*2.6. 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 [30–32]. 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).
