**1. 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 proven biological effect. On the other hand, nutraceuticals should have a proven beneficial

pharmacological effect as a requirement [5]. In practical terms, as stated by Santini and Novellino, nutraceuticals should go beyond the diet, before the drug [5].

One of the food products experiencing more dynamic changes through this healthy demand is chocolate, since it represents 60% of the world's confectionery market and is liked by adults and children due to its sweet taste and pleasant mouthfeel [3,6]. Sugarfree chocolates usually use a combination of sweeteners with high sweet power, such as stevia (Stev), and sweeteners as bulking agents, such as isomalt (Iso) [7]. Both sweeteners (Stev and Iso) are considered prebiotics [8,9]. Prebiotics are defined as non-digestible food ingredients that are metabolized by gu<sup>t</sup> microbiota, improving host health [10]. Additionally, Stev is reported to exert beneficial effects on type 2 diabetes since this molecule interacts with intestinal and pancreatic cells, improving glucose uptake and helping to maintain glucose homeostasis [2,11].

The consumption of fish oil (FO) has been related to decreasing the risk of type 2 diabetes and other coronary diseases due to its high content of ω-3 PUFAs, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [12,13]. The World Health Organization [14] recommend a consumption of 250–500 mg per day of combined EPA and DHA for healthy adults. Furthermore, several studies sugges<sup>t</sup> that dietary ω-3 PUFAs from FO improve insulin sensitivity or reduce the incidence of type 2 diabetes thorough inhibition of adipose tissue inflammation [15].

Another bioactive ingredient that can be used to improve the health of the diabetic and non-diabetic populations are probiotics, since their consumption modulates gu<sup>t</sup> microbiota [16]. Probiotics are defined as live microorganisms that confer health-promoting properties when administrated in adequate amounts to the host [17]. In this context, *Lactobacillus platarum* and *Lactobacillus acidophilus* have demonstrated to improve the health of type 2 diabetes patients by balancing the gu<sup>t</sup> microbiota [18,19].

Chocolate could be an adequate vehicle for the delivery of probiotics and ω-3 PUFAs due to its main ingredients (cocoa butter, cocoa paste, soy lecithin, and milk) that generate a food matrix with low water activity, low oxygen tension, and low moisture permeability [20]. In addition, microencapsulation of probiotics provides double protection due to the covalently or ionically crosslinked polymer networks that enclose bacterial cells [21]. However, there are few reports in the literature on the development of functional sugar-free chocolates that could be consumed by the diabetic population.

The milk chocolate system comprises solid particles (cocoa, sugar, and milk powder) dispersed in the fat phase (cocoa butter). The composition of these ingredients affects the final sensory properties and rheological behavior as a fluid mass. To obtain highquality products, the determination of these properties in chocolate manufacture must be well-defined to obtain the right palatable products and fulfill consumers' preferences [22]. Rheological properties affect the final texture of chocolates, which plays a crucial role in the confectionery industry's elaboration process [23]. For instance, if chocolate viscosity is too low, the texture would not be optimal, and if it is too high, bubbles may appear in the molded tablet. In addition to modifying texture, viscosity also affects the flavor of chocolate because the taste depends on the order and rate of contact, which is dependent on viscosity and melt rate. Chocolate rheology is usually determined by yield stress and apparent viscosity parameters. Yield stress provides information related to the transition behavior from elastic to viscous deformation. Furthermore, sensory evaluation is also a key element to evaluate the elaboration process of chocolate and ensure high-quality products that reach consumers' preferences [24].

The objective of this study was to evaluate the effect of sugar substitution, probiotics and ω-3 PUFAs addition on the physicochemical properties and consumers' acceptability of milk chocolate. Sugar was replaced by isomalt (Iso) and stevia (Stev), whereas the probiotics (Prob) strains added were *Lactobacillus plantarum* 299v (L. p299v) and *Lactobacillus acidophilus* La3 (DSMZ 17742). Furthermore, fish oil (FO) was used as a source of ω-3 PUFAs.

#### **2. Materials and Methods**

#### *2.1. Bacterial Strains and Chemicals*

Probiotic strains *Lactobacillus plantarum* 299v (L. p299v) and *Lactobacillus acidophilus* La3 (DSMZ 17742) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and the American Type Culture Collection (ATCC, Manassas, VA, USA), respectively. Sodium alginate was purchased from Deiman (Guadalajara, JAL, Mexico) and food-grade maltodextrin was obtained from Best Ingredients (Monterrey, NL, Mexico). Alkalinized cocoa paste, alkalinized cocoa, cocoa butter, whey powder, soy lecithin, polyglycerol polyricinoleate (PGPR), NaCl, vanilla, and sugar were obtained from Escuela Mexicana de Confitería y Chocolatería (San Luis Potosí, SLP, Mexico). Isomalt low moisture powder fine (LMPF) was obtained from Palsgaard Industry de México S de RL de CV (San Luis Potosí, SLP, Mexico). Stevia was obtained from Grupo Químico Amillán S.A. de C.V. (Zapopan, JAL, Mexico). Fish oil (Omega Pure®) was purchased from America Alimentos S.A. de C.V. (Zapopan, JAL, Mexico). For the fatty acid methyl esters profile determination, toluene-hexane mixture (1:1 *v*/*v*), undecanoic acid (100 ppm), and external standard fatty acid mixtures GLC 566 (39 fatty acid methyl esters) were purchased from Nu Chek Prep Inc (Elysian, MN, USA). Finally, for microbiological determinations, reconstituted skim milk (Svelty, Nestlé®) was obtained from a local market, whereas Violet Red Bile Agar (VRB agar), potato Dextrose Peptone Agar (DP agar), Xylose Lysine Deoxycholate Agar (XLD agar), Salmonella Shigella Agar (SS agar), Tetrathionate Broth Base, Rappaport Vassiliadis Broth, VRBA agar, and MRS agar were obtained from Sigma-Aldrich® (St. Louis, MO, USA).

#### *2.2. 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 (104, 106, and 108) of each replicate were plated twice on MRS agar and incubated at 37 ◦C for 48 h, aerobically.
