2.4.1. Salmonella Mutagenicity Assay (Ames Test)

An Ames test was performed to test the antimutagenicity of cocoa beans extracts. Two Salmonella strains were used: the TA98 strain to remark frame-shift mutations and TA100 to evaluate base-pair substitutions due to missense mutations [16,17]. Both strains were from the permanent collection of the Laboratory of Hygiene and Environmental Toxicology, University of Campania ‘Luigi Vanvitelli’, Italy.

For the mutagenicity test, 100 μL of extracts (1000 μg/mL, chosen as the highest concentration), 100 μL of S. typhimurium TA98 or TA100 cultured overnight (10⁸ cells), and 500 μL of 0.1 M phosphate buffer (pH 7.4) were added to 2.5 mL of 0.5 M histidine–biotin top agar and then poured into minimal glucose agar in triplicate and incubated for 72 h at 37 °C. Furthermore, saline solution (0.9% NaCl) was used as negative control while 2-nitrofluoren (2-NF) at 2.5, 5, and 10 μg/mL and sodium azide (SOD) at 5, 10, and 20 μg/mL were used as standard mutagens for TA98 and TA100, respectively. Solvent controls were prepared also. After incubation, induced His⁺ revertants were counted after 72 h instead of 48 h to simplify the reading. The mutagenic ratio (MR) was calculated as follows:

A sample was considered mutagenic when MR ≥ 2.

Differently, for the antimutagenicity assessment, the samples (10, 50, and 100 μg/mL, with the highest concentration equal to 1/10 of the concentration used to evaluate the possible mutagenic effect, 1000 μg/mL) were previously co-incubated with increasing concentrations of standard mutagens (2.5, 10 μg/mL of 2-NF for TA98 and 5, 20 μg/mL of SOD for TA100) for 2 h at 37 °C. The results, coming from three independent experiments, were expressed as the percentage of the ability of the extracts to inhibit the action of the mutagen, which was calculated as suggested by Resende and colleagues [18]:

where T is the mean number of revertant colonies in the plates containing both mutagen and test extracts, and M is the mean number of revertant colonies in the plates containing only the mutagen.

According to Resende and colleagues [18], an inhibition lower than 25% was considered to indicate no antimutagenic effect, an inhibition value between 25% and 40% was considered to indicate a moderate effect, and values greater than 40% were considered to indicate strong antimutagenicity.