Sugar-Free Milk Chocolate as a Carrier of Omega-3 Polyunsaturated Fatty Acids and Probiotics: A Potential Functional Food for the Diabetic Population

Chocolate is an adequate matrix to deliver bioactive ingredients. However, it contains high sugar levels, one of the leading causes of chronic degenerative diseases. This work aimed to evaluate the effects of milk chocolate reformulation with alternative sugar sweeteners (Sw; isomalt + stevia), probiotics (Prob), and ω-3 polyunsaturated fatty acids (PUFAs) on its physicochemical properties and consumers’ acceptability. Lactobacillus plantarum 299v (L. p299v) and Lactobacillus acidophilus La3 (DSMZ 17742) were added as Prob strains, and fish oil (FO) was added as the source of ω-3 PUFAs. Prob addition resulted in chocolates with >2 × 107 colony forming unit (CFU) per serving size (12 g). Except for Prob, aw values of all treatments were <0.46. Sw and Sw + Prob presented the nearest values to the control in hardness, whereas Sw without FO increased fracturability. FO, Sw + FO, and Sw + Prob + FO contained 107.4 ± 12.84, 142.9 ± 17.9, and 133.78 ± 8.76 mg of ω-3 PUFAs per chocolate, respectively. Prob + FO increased the resistance of chocolate to shear stress, while Sw + FO showed a similar flow behavior to the control. The consumers’ acceptability of Sw + Prob chocolate was adequate, while Sw + Prob + FO had higher acceptability than Prob + FO. Health benefits of reformulated milk chocolates requires further assessment by in vitro, in vivo and clinical studies.


Introduction
Consumption of foods high in sugar is associated with the development of metabolic syndrome, which is defined as a collection of physiological, biochemical, and clinical factors, and is one of the leading causes of death worldwide [1]. Therefore, there is a need to develop new sugar-free products. Sweeteners are sugar substitutes, with natural sweeteners being more accepted in the market [2]. Additionally, there is an interest in the addition of bioactive ingredients to food formulations, in order to obtain food products that provide health benefits, including the prevention and treatment of diseases related to metabolic syndrome [3]. The term nutraceutical was coined in 1989 by Stephen DeFelice from the words "nutrition" and "pharmaceutical", and he defined it as a food or part of a food that provides health benefits, including the prevention and treatment of disease beyond basic nutritional functions [4]. Recently, the term nutraceutical was revisited to separate the concept of food supplements and nutraceuticals [5]. Food supplements are food-derived products that compensate the lack of specific components (i.e., vitamins and minerals) in the daily diet and/or can exert a beneficial effect on health without any

Bacterial Strains' Propagation, Microencapsulation, and Viability Assessment
Bacteria were propagated by inoculating an aliquot (100 µL) from a stock of Lactobacillus plantarum 299v (L. p299v) and a stock of Lactobacillus acidophilus La3 (DSMZ 17742) in 10 mL of MRS broth, which was incubated at 37 • C in a Shel lab 1535 incubator (VWR, Randor, PA, USA) for 16 h under aerobic conditions. Then, propagation was scaled-up to a final volume of 800 mL under the same incubation conditions. Bacteria cells were harvested by centrifugation (at 10,000× g, 25 • C for 15 min). Cell pellets were washed in peptone water (0.1% peptone, 0.85% NaCl, pH 7) and resuspended in a final volume of 30 mL in peptone water.
Suspended cells were added to 750 mL of microencapsulation mix (10% w/v maltodextrin, and w/v 2% food-grade alginate) and spray-dried (ADL 311S, Yamato Scientific Co., Ltd., Santa Clara, CA, USA) at 130 • C inlet, 60 • C outlet, and 0.13 MPa. The viability of probiotics was determined by homogenizing the powder with microencapsulated probiotics (0.1 g) or the chocolates with added probiotics (1 g), with 90 mL of peptone water preheated at 37 • C in a stomacher (IUL Instruments, Barcelona, Spain) for 90 s. Proper dilutions (10 4 , 10 6 , and 10 8 ) of each replicate were plated twice on MRS agar and incubated at 37 • C for 48 h, aerobically.

