*2.9. Statistical Analysis*

The data were submitted to analysis of variance (ANOVA) and the means compared by the *t*-Student test (to compare between day 1 and day 7) or the Tukey test with a significance level of 5% using Statistica software (StatSoft, Inc., Tulsa, OK, USA).

## **3. Results and Discussion**

## *3.1. Visual Appearance, Proximal Analyses and Physicochemical Properties of Yogurts*

Figure 1 shows the visual appearance of the different yogurts. To the naked eye, all yogurts presented a similar color, with similar gel-like appearance and no evidence of whey separation. The proximate composition revealed that the yogurts were similar, with significant differences (*p* < 0.05) mainly in carbohydrate and protein content due to the incorporation of maltodextrin and hydrolysate in some yogurts.


**Figure 1.** Visual appearance and proximate composition of the different yogurts. 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 letters (a,b,c) indicate significant differences between samples within the same line (*p* < 0.05).

The pH, titratable acidity (TA), color and syneresis of the yogurts after 1 and 7 days of storage under refrigeration, are shown in Table 1. The lowest pH value after 1 and 7 days of storage was observed in the yogurt with the addition of the microencapsulated hydrolysate (YHEn) while the highest pH observed was in the yogurt with the addition of MD (YMD), (*p* < 0.05). The pH values of MD, the free hydrolysate and microencapsulated hydrolysate were 5.7, 7.03 and 6.91, respectively. Hence, it seems that the pH of the yogurts was probably not affected by the addition of these ingredients. Moreover, a decrease in pH was observed in all yogurts throughout storage, which can be mostly attributed to the production of microbial metabolites. Similarly, a decrease in pH was also observed by Abdel-Hamid et al. [9] when supplementing probiotic yogurt with 1% and 2% monk fruit extract (*Siraitia grosvenorii*). These authors reported that supplementation with the extract may have stimulated bacterial growth. Notably, fermented products with pH in the range of 4.2 and 4.4 are preferred by consumers [34]. In the present work, these pH ranges were achieved in yogurts after 7 days of storage (Table 1).

The titratable acidity of the yogurts varied from 0.73% to 1.09% (Table 1), the YH and YHEn yogurts showing the highest values, both after 1 and 7 days of storage. In addition, with the exception of YC, there was an increase in the titratable acidity with storage time (*p* < 0.05). Similar results were reported by Córdova-Ramos et al. [35] when studying the addition of jumbo squid powder (*Dosidicus gigas*) using maltodextrin as an encapsulating agent by spray drying (MD with different DE (11) and drying conditions) in yogurt. The authors verified an increase in acidity as squid powder concentration increased (1%, 3%, 5%, 7% and 10%), ranging from 0.76% to 1.05% in the yogurt without squid powder and with the highest concentration, respectively. An increase in titratable acidity was also observed in yogurts supplemented with pineapple peel powder or inulin after 28 days of storage [36]. According to the authors, the supplementation increased the acidifying capacity of the starter cultures during storage.

Color is the first characteristic perceived by consumers, and it can influence their preference, so it is an important attribute to be evaluated [37]. The color parameters (L\*, a\* and b\*) of the different formulated yogurts are shown in Table 1. A decrease in lightness values (L\*) was observed for yogurts with the addition of the different tested ingredients, and therefore, the highest values observed for YC also reflected a higher whiteness index. As also observed by Silva et al. [5] such a phenomenon may have occurred because this batch did not have any powder ingredients. Conversely, on the same evaluation day, the YH and YHEn yogurts tended to show higher values for yellowness (b\*) (*p* < 0.05), probably due to the color of the hydrolysate. Carmona et al. [38] verified that encapsulating yellow– orange cactus pear Opuntia ficus-indica pulp, using maltodextrin (MD) as an encapsulating agent, protected the quality of the pigment (thus allowing its use as a yellow colorant for yogurt) and that, additionally, maltodextrin did not negatively affect b\* and a\* parameters in the yogurt when compared with other treatments.


**Table 1.** Physicochemical properties of yogurts during storage.

TA: titratable acidity; L\*: lightness; a\*: red-green axis; b\*: blue-yellow axis; WI: whiteness index; 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,d) 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).

> The YMD sample showed differences (*p* < 0.05) in a\* values compared with YH after 1 day of storage and with YH and YHEn after 7 days of storage, but was similar to YC (*p* > 0.05). Regarding storage, in general the L\* parameter showed a slight increase over time, while a\* and b\* values did not change, except in YHEn, which did not present any change in relation to the L\* parameter and showed a slight decrease in a\* values. The whiteness index (WI) was in the same range in all samples, decreasing less than 2% compared with the control (*p* < 0.05).

> Syneresis is an important parameter in yogurt since it can affect its quality during storage through the accumulation of serum on the surface, influencing the acceptability of the product [4,28,30]. The addition of the different ingredients provided a decrease in syneresis, with the lowest values observed in YHEn, independently of storage time (*p* < 0.05) (Table 1). In general, syneresis was not influenced by storage time, with the exception of YH, which showed a small decrease after day 7 (*p* < 0.05). Córdova-Ramos et al. [35] reported a decrease in syneresis in yogurts with the addition of different concentrations (1, 3, 5, 7 and 10 g/100 mL) of jumbo squid powder (*Dosidicus gigas*) obtained by spray drying using MD as encapsulating agent. The decrease in syneresis was associated with the functional properties of the protein, since protein and MD facilitate water retention, thus preventing this phenomenon. These authors reported that the lowest syneresis values were observed in yogurts with 7 and 10 g/100 mL of the powder (4.20% and 1.0%, respectively) while the addition of 3 g/100 mL presented a syneresis percentage (9.10%) similar to that obtained in the present study in YHEn (Table 1). In addition, the lowest syneresis was

found in YHEn. This result was similar to that reported by Demirci et al. [6] in yogurts with the addition of different concentrations of rice bran (1, 2 and 3%), which ranged from 9.79% to 8.80%, while the control yogurt presented 10.29%. The authors related the decrease in syneresis to the water-retention capacity of rice bran dietary fibers. In the present work, the reduction in the syneresis of formulated yogurts may be associated with the water-retention capacity of the protein hydrolysate by ion-dipole interactions. This fact may be due to the amino acid profile, which has a predominance of hydrophilic amino acids (57.4%) and negatively charged amino acids (64.4%). Moreover, the reduction in syneresis was enhanced in the yogurt to which the microencapsulated hydrolysate was added, possibly because MD also contributed to increase water retention by hydrogen bonding and dipole–dipole interactions.
