by direct contact with the food matrix, and when thermally untreated, by the rapid release of the microencapsulated extract to the food matrix with the subsequent degradation.

In the present study there was no degradation of bioactive peptides, possibly because the antioxidant activity (ABTS and reducing power) remained stable throughout storage (*p* > 0.05). It should be noted that YH and YHEn did not differ significantly in either evaluated property (*p* > 0.05) (Figure 3). Thus, the incorporation of the microencapsulated hydrolysate may be an alternative for increasing the antioxidant activity of yogurts while possibly masking any potential changes to odor and flavor.