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. 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 13 UFC/g, resulting in chocolates with 2 × 10 7 CFU per serving size (12 g).
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].

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.

Sensory Evaluation
A sensory acceptability test was performed using the 9-point hedonic scale to assess the consumers' acceptability of chocolate formulation. A total of 223 students and staff (59% male and 41% female) from Tecnológico de Monterrey (Monterrey, NL, México) that consumed chocolate at least once a week were selected for the study, with ages ranging between 17 and 21 years old. Each chocolate sample was provided with a different random three-digit number. The samples were provided in different orders. Participants were asked to eat the chocolate samples one at a time, drink water, and eat a cookie with a plain flavor before the evaluation and between testing different samples. For each chocolate, the participants were requested to evaluate the attributes of appearance, flavor, texture, and overall acceptability using a 9-point hedonic scale ranging from 1 to 9: 1 = "dislike extremely," 2 = "dislike very much," 3 = "dislike moderately," 4 = "dislike slightly," 5 = "neither like nor dislike," 6 = "like slightly," 7 = "like moderately," 8 = "like very much," and 9 = "like extremely." To determine the microbial safety of chocolate samples before sensory evaluation, chocolate formulations were analyzed for total coliforms, yeast, molds, and Salmonella spp., according to methods previously reported in the literature [30][31][32]. Briefly, 10 g of each chocolate sample was introduced into a sample bag (Whirl-Pak ® , Nasco, Fort Atkinson, WI, USA), diluted with sterile peptone water (0.1 % peptone, 0.85% NaCl, pH 7), and homogenized for 2 min in a stomacher. Triplicate counts were performed for all dilutions. Total coliforms were determined using violet red bile agar and incubated at 37 • C for 24 h. Fungi and molds were grown in potato dextrose peptone agar and incubated at 25 • C for 5 days. All chocolates presented <10 CFU/mL for total coliforms, fungi, and molds.
For Salmonella spp. analysis, 25 g of chocolate sample was placed in 225 mL of reconstituted skim sterilized milk for 60 min at 25 • C. Then, 1 mL of each sample was put in 10 mL of Vassiliadis-Rappaport and in 10 mL of tetrathionate for 24 h. Salmonella spp. counts were performed in XLD agar and SS agar. Chocolate formulations were free of Salmonella spp., and thus, all chocolates were safe for human consumption and suitable for sensory evaluations (Ethics ID: CSERMBIGDL-002).

Statistical Analysis
Results are expressed as mean ± standard error of three independent measurements unless otherwise indicated. Data were analyzed with full factorial analysis of variance to evaluate main effects and interactions, followed by the LSD test to determine significant differences among groups (p < 0.05), using JMP software version 14.3.0 (SAS Inst. Inc., Cary, NC, USA).

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 13 CFU/g and 1 × 10 14 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 agent 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 agent 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 7 CFU per portion (12 g). This value is in the range of the minimum count of probiotic bacteria intake (≥1 × 10 6 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.

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 a w 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].

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 agreement 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.

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 agreement 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].

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].

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]. 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].

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.

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 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 agreement 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].

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 (C 22:6 , 14,122.2 ± 27.0 mg/100 g) and EPA (C 20:5 , 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].

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). 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 agreement 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.

Conclusions
In the present study, it was demonstrated that it is possible to formulate sugar-free milk chocolate formulations with added ω-3 PUFAs and probiotics, showing adequate acceptability by consumers. One of the drawbacks of the formulations evaluated was the decrease in acceptability by consumers when FO was added as an ingredient. Therefore, further studies should consider using lower concentrations of FO, or adding the ω-3 PUFAs from other sources, such as microalgae. The results presented herein support the idea that chocolate could be used as a good delivery system of bioactive ingredients, and thus further studies should evaluate the effect on these new chocolate formulations on the prevention of diseases through the evaluation of their efficacy by in vitro, in vivo and clinical studies.