*3.1. Cytotoxicity of the Biosurfactant*

Cytotoxicity tests were among the first in vitro bioassays used to predict the toxicity of substances and are performed in laboratories throughout the world to classify compounds and evaluate safety [29]. The biosurfactant produced by *Bacillus cereus* UCP 1615 was submitted to the MTT assay to monitor the response of the cells in the cultures and determine the viability of the biosurfactant for human consumption.

The results of the cytotoxicity tests regarding the viability of the L929 (mouse fibroblast) cells and the Vero (renal epithelial) cells from the African green monkey exposed to different concentrations of the biosurfactant are displayed in Table 2.

**Table 2.** Viability (percentage) of L929 and Vero cell lines after contact with the biosurfactant from *Bacillus cereus* UCP 1615 at different concentrations (data expressed as mean ± SD of the triplicate determinations).


The viability of the L929 cells was 54% when submitted to the highest concentration of the biosurfactant (400 µg/mL) but above 93% when submitted to concentrations between 3.12 and 200 µg/mL. The viability of the Vero cells was 12.13, 74.77 and 86.05% when exposed to concentrations of 400, 200 and 100 µg/mL, respectively, but above 95% when exposed to concentrations between 3.12 and 50 µg/mL (Table 2). Substances that enable an 80% or higher cell viability rate are considered to be without cytotoxic activity [30].

The MTT results revealed that the biosurfactant may have a potential application in food as it did not exhibit cytotoxicity to either cell line at concentrations of up to 100 µg/mL, equivalent to 0.1 g/L. Moreover, the viability of the L929 cell line was 100% at the relatively high concentration of 200 µg/mL. These results are compatible with those reported by Ribeiro et al. [22] for the biosurfactant produced by *Saccharomyces cerevisiae* incorporated into a cookie formulation, which exhibited no toxicity to the L929 and RAW 264.7 (mouse macrophage) cell lines.

The use of the biosurfactant produced by *Candida bombicola* URM 3718 in a cupcake formulation was successful after the determination of its toxicity to the L929 and Vero cell lines at concentrations up to 50 µg/mL [31]. The survival rate of the BHK-21 cell line (kidney cells from hamster pups) was 63% after exposure to a biosurfactant produced by *Bacillus cereus* MMC at a concentration of 10<sup>4</sup> µg/mL [32]. The biosurfactant produced by *Lactobacillus helveticus* also exhibited no cytotoxicity to the L929 cell line at concentrations of up to 25 <sup>×</sup> <sup>10</sup><sup>3</sup> <sup>µ</sup>g/mL [33].
