3.3.2. Mechanical Properties

The mechanical spectra of chocolate samples are shown in Figure 2. G is an index of a sample's elastic behavior and represents the deformation energy stored in the sample during the shear process. On the other hand, the G value measures the viscous component of a sample and compares the energy lost during the shear process [59]. The addition of Prob increased the storage modulus G over the loss modulus G". On the other hand, FO addition and Iso + Stev showed a contrast effect on G and G" at the frequency range of 0.1 to approximately 70 Hz, indicating a liquid-like behavior of a weakly structured system.

**Figure 2.** Frequency sweep test of chocolate at 35 ◦C with a linear viscoelastic region of 6 Pa. (**A**) Changes in storage modulus G. (**B**) Changes in loss modulus G. (**C**) Full factorial analysis of variance showing the main effects and interactions of the variables evaluated. Values represent the mean of 3 replicates. Asterisks indicate significant difference from a full factorial analysis of variance showing the main effects and interactions of the variables evaluated: \*\*\* *p* < 0.001. Treatments: Control = milk chocolate formulation, Prob = milk chocolate + probiotics, FO = milk chocolate + fish oil, Prob + FO = milk chocolate + probiotics + fish oil, Sw = isomalt + stevia, Sw + Prob = isomalt + stevia + probiotics, Sw + FO = isomalt + stevia + fish oil, Sw + Prob + FO = isomalt + stevia + probiotics + fish oil.

Prob addition generated a structured system to the chocolate emulsion due to the results of G over G" presented herein. This behavior has been previously reported for milk chocolate [22,59]. Additionally, the combination of Prob with FO (Prob + FO) showed a similar result to Prob, meaning that Prob as an ingredient increased the stability of chocolate's mechanical properties. Although the addition of Prob generated a strong matrix, when Prob was mixed with Sw (Sw + Prob) or Sw + FO (Sw + Prob + FO), a solidlike and more elastic formulation was observed mainly due to a higher solid fraction. On the other hand, the addition of FO generated a liquid-like behavior when it was combined with Sw. These results indicate that FO addition dominates the mechanical properties in sugar-free chocolates, such as Sw + FO and Sw + Prob + FO, because FO increased the number of fatty acids in the chocolate's emulsion [53]. The fat content of chocolate determines the mass fraction of particulates, which governs the proximity of those particles to each other. Thus, if fat content increases, the distance between particles increases, resulting in a lower viscosity [47]. These observations are in agreemen<sup>t</sup> with the results obtained for chocolates with added FO. Furthermore, results of the substitution of sucrose by Sw in chocolates showed an unstable chocolate matrix, generating a deep increase of G and G, as observed in Figure 2. This behavior is attributed to the higher solid volume fraction and lower density of isomalt, resulting in more flexible chocolates [28].

#### *3.4. Fatty Acid Methyl Esters (FAMEs) Profile*

The most widely available dietary source of EPA and DHA is cold-water oily fish or fish oil offered to consumers as a dietary supplement [55]. The chocolates formulated herein had 790 mg of FO added per serving size (12 g) of chocolate, expecting to obtain 200 mg of ω-3 PUFAs.

The fatty acid composition of chocolates with and without added FO are shown in Table 3. Likewise, the fatty acid composition of FO used as an ingredient for chocolate formulations is shown in the Supplementary Material (Table S1). FO contains a high amount of PUFAs (38,746.6 ± 45.8 mg per 100 g fish oil), of which ω-3 were the most abundant (34,712.6 mg ± 0.06 g per 100 g fish oil), with DHA (C22:6, 14,122.2 ± 27.0 mg/100 g) and EPA (C20:5, 12,862.1 ± 17.8 mg/100 g) being the major ω-3 PUFAs.

Chocolates without FO showed a low concentration of ω-3 PUFAs, mainly due to the presence of alfa linolenic acid in cocoa butter [53]. Additional fatty acids detected in the control and Prob treatment included linoleic acid, palmitic acid, stearic acid, and oleic acid, which are the primary fatty acids of cocoa butter [6,53,60]. FO addition resulted in a chocolate formulation with 107.4 ± 12.84 mg of ω-3 PUFAs per serving size (12 g). Interestingly, higher ω-3 PUFAs content was quantified when FO was added in sugar-free chocolate formulations (Sw + FO and Sw + Prob + FO) as compared with FO added alone, showing ω-3 PUFAs levels of 141.9 ± 17.9 mg and 133.8 ± 8.76 mg per 12 g of Sw + FO and Sw + Prob + FO formulations, respectively.

FO was added to the chocolate formulation to obtain 200 mg of ω-3 PUFAs per portion (12 g). However, results indicate that lower amounts were detected, indicating that ω-3 PUFAs were degraded during the chocolate-making process. Fatty acid degradation during the chocolate-making process can be attributed to lipid oxidation induced by low water activity and thermal treatment [3], which degrades EPA and DHA by breaking down the double bonds by oxidation [61,62].




**Table 3.** *Cont.*

stevia + probiotics, Sw + FO = isomalt + stevia + fish oil, Sw + Prob + FO = isomalt + stevia + probiotics + fish oil. Values with different letters within the same row indicate statically significant difference by the LDS test (*p* < 0.05). Values represent the mean of 3 replicates with their standard error. Sw, sweetener; FO, fish oil; Prob, probiotic; NS, non-significant.

## *3.5. Consumers' Acceptability*

A consumer acceptability test was performed to evaluate the appearance, taste, texture, and overall acceptability of chocolates, using a 9-point hedonic scale (Table 4).

**Table 4.** Sensory acceptability values of milk chocolate and sugar-free milk chocolate formulations with added probiotics and fish oil.


Treatments: Control = milk chocolate formulation, Prob = milk chocolate + probiotics, FO = milk chocolate + fish oil, Prob + FO = milk chocolate + probiotics + fish oil, Sw = isomalt + stevia, Sw + Prob = isomalt + stevia + probiotics, Sw + FO = isomalt + stevia + fish oil, Sw + Prob + FO = isomalt + stevia + probiotics + fish oil. a Values with different letters within the same column indicate statically significant difference by the LSD test (*p* < 0.05). b Asterisks indicate significant difference from a full factorial analysis of variance showing the main effects and interactions of the variables evaluated: \*\* *p* < 0.01, \*\*\* *p* < 0.001. Sw, sweetener; FO, fish oil; Prob, probiotic; NS, non-significant.

FO addition significantly reduced the acceptability of chocolate for all the parameters evaluated. On the other hand, Prob did not affect the acceptability by consumers. These results are in agreemen<sup>t</sup> with previous reports, where probiotics' addition did not affect the acceptability of chocolate [25,27,49]. FO addition mainly affected the acceptability of flavor and texture in FO and Prob + FO chocolates. This may be related to the fish odor present in fish oil. Interestingly, when FO was added to sugar-free formulations (Sw + FO and Sw + Prob + FO), chocolates showed higher acceptability as compared with formulations containing sugar and FO (FO and Prob + FO). This behavior can be explained by the fact that sugar can enhance flavors [63], and Sw chocolates have antioxidant properties (due to isomalt) that could protect FO from lipid oxidation [64].

Sugar-free chocolates showed lower flavor, texture, and overall acceptability values as compared with the control. The lower acceptability scores could be attributed to stevia's bitter taste and to the changes in rheological and mechanical properties induced by Sw addition [65,66]. It is important to point out that sugar-free chocolates, and sugar-free chocolates with added probiotics, showed values in the acceptable range, indicating that they could be excellent candidates for commercialization.
