*2.3. Chocolate Preparation*

Milk chocolates were prepared in a confectionery pilot plant factory as previously described [25]. Chocolates were formulated to develop a sugar-free product rich in *Lactobacillus plantarum* 299v (L. p299v)*, Lactobacillus acidophilus* La3 (DSMZ 17742), and ω-3 PUFAs (EPA and DHA). Eight milk chocolate formulations were tested using the same base (alkalized cocoa paste 12%, natural cocoa powder 3%, cocoa butter 26%, whole milk powder 13%, skim milk powder 10%, soy lecithin 0.3%, PGPR 0.2%, NaCl 0.08%, and vanilla 0.003% *w/w*). Sugar was replaced with a mixture of Iso and Stev as sweeteners (Table 1). Likewise, FO and probiotics (*L. plantarum* 299v and *L. acidophilus* La3) were added as indicated in the formulations shown in Table 1.


**Table 1.** Milk chocolate formulations added with probiotics and fish oil.

Abbreviations: Prob, probiotics; FO, fish oil; Sw, sweeteners; PGPR, polyglycerol polyricin; LMPF: low moisture powder fine.

> Each chocolate formulation was produced by the following procedure: (1) melting and heating, (2) coaching, (3) refining, (4) tempering, and (5) molding. In the melting step, a water bath at 40 ◦C was used; for the coaching and refining steps, the temperature was 25 ◦C, and the duration was 24 h using a chocolate refiner (Premier, Diamond Custom Machines Corp., Hillsborough Township, NJ, USA). The tempering step followed three changes of temperature. The first stage of tempering was maintained at 45 ◦C to melt fat crystals (3–5 min); then, in the second stage, chocolate was cooled at 27 ◦C under manual agitation using a spatula (3–5 min), and finally, chocolate was reheated to 29 ◦C. Chocolate formulations were molded at 14 ◦C for 1 h and stored at 11 ◦C until analysis. FO and microencapsulated probiotics were added to chocolate after tempering at 29 ◦C at a ratio of 1 × 10<sup>13</sup> UFC/g, resulting in chocolates with 2 × 10<sup>7</sup> CFU per serving size (12 g).

#### *2.4. Water Activity, Color, Texture, and Rheological Determinations*

Water activity a w of chocolate samples was measured using a water activity meter (Aqualab CX-2, Decagon Divices Inc., Pullman, WA, USA) at 25 ◦C using 3.0 g of the samples previously homogenized with a grinder (80350R, Hamilton Beach, Glen Allen, VA, USA). The color was determined with a spectrophotometer cm-600d (Konica Minolta Inc., Tokyo, Japan). Colorimetric parameters obtained (CIE *L\**, *<sup>a</sup>\**, and *b\**) were used to calculate the whiteness index (*WI\**) value, as indicated in Equation (1):

$$\mathcal{W}I^\* = 100 - \left[ (100 - L^\*)^2 + a^{\*2} + b^{\*2} \right]^{1/2} \tag{1}$$

Treatments: Control = milk chocolate formulation, Prob = milk chocolate + probiotics, FO = milk chocolate + fish oil, Prob + FO = milk chocolate + probiotics + fish oil, Sw = sugarfree chocolate formulation (with added isomalt + stevia as sweeteners), Sw + Prob = sugarfree chocolate + probiotics, Sw + FO = sugar-free chocolate + fish oil, Sw + Prob + FO = sugarfree chocolate + probiotics + fish oil.

Hardness and fracturability (N) of the samples were determined using a texture analyzer (TVT 6700, Perten Instruments, Sydney, NSW, Australia) equipped with a cylinder probe (height 45 mm, diameter 3 mm). The conditions used were: sample height: 8 mm; starting distance from sample: 5 mm; compression: 2 mm; initial speed: 0.5 mm/s; test speed: 0.5 mm/s; retract speed: 10 mm/s; trigger force: 5 g; data rate: 500 pps, at 25 ◦C [25–27]. Five replicates of each treatment were evaluated.

Rheological experiments (flow behavior, stress sweep, and frequency sweep test) were carried out with a previously reported protocol [25,28]. A controlled stress rheometer (Physica MCR 101, Anton Paar, Ostfildern, Germany) fitted with a parallel plate geometry (PP25/S, 24.973 mm diameter, 1.0 mm gap) was used. Chocolate samples were melted in a water bath at 35 ◦C and poured on the bottom plate based on the methodology previously reported [25,28].

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

Chocolate fat was extracted following the AOAC 948.22 Soxhlet method, using ethyl ether as the extraction solvent [29]. For each formulation, fat extraction was performed in triplicate from the chocolate bars (12 g). A sample of extracted fat (5 mg) was dissolved in a toluene-hexane mixture (0.6 mL, 1:1 *v*/*v*). Undecanoic acid (100 ppm) was added to samples as an internal standard for quantification. Subsequently, samples were derivatized using methanol-sulfuric acid (1 mL, 93:7 *v*/*v*) in capped vials placed in a water-bath (80 ◦C, 60 min). Thereafter, the samples were chilled, and the FAMEs were extracted with hexane and volume-adjusted (2 mL) for chromatographic analysis.

FAMEs profile was determined on a GC Agilent 6850A gas chromatograph coupled with a flame ionization detector (GC-FID, Agilent Technologies Inc., Santa Clara, CA, USA). The chromatography column employed was a fused-silica SP-2380 capillary column (100 m × 0.25 mm i.d., 0.2 μm film thickness, Supelco, Bellefonte, PA, USA). The chromatographic setup and FAMEs' identification and quantification were performed as previously reported by Faccinetto-Beltrán et al. [25]. Quantification for each compound and the total amount of fatty acids (FAs) were calculated by the AOAC method 996.06. Concentration of FAs were expressed as mg of each individual FA per 100 g of product on a fresh weight (FW) basis.
