*3.1. Probiotics Viability*

Microencapsulation by spray-drying is a common technology to protect the viability of probiotics [33]. In the present study, maltodextrin (10%, *w*/*v*) and sodium alginate (2%, *w*/*v*) were used as bacteria-protecting ingredients to generate powders with microencapsulated probiotics. Spray-drying microencapsulation resulted in powders with 7 × 10<sup>13</sup> CFU/g and 1 × 10<sup>14</sup> CFU/g for *Lactobacillus plantarum* L299v and *Lactobacillus acidophilus* La 3, respectively. These results agree with previous reports that evaluated microencapsulation of probiotics with sodium alginate, demonstrating that it can be used as a heat protector agen<sup>t</sup> for different probiotic strains, such as *L. rhamnosus, B. longum, L. salivarius, L. plantarum, L. acidophilus, L. paracasei, B. lactis* B1-O4, *B. lactis* Bi-07 [34], and *L. casei* [35]. Furthermore, the use of prebiotic agents such as maltodextrin, in addition to alginate, is recommended to generate a physical barrier with a symbiotic relationship [36]. In this tenor, previous reports have demonstrated that maltodextrin can be used as an effective microencapsulating protective agen<sup>t</sup> for probiotics, reducing the caking and stickiness to the spray-dryer's wall, increasing the free-flowing nature of the spray-dried powder [37], and exerting heat protection [38].

For all chocolate formulations, the addition of microencapsulated probiotics resulted in a product with ≥2 × 10<sup>7</sup> CFU per portion (12 g). This value is in the range of the minimum count of probiotic bacteria intake ( ≥1 × 10<sup>6</sup> CFU) recommended to have a beneficial effect [39,40]. Prior reports have shown that chocolate ingredients are suitable as a vehicle for probiotics [20,25,27]. For instance, the high total solids in milk chocolate, including fat and protein, generate a protective matrix for probiotics [40]. Furthermore, the low water activity (a w) and fat concentration in chocolate aid in preserving the viability of probiotic bacteria in an inactive state.

#### *3.2. Physicochemical Properties of Sugar-Free Milk Chocolate Formulations with Added Probiotics and Fish Oil*

#### 3.2.1. Water Activity (a w)

Water activity has an important role in the safety, quality, processing, shelf-life, texture, and sensory characteristics of confectionary products [3]. The a w values for milk chocolate formulations are shown in Table 2. The control showed a w = 0.46, which is in the threshold for a w values of pathogenic microbial growth in foods. Sweetener addition (Sw), FO addition, and their combination (Sw\*FO) showed a significant reduction in a w, whereas Prob addition impeded this effect. Water activity reduction by isomalt addition has been previously reported for sugar-free milk chocolate formulations, which has been attributed to its hygroscopic property [41]. FO addition generated chocolate formulations with lower aw values. This phenomenon could be attributed to the degree of unsaturation in fatty acid, generating electric charges that affect the molecular interaction with water molecules [42].


**Table 2.** Water activity (a w), whiteness index (WI), and texture parameters' (hardness and fracturability) values of sugar-free milk chocolate formulations with added probiotics and fish oil.

W.I., white index; aw, water activity. 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. Values with different letters within the same column indicate a statistically significant difference by the LSD test (*p* < 0.05). a Values represent the mean of 3 replicates with their standard error. b Values represent the mean of 5 replicates with their standard error. c Asterisks indicate significant difference from a full factorial analysis of variance showing the main effects and interactions of the variables evaluated: \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001. Sw, sweetener; FO, fish oil; Prob, probiotic; NS, non-significant.

#### 3.2.2. Whiteness Index (WI)

The WI indicates fat bloom formation [43]. Fat blooming plays a crucial role in the final structure, mechanical properties, appearance, quality, and marketability of chocolate products [44]. The effects of Sw, FO, and Prob addition as well as their interactions in the WI values of chocolate are shown in Table 2. Sw and Prob added alone (without sugar replacement) showed a significant increase on the WI value, whereas FO added alone did not affect the WI value. However, when FO was added to sugar-free chocolate (Sw + FO and Sw + Prob + FO), the individual effect of Sw and Prob on the increase in WI value was impeded.

The lower WI values observed in Sw treatments indicate that sugar replacement by sweeteners generates darker chocolates less prone to fat blooming. This result is in agreemen<sup>t</sup> with previous reports, where sucrose replacement with polyols, such as maltitol, xylitol, isomalt, and stevia, generated darker chocolates compared to their reference chocolate [45,46]. Particle size and distribution play an important role in instrumental color measurements. The tempering process of Sw and Sw + Prob chocolates could be responsible for the development of appropriate cocoa butter nucleation, generating more stable microparticle interaction due to the generation of adequate amounts and sizes of β V polymorphic form crystals [47].

On the other hand, Prob addition increased fat blooming predisposition in the formulation. These agreed with a report where the incorporation of *L. paracasei* to white chocolate formulation generated brighter chocolates as compared to the control [48]. The color of microencapsulated probiotic powder can explain this increase since probiotics could affect the particle size distribution in the chocolate matrix [49]. Since microcapsules are composed of carbohydrates such as maltodextrin, sugar bloom and fat bloom could be occurring. Sugar bloom is caused by absorption of moisture solubilizing sugar and then re-crystallized at the surface as a thin film of sugar crystals [50]. The fat bloom is distinguished from loss of gloss caused by larger crystals' growth, causing scattering of the light, and the surface appears dull, due to an incorrect tempering [51]. Similar results from chocolate with added probiotics were obtained by Silva et al. [52]. The authors attributed this phenomenon to the

addition of probiotics during the tempering process, which influences the recrystallization of lipids. However, the interaction of Sw and Prob shows a decrease in WI values, which could be related to the microstructure interaction between sweeteners, Prob, and other ingredients in the chocolate formulation.
