3.2.3. Texture

Hardness and fracturability are two texture parameters that have a direct correlation with the acceptability by consumers. Hardness represents the physical rigidity, whereas fracturability is associated with the maximum force for penetration [6,7]. Prob, FO, and sugar replacement (Sw) either evaluated alone or combined significantly reduced the hardness value of chocolate. The hardness decrease by FO addition could be attributed to the increase of PUFAs in the chocolate matrix, yielding a softer product that melts easier [53]. Lipids in chocolate represent the continuous phase in the chocolate emulsion, which governs the physical and the textural properties. The hardness of chocolate is affected by the extent and nature of the crystalline lipid phase, linked to the control of the proper polymorphic form controlled by tempering [47].

Probiotics' addition also decreased the hardness value of chocolate. This result is in agreemen<sup>t</sup> with a previous report where the hardness of chocolate was evaluated in dark chocolates with and without probiotics [54]. The authors attributed the lower hardness values to the effect that microencapsulated probiotics' addition could have on crystal formation during the tempering process. Furthermore, sugar replacement (Sw) also generated chocolates with lower hardness values. Polyols sweeteners such as isomalt and high-intensity sweeteners such as stevia affect the texture of chocolate due to their hydroxyl sites, which interact with intermolecular bonds between particles in chocolate [45,46].

Fracturability of chocolate was increased only when Prob and FO were added either alone or combined in sugar-free chocolates. For instance, treatments without sugar and without FO showed higher fracturability values as compared with the control. Interestingly, FO addition in sugar-free chocolates decreased fracturability, showing the lowest values among treatments (Table 2). Previous studies on physical analyses of chocolate formulations with added EPA/DHA in the triglyceride form resulted in a softer product as compared with the control, attributed to the high content of PUFAs, which contributes to the generation of a softer product with lower fracturability when sugar is replaced [55]. Other authors have suggested that process and product optimization could improve the texture of chocolates when the formulation has added FO or EPA/DHA as microencapsulated oil and powder, overcoming undesirable textural and physiological effects of FO addition [3,55].

Texture values presented herein are influenced by the tempering process, since properly tempered chocolate contains numerous β V polymorph crystals of cocoa butter that form a tight crystalline matrix, giving a high degree of hardness and fracturability. Besides, in milk chocolates, it is important to consider the effect of milk fat on cocoa butter crystallization since it can influence the modification of β V crystals to β'polymorph, which foment disorder in the emulsion matrix [6,47]. Therefore, the fatty acid composition affects liquid fat solidification, and thus the texture properties. The addition of isomalt in chocolate has been reported to increase hardness and fracturability [56]. However, the interaction between Sw and FO treatments decreased fracturability values, likely due to the increased concentration of PUFAs [3,55].

#### *3.3. Rheological Analysis: Shear Stress, Apparent Viscosity, and Frequency Sweep Test* 3.3.1. Flow Behavior

Rheological characteristics of chocolate are directly related to the quality attributes of the product [23]. Viscosity plays an important role in texture, flavor, and mouthfeel. Likewise, flow properties can be perceived by consumers in flavor and mouthfeel, since the perceived taste depends on the melting rate [6].

The variations of shear stress versus shear rate as well as apparent viscosity versus shear stress of milk chocolate formulations are shown in Figure 1. Probiotics' addition induced a significant increase in shear stress and apparent viscosity values, whereas sugar replacement (Sw) and FO addition evaluated individually did not affect shear stress values or apparent viscosity values. However, the Sw\*FO interaction significantly modified the rheological behavior of chocolate. Chocolates with added Prob showed a plunge more stable than the control (Figure 1A). Likewise, FO combined with Sw significantly modified the shear stress. For instance, Sw + FO treatment showed a similar flow behavior as compared with the control. Additionally, Prob showed the highest apparent viscosity values compared with the control and the other sucrose milk chocolate formulations (Figure 1B). Nevertheless, FO addition affected the apparent viscosity as well as the use of Sw. As the apparent viscosity decreased, the shear rate increased, which agrees with the pseudoplastic or shear-thinning nature of chocolate [22].

**Figure 1.** (**A**) Shear stresses were at a range of steady-shear rate 0.1 to 100 s<sup>−</sup><sup>1</sup> and temperature 35 ◦C. (**B**) Apparent viscosity was at a range of steady-shear rate 0.1 to 100 s<sup>−</sup><sup>1</sup> and temperature 35 ◦C. (**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* < 0.05, \*\*\* *p* < 0.001. Sw, sweetener; FO, fish oil; Prob, probiotic; NS, non-significant. 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.

The higher apparent viscosity observed in the Prob treatment could be attributed to an increase in the size and number of solid particles in the chocolate formulation. A study conducted by Afoakwa et al. [49] showed that the increase of an average particle size resulted in a decrease of Casson plastic viscosity, shear stress, yield stress, and apparent viscosity. Furthermore, previous reports have demonstrated that the addition of lyophilized probiotics increased rheological parameters and negatively affected chocolate flow properties [57,58].

As described earlier, the content and type of ingredients, such as the incorporation of PUFAs, have a critical role in chocolate viscosity. For instance, FO addition in chocolates induced a decrease in the shear stress since the fat in chocolate recovers solid particles, allowing an easy flow [23]. Similar observations were reported by Konar et al. [3], who

evaluated the addition of different sources of DHA/EPA, and the authors reported a decrease in shear stress.

On the other hand, sweeteners induced an increase in shear stress, indicating that sugar-free chocolate formulations did not reach a steady condition in their rheology (Figure 1A). Previous authors studied the rheology of chocolates with different added bulk sweeteners, including isomalt, and observed that the shear-thinning index changes between the control (chocolate with sucrose) and the different bulk sweeteners. Likewise, the authors concluded that each sweetener's structure interacts with other particles in the chocolate matrix in each void space [28,47]. Void spaces between cocoa particles and cocoa butter allow optimal rheology. When the void space is too tight, the shear-thinning index is increased, and the viscosity is reduced. Similar behavior occurs when adding isomalt. However, the special molecular conformation of isomalt allows more void spaces, reducing the shear-thinning index [28].
